Citation
Isolation and Characterization of Equine Umbilical Cord Blood Derived Stem Cells

Material Information

Title:
Isolation and Characterization of Equine Umbilical Cord Blood Derived Stem Cells
Creator:
Reed, Sarah
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (171 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Animal Molecular and Cellular Biology
Committee Chair:
Johnson, Sally
Committee Members:
Ealy, Alan
Moore, Karen
Brown, Murray P.
Graduation Date:
8/8/2009

Subjects

Subjects / Keywords:
Blood ( jstor )
Bone marrow ( jstor )
Cells ( jstor )
Cultured cells ( jstor )
Embryonic stem cells ( jstor )
Horses ( jstor )
Mesenchymal stem cells ( jstor )
Stem cells ( jstor )
Tendons ( jstor )
Umbilical cord ( jstor )
Animal Molecular and Cellular Biology -- Dissertations, Academic -- UF
foal, horse, oct4, stem, tendon, umbilical
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Animal Molecular and Cellular Biology thesis, Ph.D.

Notes

Abstract:
Musculoskeletal injuries are responsible for a large portion of wastage in sport horses. Mesenchymal stem cells (MSCs) offer promise as therapeutic aids in the repair of tendon, ligament, and bone damage suffered by these horses. The objective of these studies was to identify and characterize stem-like cells from newborn foal umbilical cord blood (UCB). UCB was collected and MSC isolated using human reagents. The cells exhibit a fibroblast-like morphology and express the stem cell markers Oct4, SSEA-1, Tra1-60 and Tra1-81. UCB express transcripts implicated in embryonic stem (ES) cell pluripotency, namely Oct4, nanog, Sox2, Klf4 and c-myc. Culture of the cells in tissue-specific differentiation media leads to the formation of cell types characteristic of mesodermal and endodermal origins including chondrocytes, osteocytes, and hepatocytes. Limited adipogenic and myogenic differentiation occurred. Population doubling time and the presence of Oct4, nanog and Sox2 transcripts were used to determine culture conditions that promoted the proliferation of a stem cell population. Culture on a protein matrix (gelatin, collagen or fibronectin) shortened population doubling time compared to growth on uncoated plasticware. Inclusion of fibroblast growth factor 2 (FGF2) in the growth media slowed proliferation. The persistence of Oct4, nanog and Sox2 expression was monitored over time in culture in UCB stem cells. Oct4 was detected throughout the duration of the experiment. Sox2 and nanog expression declined with time in culture. Finally, the effect of culture conditions on expression of tendon markers was assessed. Initial stem cell populations express scleraxis, an early marker of tenocytic differentiation. Culture on collagen coated beads did not affect scleraxis levels; however culture in 30% Matrigel significantly increased levels of the transcript. This change was accompanied by differences in morphology. Cells grown in Matrigel formed tight colonies reminiscent of ES cell colonies while those on collagen coated beads maintained a fibroblast-like morphology. In conclusion, we have isolated a population of stem cells from equine umbilical cord blood that possess a number of stem cell markers, can be expanded in culture, and can differentiate into a variety of potentially useful cell types. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2009.
Local:
Adviser: Johnson, Sally.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31
Statement of Responsibility:
by Sarah Reed.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Reed, Sarah. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
8/31/2010
Resource Identifier:
489258205 ( OCLC )
Classification:
LD1780 2009 ( lcc )

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Full Text





higher levels of sex (p<0.0001; Figure 5-9A). However, Sex tended to be decreased by

supplementation with FGF2. Levels of TnC mRNA are unaffected by culture on different

matrices or the inclusion of FGF2 or FGF5 (Figure 5-9B).

In AdMSC, sex expression was increased by culture in matrigel but decreased by culture

on collagen beads (p<0.0001; Figure 5-9C). When cultured on gelatin or matrigel, FGF5

supplementation increased TnC transcription in AdMSC (p<0.0001). Inclusion of FGF2 in the

culture media of cells grown in matrigel increased TnC transcription but to a lesser extent than

caused by FGF5 (Figure 5-9D). Inhibition of the MAPK signaling pathway by PD98059 did not

reverse the changes in scleraxis or tenascin C mRNA caused by FGF2 or FGF5 stimulation in

adipose or umbilical cord blood derived stem cells (data not shown).

Effect of FGF5 Supplementation on Actin Structure

To determine if FGF5 supplementation resulted in changes in cell morphology and actin

structure, UCB were stripped of extracellular signaling molecules using 10 [tg/ml protamine

sulfate for 10 minutes. Cells were placed in serum free media for one hour prior to the addition

of 10ng/ml rhFGF5 or growth media. Those cells receiving neither growth media nor FGF5

were used as controls. UCB stem cells were fixed in 4% paraformaldehyde at times 0, 10, and

30 minutes of treatment prior to permeabilization with 0.1% TritonX100 in phosphate buffered

saline (PBS) supplemented with 5% FBS. Actin filaments were immunostained with fluorescein

conjugated phalloidin and Hoechst 33342. Morphology was visualized using a Nikon T200

microscope equipped with epifluorescence. Images were captured with NIS Elements (Nikon

Instruments, Melville, NY) software and compiled with Adobe PhotoShop CS.

No appreciable differences were visible in UCB receiving any treatment (Figure 5-10). All

cells maintained similar sizes. No treatment resulted in the formation of extensive filopodia or









and coordinated with the body plan. Cell fate determinants are localized asymmetrically along

this axis. During mitosis, the mitotic spindle is oriented along the axis such that cytokinesis

creates two daughter cells containing different concentrations of these determinants. In

Drosophila germ line stem cells, differential positioning of the mother and daughter centrosomes

during mitosis determines the fate of each resulting cell. The mother centrosome is anchored to

the niche by astral microtubules. The cell containing the daughter centrosome migrates away

from the niche following cytokinesis and differentiates (Yamashita et al., 2003; Spradling and

Zheng, 2007; Yamashita et al., 2007). In neural stem cells, asymmetrical localization of cellular

proteins influences cell fate. Numb, a negative regulator of Notch signaling is localized

asymmetrically at one pole of mitotic spindle in the sensory organ precursor cell of the

Drosophila peripheral nervous system such that only one daughter cell inherits the protein (Rhyu

et al., 1994). In this system, Numb influences cell fate by repressing Notch signaling. The

primary function appears to be the creation of two daughter cells that can respond differently to

external cues rather than to specify a specific cell fate (Rhyu et al., 1994). Using

videomicroscopy, Li et al report that 30-40% of mouse neuroepithelial cells segregate Numb to

one daughter cell during division (Li et al., 2003). This is correlated with asymmetric division to

one neuroepithelial cell and one neuron. In addition, 18% of cortical precursor cells divided and

expressed Notchl asymmetrically. Notchl was associated with the basal cell of the dividing pair

(Chenn and McConnell, 1995).

Hematopoietic stem cells (HSC) provided the first indication that stem cells were capable

of self renewal through asymmetric division. Colony formation assays of HSC demonstrated

that approximately 20% of progenitors divide asymmetrically (Suda et al., 1984a; Suda et al.,

1984b). In a population of CD34+ HSC, one daughter remained quiescent or divided very









slowly while the other multiplied quickly and yielded committed progenitors (Huang et al.,

1999). Slow dividing fractions of HSC are associated with primitive function and self renewal

while the fast dividing fraction proceeds to differentiation (Huang et al., 1999; Cheng et al.,

2000; Punzel et al., 2002). Using time lapse microscopy, Wu et al showed that hematopoietic

precursor cells are capable of symmetric commitment, symmetric self renewal, and asymmetric

division (Wu et al., 2007c). Additionally, overexpression of Numb in these cells resulted in fewer

lineage negative cells. In addition, HSC self renewal is mediated at least in part by the stem cell

niche. Long term repopulating cells (LTRC; CD34+CD38-) are located in the HSC niche (the

endosteal region associated with bone lining osteoblasts or endothelial cells) in a quiescent state

(Yahata et al., 2008). Approx 75% of the most primitive long term repopulating hematopoietic

stem cells are resting in GO (Cheshier et al., 1999). Following isolation, these cells were capable

of successful engraftment and reconstitution of hematopoiesis (Yahata et al., 2008). Clonally

distinct LTRC controlled hematopoietic homeostasis and created a stem cell pool hierarchy by

asymmetric self renewing division that produced both lineage restricted, short term repopulating

cells and LTRCs (Yahata et al., 2008). Quiescent LTRC clones can expand to reconstitute the

hematopoiesis of the secondary recipient (Yahata et al., 2008).

More restricted adult stem cells are also capable of self renewal. Muscle satellite cells that

are Pax7+/Myf5- can give rise to Myf5+ cells when dividing in the basal/apical orientation

(Kuang et al., 2007). Myf5 expressing cells lay next to fiber in a position to differentiate and fuse

while null cells remain next to basal lamina in a position to remain as a precursor cell.

Committed cells expressed Delta], while the more naive progenitor cells did not. Conversely,

Notch3 was highly expressed in the progenitor but not committed cells. Notch 1, 2 and Numb

levels were equal in both populations. Using videomicroscopy, Shinin et al examined the





















603bp-
310 bp





603bp-
310 bp


I



...





g em:


Figure 4-3. UCB and AdMSC express a limited number of molecules in the Notch signaling
pathway. RT-PCR was performed for the transcripts listed. mES = mouse embryonic
stem cells, UCB = umbilical cord blood derived stem cells, AdMSC = adipose
derived stem cells









Finally, I am supremely thankful to my husband, Jared, who has been there through all of

the tears, successes, late night blood collections and long work hours. He had faith that I would

succeed even when I lost mine. Even when he didn't understand what, he understood why and

stood behind me the whole way. His unfailing support is the only reason I stand where I do

today. He is the love of my life, my partner, my other half, my husband and I am the luckiest

girl in the world to have found him.









Cheshier, S.H., Morrison, S.J., Liao, X., and Weissman, I.L. (1999). In vivo proliferation and
cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proceedings of the
National Academy of Sciences of the United States of America 96, 3120-3125.

Chew, J.L., Loh, Y.H., Zhang, W., Chen, X., Tam, W.L., Yeap, L.S., Li, P., Ang, Y.S., Lim, B.,
Robson, P., and Ng, H.H. (2005). Reciprocal transcriptional regulation ofPou5fl and Sox2
via the Oct4/Sox2 complex in embryonic stem cells. Molecular and cellular biology 25,
6031-6046.

Chiquet, M., Sarasa-Renedo, A., and Tunc-Civelek, V. (2004). Induction oftenascin-C by cyclic
tensile strain versus growth factors: distinct contributions by Rho/ROCK and MAPK
signaling pathways. Biochimica et biophysica acta 1693, 193-204.

Choi, S.C., Kim, S.J., Choi, J.H., Park, C.Y., Shim, W.J., and Lim, D.S. (2008). Fibroblast
growth factor-2 and -4 promote the proliferation of bone marrow mesenchymal stem cells
by the activation of the PI3K-Akt and ERK1/2 signaling pathways. Stem cells and
development 17, 725-736.

Clase, K.L., Mitchell, P.J., Ward, P.J., Dorman, C.M., Johnson, S.E., and Hannon, K. (2000).
FGF5 stimulates expansion of connective tissue fibroblasts and inhibits skeletal muscle
development in the limb. Dev Dyn 219, 368-380.

Clegg, P.D., Strassburg, S., and Smith, R.K. (2007). Cell phenotypic variation in normal and
damaged tendons. International journal of experimental pathology 88, 227-235.

Clements, D.A., Wang, J.K., Dionne, C.A., and Goldfarb, M. (1993). Activation of fibroblast
growth factor (FGF) receptors by recombinant human FGF-5. Oncogene 8, 1311-1316.

Crovace, A., Lacitignola, L., De Siena, R., Rossi, G., and Francioso, E. (2007). Cell therapy for
tendon repair in horses: an experimental study. Veterinary research communications 31
Supply 1, 281-283.

Cserjesi, P., Brown, D., Ligon, K.L., Lyons, G.E., Copeland, N.G., Gilbert, D.J., Jenkins, N.A.,
and Olson, E.N. (1995). Scleraxis: a basic helix-loop-helix protein that prefigures skeletal
formation during mouse embryogenesis. Development (Cambridge, England) 121, 1099-
1110.

Cummings, B.J., Uchida, N., Tamaki, S.J., Salazar, D.L., Hooshmand, M., Summers, R., Gage,
F.H., and Anderson, A.J. (2005). Human neural stem cells differentiate and promote
locomotor recovery in spinal cord-injured mice. Proceedings of the National Academy of
Sciences of the United States of America 102, 14069-14074.

Dani, C., Chambers, I., Johnstone, S., Robertson, M., Ebrahimi, B., Saito, M., Taga, T., Li, M.,
Burdon, T., Nichols, J., and Smith, A. (1998). Paracrine induction of stem cell renewal by
LIF-deficient cells: a new ES cell regulatory pathway. Developmental biology 203, 149-
162.









Aim 1: Stem cells can be isolated from equine umbilical cord blood and cultured using methods

currently used in human medicine and research. Equine UCB stem cells express markers and

morphology similar to other mesenchymal stem cells.

Aim 2: Equine UCB have the potential to contribute to tendon injury and repair. Stem cells were

examined for expression of early tenocytes. Tenocytic differentiation was promoted by culture

on a variety of protein matrices and in growth factor containing media.









Protein Isolation and Evaluation

Equine UCB and AdMSC were treated with 10 pg/ml protamine sulfate for 10 minutes to

remove signaling molecules from the extracellular matrix. Cells were placed in serum free

media for one hour prior to stimulation with FGF2, FGF4, or FGF5 in the presence or absence of

the MEK inhibitor PD98059. Cells were lysed directly into SDS PAGE sample buffer. Protein

was loaded based on equal cell number and electrophoresed across a 10% polyacrylamide gel.

ERK1/2 activity was assessed using phospho- and total-ERK1/2 antibodies (Cell Signaling

Technologies, Danvers, MA). Briefly, proteins were transferred to nitrocellulose and non-

specific binding sites were blocked with 10% non-fat dry milk in TRIS-buffered saline

supplemented with 0.1% Tween20 (TBS-T). Blots were incubated overnight at 4 OC with

primary antibody (1:1000) in blocking solution. Following extensive washing, blots were

incubated with secondary antibody (1:2000). Equal protein loading was ensured by probing

membranes with anti-tubulin (Abcam, Cambridge, MA) for one hour (1:2000) followed by

incubation in secondary antibody for one hour (1:5000) prior to visualization. Immune

complexes were visualized by chemiluminescence (ECL; GE Life Sciences, Piscataway, NJ) and

autoradiography.

Assessment of Proliferation

Cells were seeded at equal density and cultured in low serum medium (2% FBS)

supplemented with FGF2, FGF4 or FGF5 in the presence or absence of PD98059 for 48 hours.

Prior to fixation, cells were pulsed with 10 [iM bromodeoxyuridine (BrdU) for two hours.

Proliferation index was determined as the proportion of cells expressing BrdU:total cell number.

All experiments were performed on UCB collections from four horses in duplicate wells with

two replicate experiments.









Fibroblast Growth Factors Elicit Differing ERK1/2 Responses in UCB and AdMSC

Not only can FGF stimulation promote mitogenesis in stem cells, but FGF signaling

through the MAPK pathway can lead to downstream regulation of scleraxis and tenascin C.

Thus, the ERK1/2 response was examined in UCB and AdMSC following stimulation by FGF2,

FGF4, or FGF5. FGF2 elicited ERK1/2 phosphorylation in both cell types, albeit with differing

activation kinetics (Figure 5-7). FGF4 did not activate pERK1/2 in UCB, however was

responsible for a slight activation in AdMSC. Further experiments with FGF4 were discontinued

due to lack of a MAPK response in UCB. Stimulation with FGF5 elicited a transient response in

both AdMSC and UCB with similar kinetics. These results indicated that both UCB and AdMSC

are capable of downstream signaling elicited by the fibroblast growth factors.

The mitogenic effects of the fibroblast growth factors have been shown in adipose and

bone marrow derived stem cells. To clarify the effect of the fibroblast growth factors on UCB

and AdMSC proliferation, cells were cultured for 48 hours in media supplemented with FGF2 or

FGF5 in the presence or absence of the MEK inhibitor PD98059 (Figure 5-8). Cells were pulsed

with BrdU prior to fixation and quantification. Similar to bone marrow derived stem cells,

AdMSC increased proliferation when cultured in the presence of FGF2. This effect was

abrogated by the presence of PD98059. In contrast, supplementation of FGF2 to UCB retarded

BrdU incorporation in a MAPK dependent manner. Supplementation of AdMSC with FGF5

inhibited proliferation, contrasting with the increased proliferation of UCB. Inclusion of the

MEK inhibitor PD98059 reversed the effects of FGF5 in both cell types.

To determine the combined effects of three dimensional culture and stimulation of the

fibroblast growth factors on stem cell identity, UCB and AdMSC were cultured on gelatin,

collagen beads and matrigel and stimulated with FGF2 or FGF5 for 48 hours. Real-time PCR

was used to quantify changes in sex and TnC expression. UCB cultured on matrigel express














--U
















Figure 3-1. Foal UCB cells express stem cell marker proteins. UCB stem cells (passage 5) were
fixed and incubated with antibodies directed against Oct4, SSEA-1, Tral-60 or Tral-
81. Immunoreactivity was detected with goat anti-mouse AlexaFluor 488 or anti-
rabbit AlexaFluor 568 (Oct4). Nuclei were counterstained with Hoechst 33245. Scale
bar= 10 tm.









ERK1 than AdMSC which may allow them to better respond to other mitogenic factors. This

may suggest that a more naive stem cell may have a higher ratio of ERK1 :ERK2 than more

differentiated cell types. These cells may be poised to respond to extracellular factors differently

than AdMSC.

The expression of low levels of scleraxis in immature cells is not surprising, as many stem

cells exist in a state poised for differentiation and transcribe low levels of mRNAs required for

that transformation. Not only is scleraxis upregulated in cells grown on Matrigel, but the

morphology of the cells changes drastically. The response of UCB to Matrigel is exciting and

presents many possibilities for future research. The changes in gene transcription may or may

not be directly related to the change in morphology. Integrin signaling and interaction with other

ECM components likely result in changes that may allow differentiation. The growth factor

concentrations and/or combinations present in Matrigel may signal for transcriptional changes

independent of the structure of the extracellular matrix. Alternatively, the changes may result

from changes in tensional stress, ligand:receptor interactions with the extracellular matrix, or

differences in cell:cell contact. Likely, a combination of stimuli allow for the formation of a

cluster of cells similar to tendon precursor cells. Complete differentiation is not recorded at this

stage, likely for a variety of reasons. The combination of growth factors that initiates early

tendon precursor development is likely not sufficient for complete maturation. Another

combination or different ratios of growth factors could help complete the transition. In vivo,

tendon cells are subjected to a high degree of strain on a daily basis as a result of movement.

Many cell types require cyclic or tensional stress to completely differentiate and it would be

extremely surprising were it not also true for tendon cells. Adding strain to UCB cultured in









Control


BMP6 L685,458 BMP6 + L685,458


SMyHC


E Hoechst


76% 7.9% 86% 35% %MyH
(+)nuclei

Figure 4-6. BMP6 inhibits myoblast differentiation in a Notch dependent manner. 23A2
myoblasts were placed in differentiation media in the presence or absence of 50 ng/ml
BMP6 and/or the Notch inhibitor L685,458. Cells were fixed after 48 hours and
immunostained for myosin heavy chain. Images were captured at 200x. The percent
of cells expressing myosin heavy chain was determined and is reported below its
respective panel. MyHC = myosin heavy chain









cell type, (2) a small population of naive stem cells express these markers and this cell type is

essentially diluted out in culture due to slower proliferation, (3) a small population of naive stem

cells exist but are not maintained due to inappropriate culture conditions. High density, early

passage cultures contain a small number of dense colonies with morphology reminiscent of ES

cell colonies. These colonies may contain cells that are more naive than those that grow in

monolayer culture. It is tempting to speculate that the cells which form these colonies are those

that contain the stem cell markers found in early cultures, but with passage are greatly

outnumbered by other cell types. This is further supported by the notion that generally more

naive stem cells proliferate slowly and thus would not be expected to create as many daughter

cells as the cells which grow on a monolayer. Thus, the colony forming cells may be the true

stem cells while those which grow in a monolayer are more limited precursor cells. Further

work should evaluate the subpopulations within UCB stem cells to identify cells that may be

more or less useful in various therapeutic settings or as a model for human medicine.

The differential response of AdMSC and UCB to stimulation by fibroblast growth factors

further highlights the differences between the two cell types. It is interesting to note that while

both respond to FGF2 and FGF5 stimulation by phosphorylating ERK1/2 and activating the

MAPK pathway, there are some significant differences in the type of response. In general,

AdMSC appear to have less total ERK1 than UCB, resulting in a different ratio of the two

kinases. This may be a reflection of cells poised either for terminal differentiation or for

proliferation prior to differentiation. In other cell types, strong phosphorylation of ERK2 occurs

in response to differentiation signals, while ERK1 responses are associated with proliferation.

The response of ERK1 to stimulation by FGFs was weak in both cell types; ERK2

phosphorylation in response to FGF was predominant. However, UCB retain higher amounts of









Kuroda, T., Tada, M., Kubota, H., Kimura, H., Hatano, S.Y., Suemori, H., Nakatsuji, N., and
Tada, T. (2005). Octamer and Sox elements are required for transcriptional cis regulation
ofNanog gene expression. Molecular and cellular biology 25, 2475-2485.

Le Guen, L., Notamicola, C., and de Santa Barbara, P. (2009). Intermuscular tendons are
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conditions and over time in culture. Cells maintained Oct4 expression throughout the duration of

culture, regardless of media or substrata used. In contrast, the presence of both Nanog and Sox2

decreased with time in culture. The presence of a protein matrix prolonged expression of both

nanog and Sox2 transcripts. This was not unexpected, as most stem cells reside in a very specific

niche, a large part of which is composed of extracellular matrix. Extracellular matrix in the stem

cell niche provides not only support for adhesion, but instructive signals to maintain the naive

state (Bi et al., 1999; Chen et al., 2007). Additionally, UCB cultured on various substrata had

decreased population doubling times compared to those on uncoated plasticware. As stem cells

divide, one daughter cell may proliferate rapidly to expand the progenitor cell population while

the other divides much more slowly or not at all to maintain the stem cell population. Thus,

slowly dividing stem cells are considered to be the more naive population. However, when

cultured for use as a therapeutic aid, cultivation of UCB on a protein substrate decreased

population doubling time. This is a beneficial property when considering the expansion of these

cells in culture prior to use as a therapeutic aid. The loss of Nanog and Sox2 during extended

culture highlights the need for short expansion periods prior to use as a cytotherapeutic tool.

Interestingly, despite the increase in population doubling time of cells cultured in media

containing FGF2, there was no effect of FGF2 on the transcript profile of UCB. While FGF2

appears dispensable for the maintenance of pluripotency markers, its negative effects on

population doubling time preclude its use in media for UCB expansion.

In summary, we defined culture conditions sufficient to ensure ES-like gene expression

patterns for early passage equine UCB stem cells. These include maintenance on matrix protein

coated surfaces and cultivation in fetal bovine serum. Genetic markers of pluripotency declined









injured in traumatic spinal cord injuries. Using a porous scaffold of PTCP, autologous bone

marrow derived MSC and autografts from the iliac crest fused the L4 and L5 vertebrae similarly

in the Cynomolgus monkey (Orii et al., 2005). However, bone formation assessed by micro

computed tomography showed greater new bone formation at 12 weeks in BMSC engrafted

vertebrae than those receiving autografts. Likewise, in an ovine model of vertebrae injury,

higher fusion was apparent in implants of BMSC on a PTCP scaffold than autografts (Gupta et

al., 2007). Newly formed woven and lamellar bone trabeculae in a cancellous organization with

evidence of remodeling was observed in BMSC/PTCP grafts. Formation of hematopoietic and

fatty marrow tissue was also apparent. Using a canine cancellous bone matrix transplanted with

BMSC, superior fusion volume and fusion area for mineralized and demineralized matrix was

observed over graft alone (Muschler et al., 2003). In a clinical trial, bone grafts saturated with

uncultured autologous BMSC were implanted into acute thoracolumbar fractures (Faundez et al.,

2006). The resorbable matrix was replaced with new bone containing several active foci of

membranous and/or endochondral ossification.

Cardiovascular Repair

In rabbits with surgical myocardial infarctions, hBMSC were injected into the border area

of myocardial ischemia (Wang et al., 2005a). Lower mortality was reported in the cell transplant

group compared to controls. Surviving patients had higher ejection fractions. Engrafted cells

differentiated into cardiac troponin I expressing cells. In a canine model of cardiovascular

disease, labeled canine BMSC were injected into a chronically infarcted myocardium due to a

permanently ligated coronary (Bartunek et al., 2007). Labeled cells expressed cardiac specific

myosin or troponin I by four and twelve weeks after injection. Newly differentiated

cardiomyocyte-like cells were observed within the fibrotic area of infarction. Transplanted

BMSC expressed connexin43, consistent with the integration into the host tissue. No









transplants and 67% in bone marrow grafts (Brodke et al., 2006). Goats with tibial bone defects

that received autologous BMSC on a beta-tricalcium phosphate scaffold achieved full union by

32 weeks post surgery (Liu et al., 2008). At this point, the ceramic scaffold was almost

completely resorbed and the engrafted bone had reached similar biomechanical properties as a

normal tibia. Little callus was observed at 16 or 32 weeks in acellular grafts, which were not

resorbed and did not heal during the 32 week trial. hBMSC implanted into a rat femur defect on

hydroxyapatite/pTCP cubes exhibited more bone formation than grafts alone (Bruder et al.,

1998). By eight weeks, the BMSC seeded graft had significant bone formation within the pores

of the scaffold and were integrating into the ends of the host bone. Union was complete by

twelve weeks. New woven and lamellar bone were detected in close contact with the edges of the

defect and were directly contiguous. Defects corrected with cell seeded grafts exhibited

increased strength and stiffness in biomechanical testing compared to grafts alone. Pores of the

acellular grafts were primarily filled with fibrous tissue at twelve weeks. Following lunate

arthroplasty, autologous rabbit BMSC were seeded on a scaffold of hyaluronan and gelatin prior

to transplantation into the surgical defect (Huang et al., 2006). Removal of the entire lunate

resulted in carpal collapse. However, rabbits receiving the implant presented evidence of new

bone formation twelve weeks after surgery. Scaffold only implants showed no bone formation.

Repaired tissue contained intensely stained metachromatic matrix and islands of ossicles within

the lunate space. Endochondral ossification had occurred in central areas of the scaffold seeded

with BMSC by twelve weeks with chondrocytes being replaced by woven bone. In a clinical

trial, Marcacci et al report that patients injected with autologous BMSC into bone defects

resulting from trauma exhibit callus formation between the host bone and hydroxyapatite

cylinder implant after one to two months (Marcacci et al., 2007). Complete resorption occurred









is performed as soon as possible after collection, generally within 15 hours (Bieback et al.,

2004). Prolonged time from collection to processing may decrease the number of cells obtained.

Blood is diluted in a buffered saline solution prior to separation of the mononuclear layer by

density gradient centrifugation (Sparrow et al., 2002; McGuckin et al., 2003; Romanov et al.,

2003; Bieback et al., 2004; Gang et al., 2004a; Lee et al., 2004a; Chang et al., 2006; Kern et al.,

2006). The buffy coat, containing the mononuclear cells, is collected and subjected to at least

two further washes in saline solution or medium. Lysing of the red blood cells may be

performed at this time. Cells are then plated in expansion medium which is typically composed

of Dulbecco's modified eagle medium (DMEM), 10-20% fetal bovine serum (FBS), and

penicillin/streptomycin. Some researchers suggest the addition of various growth factors and

supplements (Sparrow et al., 2002; Romanov et al., 2003; Bieback et al., 2004; Gang et al.,

2004a; Lee et al., 2004a; Kang et al., 2005; Wagner et al., 2005; Chang et al., 2006; Kern et al.,

2006). Fibroblast like colonies can be seen two to four weeks after plating (Bieback et al., 2004;

Kern et al., 2006). Upon confluency, UCB can be detached with trypsin-EDTA and subcultured.

In contrast to stem cells derived from bone marrow, most UCB populations show an increased

(but not indefinite) potential for proliferation (Bieback et al., 2004).

Differentiation

In an effort to prove the plasticity of umbilical cord blood derived stem cells in relation to

other stem cell populations, in vitro and in vivo differentiation protocols have been performed.

UCB have been successfully differentiated into cells from all three germ layers. Mesodermal

cell types, such as osteoblasts, chondrocytes, and adipocytes, are most commonly used to

identify a stem cell's ability to differentiate. However, differentiation into cells of the

ectodermal or endodermal layer tends to be more difficult.









regardless of substrata or media (Table 4-3). However, nanog and Sox2 mRNA decline with

serial passage. Expression of both genes is affected by substrata, maintaining expression longer

on a protein matrix (particularly collagen) than on uncoated plasticware. Media containing

FGF2 does not affect the maintenance of either nanog or Sox2 mRNA. The presence of nanog

transcripts was significantly different among horses, suggesting an innate heterogeneity among

animals.

Notch Signaling in UCB Stem Cells

Notch signaling plays a crucial role in cell:cell communication among mature and naive

cells. In human ES cells, constitutive Notch signaling promotes differentiation, particularly to

neural cell lineages (Lowell et al., 2006). In hematopoietic stem cells (HSCs), constitutive

activation of the Notch pathway including downstream target Hes led to an increased self-

renewal capacity of long term in vivo repopulating HSCs (Stier et al., 2002; Kunisato et al.,

2003). The Notch signaling pathway consists of a transmembrane receptor, Notch, which is

cleaved upon the binding of an extracellular ligand. The ligands for Notch are generally also

membrane bound to adjacent cells. There are three Jagged proteins and two Delta-like ligand

(Dll) proteins that serve to activate Notch signaling. Upon ligand binding, Notch undergoes a

series of proteolytic cleavages, resulting in the release of the Notch intracellular domain (NICD)

which relocates to the nucleus and acts as a transcriptional regulator.

To determine what members of the Notch pathway were present in UCB and AdMSC, and

to compare that expression with the pathway present in mES, RT-PCR was performed for

jagged-], jagged-2, notch-], notch-2, notch-2, Dll-1, and Dll-2. Mouse ES exhibited amplicons

for all transcripts examined (Figure 4-3). Notch-], Notch-3 and Jagged-] were present in UCB

stem cells, indicating the possibility for a functional pathway in these cells. However, only the
















Collagen Bead


Figure 5-5. UCB and AdMSC attach to various culture surfaces. UCB and AdMSC were
cultured on gelatin coated plasticware, 30% Matrigel, or on collagen coated beads for
48 hours prior to fixation. Actin structures were stained with fluorescein conjugated
phalloidin and nuclei were identified with Hoechst 33342 dye. Immunofluorescence
was visualized using a TCS SP5 Laser Scanning Confocal Microscope running Leica
LAS-AF. Scale bar = 10 [m


Gelatin


30% Matrigel









Implants containing gel alone had much less area of regeneration coupled with the presence of

fibrous tissue and less intense staining of extracellular matrix for Safranin O. BMSC injected

directly into a full thickness defect of the articular cartilage of the left distal femur in 16 New

Zealand white rabbits showed smooth, consistent white tissue resembling articular cartilage in

the regenerating area (Im etal., 2001). In contrast, control animals exhibited red, irregular

tissue with the margin of the defect sharply differentiated from the surrounding normal cartilage.

Defects in the group receiving BMSC had relatively normal surfaces with adjacent normal

cartilage showing little degenerative change. Control animals showed degenerative changes in

adjacent cartilage, thin and undifferentiated defects, and decreased cellularity. Collagen matrices

containing type I, type II and type III collagen seeded with ovine BMSC were used to fill

surgical defects in the medial condyle of the femur (Dorotka et al., 2005). Defects treated with

cell seeded matrices appeared more filled than control defects and had the largest quantity of

hyaline cartilage. Autologous BMSC were implanted into full thickness cartilage lesions in the

femoropatellar articulation in mature horses (Wilke et al., 2007). After thirty days, defects

repaired with BMSC appeared more completely filled with more homogenous tissue.

Arthroscopic scores were significantly higher in the BMSC seeded defects. No consistent

differences between control and cell seeded scaffolds could be determined eight months after

surgery. The authors concluded that early repair was improved by the presence of BMSC but no

long term benefits were apparent. Yan and Yu compared the effects of chondrocytes, BMSC,

fibroblasts and hUCB seeded on a polylactic acid scaffold on a full thickness cartilage defect in

rabbits (Yan and Yu, 2007). While no apparent visible difference in the defects were found after

twelve weeks, the fibroblast and control scaffolds were filled with non-cartilaginous tissue while

hUCB and BMSC seeded scaffolds housed tissue indistinguishable from host hyaline cartilage.









specific genomic regions including chromatin remodeling, histone modifications, histone

variants, and DNA methylation. For example, trimethylation oflysine 9 and lysine 27 on histone

3 (H3K9 and H2K27, respectively) correlate with inactive regions of chromatin. However,

trimethylation of H3K4 and acetylation of histones three and four are associated with active

areas of transcription (Jenuwein and Allis, 2001). Generally, methylation is considered a

repressive mark (Santos and Dean, 2004). Polycomb group proteins (PcG) also silence

developmental regulators, aiding in the maintenance of a plastic state (Boyer et al., 2006; Lee et

al., 2006). PcG proteins form two repressor complexes, PRC1 and PRC2. PRC2 inhibits

initiation of transcription, while PRC1 maintains the repressed state (Boyer et al., 2006). Genes

that are co-occupied by PRC 1 and PRC2 also exhibit H3K27 trimethylation which is catalyzed

by PcG proteins (Cao et al., 2002; Ringrose et al., 2004). Recently, a configuration of epigenetic

modification has been described on target genes poised for transcription but not yet active.

These bivalent histones contain both repressive and active histone markers, with large regions of

H3K27 trimethylation interrupted by a smaller region of H3K4 trimethylation. This

configuration is frequently associated with developmentally regulated factors that may be

expressed at low levels in stem cells. Upon differentiation, most bivalent domains become H3K4

or H3K27 methylated (Azuara et al., 2006; Bernstein et al., 2006).

BAF, a member of the SWI/SNF family of ATP dependent chromatin remodeling

complexes, is expressed abundantly in ES cells. BAF250a is a member of the BAF complex

which can target and antagonize PcG proteins. Absence of BAF250a results in embryos that fail

to undergo gastrulation and proper germ layer development (Gao et al., 2008). Null ES cells

differentiate into cells with endoderm-like morphology. These cells express a marked reduction









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ISOLATION AND CHARACTERIZATION OF EQUINE UMBILICAL CORD BLOOD
DERIVED STEM CELLS




















By

SARAH ANN REED


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2009









Aggregation into EBs produced cells with morphologies that represented cells from all three

germ layers.

In an effort to avoid the ethical controversies surrounding embryonic stem cells and to

make pluripotent stem cells that matched the unique identity of each individual, Takahashi and

Yamanaka sought to find a set of factors that would induce a pluripotent state in a somatic cell

(Takahashi and Yamanaka, 2006). They identified four factors which, when transduced into

mouse embryonic fibroblasts, elicited a naive state with ES cell morphology and the ability to

produce teratomas in nude mice that contained cell types from all three germ layers. Oct4, Sox2,

Klf4, and c-Myc induced the expression ofnanog and other pluripotency factors. The induced

pluripotent stem cells (iPS) showed increased acetylation of histone H3 of the Pou5fl and nanog

promoters as well as decreased methylation of lysine 9 of histone H3, indicating a permissive

state for Pou5fl and nanog transcription. However, the CpG dinucleotides remained partially

methylated. Additionally, iPS cells exhibited higher telomerase activity at levels similar to ES

cells. Human embryonic fibroblasts respond similarly to induction with Oct4, Sox2, Klf4 and c-

Myc, expressing ES cell markers at both the protein and mRNA level (Park et al., 2008).

However, recovery of iPS from postnatal human cells was successful only when hTERT and

SV40 large T were included in the retroviral cocktail. The authors speculate that these factors

are required to act on supportive cells in the culture to enhance the efficiency with which the

reprogrammed colonies can be selected (Park et al., 2008). Further work has indicated that only

two factors, Oct4 and Sox2 may be necessary for induction of iPS cells from human fibroblasts

when coupled with valproic acid (Huangfu et al., 2008). These cells are morphologically similar

to hES cells and express nanog, Oct4, Sox2, SSEA4, Tral-60 and Tral-81. iPS cells have been

postulated to be functionally equivalent to mouse ES cells in that they express the same markers,










Table 5-1. Real-time PCR primers
Expected Standard Primer
Product Curve Efficiency,
Primer Sequence Size, bp Slope %
CollA2 F GCACATGCCGTGACTTGAGA 127 -3.39 97.24
R-CATCCATAGTGCATCCTTGATTAGG
TnC1 F GGGCGGCCTGGAAATG 70 -3.34 99.25
R CAGGCTCTAACTCCTGGATGATG
ScxB1 F TCTGCCTCAGCAACCAGAGA 59 -3.35 98.84
R TCCGAATCGCCGTCTTTC
Scx2 F AGGACCGCGACAGAAAGAC 261 n/a n/a
R CAGCACGTAGTGACCAGAAGAA
18S1 F GTAACCCGTTGAACCCCATT 151 -3.35 98.84
R -CCATCCAATCGGTAGTAGCG
Pea32 F GTGGCAGTTTCTGGTGGCCCTG n/a n/a
R GACTGGCCGGTCAAACTCAGCC
Erm2 F GAGAGACTGGAAGGCAAAGTC n/a n/a
R CCCAGCCACCTTCTGCATGATGC
(Taylor et al., 2009)
2 Used for endpoint PCR only.



















(+ ~F4


500bp.


200bp.





500bp-


200bp.


Figure 4-2. Equine UCB stem cells express markers of embryonic stem cell pluripotency. RT-
PCR was performed on mouse embryonic (n=l, in triplicate) and UCB stem cell
(n=4, in duplicate) total RNA using primers specific for Oct4, nanog, Sox2, KLF4
and c-myc transcripts. Both populations of stem cells express transcripts for the
reprogramming genes. UCB = umbilical cord blood stem cells; mES = mouse
embryonic stem cells. Representative photo shown.









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ISOLATION AND CHARACTERIZATION OF EQUINE UMBILICAL CORD BLOOD
DERIVED STEM CELLS

By

Sarah Ann Reed

August 2009

Chair: Sally E Johnson
Major: Animal Molecular and Cellular Biology

Musculoskeletal injuries are responsible for a large portion of wastage in sport horses.

Mesenchymal stem cells (MSCs) offer promise as therapeutic aids in the repair of tendon,

ligament, and bone damage suffered by these horses. The objective of these studies was to

identify and characterize stem-like cells from newborn foal umbilical cord blood (UCB). UCB

was collected and MSC isolated using human reagents. The cells exhibit a fibroblast-like

morphology and express the stem cell markers Oct4, SSEA-1, Tral-60 and Tral-81. UCB

express transcripts implicated in embryonic stem (ES) cell pluripotency, namely Oct4, nanog,

Sox2, Klf4 and c-myc. Culture of the cells in tissue-specific differentiation media leads to the

formation of cell types characteristic of mesodermal and endodermal origins including

chondrocytes, osteocytes, and hepatocytes. Limited adipogenic and myogenic differentiation

occurred. Population doubling time and the presence of Oct4, nanog and Sox2 transcripts were

used to determine culture conditions that promoted the proliferation of a stem cell population.

Culture on a protein matrix (gelatin, collagen or fibronectin) shortened population doubling time

compared to growth on uncoated plasticware. Inclusion of fibroblast growth factor 2 (FGF2) in

the growth media slowed proliferation. The persistence of Oct4, nanog and Sox2 expression was

monitored over time in culture in UCB stem cells. Oct4 was detected throughout the duration of









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571-579.

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Hough, S.R., Clements, I., Welch, P.J., and Wiederholt, K.A. (2006). Differentiation of mouse
embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog.
Stem cells (Dayton, Ohio) 24, 1467-1475.

Huang, J.I., Durbhakula, M.M., Angele, P., Johnstone, B., and Yoo, J.U. (2006). Lunate
arthroplasty with autologous mesenchymal stem cells in a rabbit model. The Journal of
bone and joint surgery 88, 744-752.

Huang, S., Law, P., Francis, K., Palsson, B.O., and Ho, A.D. (1999). Symmetry of initial cell
divisions among primitive hematopoietic progenitors is independent of ontogenic age and
regulatory molecules. Blood 94, 2595-2604.

Huangfu, D., Osafune, K., Maehr, R., Guo, W., Eijkelenboom, A., Chen, S., Muhlestein, W., and
Melton, D.A. (2008). Induction of pluripotent stem cells from primary human fibroblasts
with only Oct4 and Sox2. Nature biotechnology 26, 1269-1275.

Hyslop, L., Stojkovic, M., Armstrong, L., Walter, T., Stojkovic, P., Przyborski, S., Herbert, M.,
Murdoch, A., Strachan, T., and Lako, M. (2005). Downregulation of NANOG induces
differentiation of human embryonic stem cells to extraembryonic lineages. Stem cells
(Dayton, Ohio) 23, 1035-1043.

Im, G.I., Kim, D.Y., Shin, J.H., Hyun, C.W., and Cho, W.H. (2001). Repair of cartilage defect in
the rabbit with cultured mesenchymal stem cells from bone marrow. J Bone Joint Surg Br
83, 289-294.

Jaenisch, R., and Bird, A. (2003). Epigenetic regulation of gene expression: how the genome
integrates intrinsic and environmental signals. Nature genetics 33 Suppl, 245-254.

Jenuwein, T., and Allis, C.D. (2001). Translating the histone code. Science (New York, N.Y 293,
1074-1080.

Jeong, J.A., Gang, E.J., Hong, S.H., Hwang, S.H., Kim, S.W., Yang, I.H., Ahn, C., Han, H., and
Kim, H. (2004). Rapid neural differentiation of human cord blood-derived mesenchymal
stem cells. Neuroreport 15, 1731-1734.

Johnson, D.E., Lee, P.L., Lu, J., and Williams, L.T. (1990). Diverse forms of a receptor for
acidic and basic fibroblast growth factors. Molecular and cellular biology 10, 4728-4736.









CHAPTER 4
REFINEMENT OF CULTURE CONDITIONS TO PROMOTE THE MAINTENANCE OF
EQUINE UMBILICAL CORD BLOOD DERIVED STEM CELLS

Introduction

Cytotherapeutic repair in horses is a proposed means of decreasing the prescribed stall

confinement period. Current strategies involve isolation of mesenchymal stem cells (MSCs)

from bone marrow (BM) aspirates or adipose tissue coupled with autologous engraftment into

the site of damage (Richardson et al., 2007; Taylor et al., 2007). Tendon lesions in horses

treated with BM MSC retained a portion of the cells at the lesion site, exhibited properly oriented

collagen fibrils and returned to exercise sooner (Pacini et al., 2007). Injection ofBM-MSCs into

damaged superficial digital flexor tendons (SDFT) demonstrates that a portion of the cells is

retained at the lesion site indicating that the improved healing is likely a product of the engrafted

MSCs (Guest et al., 2008).

MSC from equine adipose, bone marrow and umbilical cord blood efficiently differentiate

into chondrocytes and osteocytes in vitro (Fortier et al., 1998; Arnhold et al., 2007; Koch et al.,

2007; Stewart et al., 2007; Kisiday et al., 2008; Reed and Johnson, 2008). Their ability to

transform into cell types of additional lineages is limited. Under the appropriate conditions,

UCB stem cells differentiate into adipocytes (Koch et al., 2007). A limited set of hepatogenic

and myogenic markers was reported following UCB stem cell differentiation (Reed and Johnson,

2008). The reason for the restricted plasticity is unknown, but may be associated with

suboptimal culture conditions.

Implicit to the pluripotent nature of human and rodent embryonic stem (ES) cells is

expression of Oct4 and nanog (Chambers et al., 2003; Yates and Chambers, 2005; Wang et al.,

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while not affecting proliferation suggesting a role in cell attachment and spreading. Exogenous

FGF2 enhances growth of bone marrow mesenchymal stem cells (Solchaga et al., 2005). It

prolongs the life span of bone marrow stromal cells by stimulating telomere elongation (Bianchi

et al., 2003). Murine bone marrow derived Scal-positive stem cells proliferated in response to

FGF4 and FGF2 under low serum conditions and exhibited a simultaneous phosphorylation of

ERK1/2 and Akt. Changes in proliferation were abrogated by the inclusion of MEK and PI3K

inhibitors. The up-regulation of the immediate early gene c-jun was also apparent following

ERK1/2 activity (Choi et al., 2008).

FGF4 is secreted from undifferentiated hES cells and promoted self-renewal. Not only did

expression of FGF4 decline with differentiation, but knockdown of the growth factor with

siRNA resulted in differentiation, indicating a role for FGF4 in the maintenance of the

undifferentiated state in human ES cells (Mayshar et al., 2008). Accordingly, Sox2 and Oct4 can

transactivate the FGF4 enhancer in F9 embryonal carcinoma cells suggesting a circular feedback

mechanism (Yuan et al., 1995). Alternatively, Fgf4-/- mES cells do not undergo differentiation

upon removal of LIF, but will differentiate along the neural lineage if further supplemented with

exogenous FGF4. Cells null for FGF4 exhibit a reduced ability to enter neuronal and

mesodermal lineages which does not stem from a lack of proliferation or death of precursor cells.

Null cells do not select alternative commitment programs, rather they remain in an

undifferentiated state (Kunath et al., 2007).

Embryonic Stem Cells

Characteristics

Embryonic stem cells were first derived from the inner cell mass of a mouse blastocyst in

1981 (Evans and Kaufman, 1981). Established human lines followed in the late 1990's

(Thomson et al., 1998). The immortal cells derived from the inner cell mass are an in vitro









Table 5-2. Cycle threshold ranges
Cell Type Transcript Mean SEM Min Max Median
UCB 18S 13.72 0.06 13.15 15.59 13.59
UCB Scx 30.72 0.21 27.13 33.77 30.71
UCB TnC 21.89 0.29 18.67 28.20 21.27
AdMSC 18S 13.59 0.06 13.13 14.18 13.57
AdMSC Scx 31.19 0.55 27.10 34.31 31.54
AdMSC TnC 22.33 0.64 18.44 25.65 23.10














10= 70


L 6o 40
60




aj 1
i c 0 >
0- 0I1



Gelatin Beads Matrigel Gelatin Beads Matrigel
8- 16-

S6- 12
8


0 O 0
Gelatin Beads Matngel Gelatin Beads Matrigel




Figure 5-6. Culture in matrigel increases tenocyte gene expression. AdMSC and UCB stem cells
were cultured on gelatin coated plasticware, collagen coated beads, or 30% matrigel
for 48 hours prior to RNA isolation and real-time PCR with gene-specific primers.
Culture on matrigel increased scleraxis expression in AdMSC and UCB. Tenascin C
mRNA was also increased by culture on matrigel. Asterisk indicates p<0.05.









myc is sufficient to reprogram somatic cells into ES-like cells (Takahashi and Yamanaka, 2006;

Takahashi et al., 2007; Aoi et al., 2008; Lowry et al., 2008). Oct4 mRNA is present in MSCs

isolated from multiple tissue sources suggesting that the transcription factor participates in the

global inhibition of stem cell differentiation (Tondreau et al., 2005; Ren et al., 2006; Greco et

al., 2007). Undifferentiated equine UCB stem cells express Oct4 in the nucleus, which is lost

upon introduction of lineage decisions (Reed and Johnson, 2008).

The objective of the experiment was to refine culture conditions of equine UCB stem cells

and examine the effects of continuous culture on genetic markers of ES cell identity. Results

indicate improved UCB stem cell population doubling times (PDTs) with cultivation on matrix-

associated protein surfaces. UCB stem cells express Oct4, nanog and Sox2 immediately upon

establishment in vitro. Serial passage is associated with decreased expression of both nanog and

Sox2.

Materials and Methods

UCB Collection and Stem Cell Isolation

Cord blood was collected from the intact umbilicus of Thoroughbred foals (N= 4) at

foaling into a sterile 50 ml centrifuge tube containing EDTA (1 mg/ml) as an anti-coagulant.

UCB was stored at 40 C and putative stem cells isolated within 12 hours of collection. Samples

were incubated for 20 minutes with RosetteSep Human Cord Blood Progenitor Enrichment

Cocktail (50 pl/ml blood; Stem Cell Technologies, Seattle, WA). An equal volume of

phosphate-buffered saline (PBS) containing 2% FBS was added and the mixture was layered on

a bed of Ficoll-Paque (Sigma, St. Louis, MO). Cell aggregates were sedimented through the

density gradient by centrifugation at 1,200 X G for 20 minutes. Mononuclear cells at the

gradient interface were collected, washed with PBS and placed into culture.









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In conclusion, we have further delineated the differences between adipose and umbilical

cord blood derived stem cells. While both cell types upregulate scleraxis expression in response

to culture on matrigel, they respond very differently to stimulation with fibroblast growth factors.

We have established culture conditions appropriate to induce an early tenocytic lineage. Further

work should clarify the mechanisms behind such changes.









maintaining growth kinetics in this population. Culture in conditioned media proved deleterious

to UCB stem cells, evidenced by poor growth kinetics and morphology suggestive of replicative

senescence. This suggests that the improvement seen in growth kinetics at higher cell densities

may be a reflection of cell:cell and extracellular matrix interactions rather than the paracrine

action of secreted factors.

Similar to conditioned media, FGF2 appears to be a hindrance to UCB stem cell

proliferation. FGF2 is mitogenic for MSCs isolated from adipose and bone marrow tissues

(Baddoo et al., 2003; Benavente et al., 2003; Rider et al., 2008). Treatment of mouse BM-MSC

with FGF2 causes an increase in proliferation without induction of differentiation (Baddoo et al.,

2003). Indeed, FGF2 reversibly inhibited MSC differentiation toward the adipogenic and

chondrogenic lineages. However, treatment of equine UCB stem cells with FGF2 resulted in

longer PDTs but no negative effects on the ES marker profile. The contrasting results may

represent specie and tissue source differences.

Nanog, Oct4, and Sox2 are key factors in maintaining ES cell pluripotency in addition to

being capable of inducing pluripotency in somatic cells (Takahashi and Yamanaka, 2006). In the

absence of nanog, mouse ES cells differentiate into presumptive endoderm lineages (Mitsui et

al., 2003). Oct4 is required to maintain pluripotency of the inner cell mass; loss of function

results in differentiation to trophectodermal lineages (Zaehres et al., 2005). The requirement of

Sox2 to maintain the expression of Oct4 has been demonstrated (Masui et al., 2007). These

transcripts, along with c-myc, are capable of inducing a pluripotent, ES-like state in somatic cells

(Takahashi and Yamanaka, 2006). Notably, the more differentiated adipose derived stem cells

lack expression of Nanog and Sox2 (data not shown). Expression of this panel of transcripts, as

well as population doubling time was used to monitor the stemness of UCB in different culture









UCB


23A2


Con NI Con NI
N C
c~'~I c~-2~ R


500bp
300bp-


Figure 4-5. UCB and 23A2 myoblasts express hes. RT-PCR for the transcripts listed was
performed on RNA isolated from cells treated with the Notch inhibitor, L685,458.
UCB = umbilical cord blood derived stem cells, Con = control, no inhibitor, NI =
cells supplemented with L685,458.











A B
0 CTL 0 CTL
S8 FGF2 W 8 FGF2
I FGF5 I FGF5




0 0
Gelatin Beads Matrigel Gelatin Beads Matrigel

C D
45 g CTL 45 0 CTL
5 FGF2 FGF2
35 bo 3 M FGF5
35 g FGF53 FGF5

75
5k

3 4
0 0n
Gelatin Beads Matrigel Gelatin Beads Matrigel


Figure 5-9. Culture conditions affect tenocyte gene expression in AdMSC and UCB. AdMSC
and UCB stem cells were cultured for 48 hours in low serum media containing 10
ng/ml FGF2 or FGF5. Total RNA was isolated and subjected to real-time PCR with
gene specific primers for scleraxis and tenascin C. Matrigel increased expression of
scleraxis in UCB stem cells (A). There was no effect of matrix or FGF stimulation on
TnC in UCB (B). Scleraxis expression in AdMSC was increased by culture on
matrigel (C). Matrigel increased Tenascin C mRNA in AdMSC (D). This was further
increased by supplementation with FGF2 or FGF5.









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Transfusion 44, 1087-1097.









ligand Jagged-1 is found in AdMSC. This suggests that adipose derived stem cells may not be

fully capable of Notch signaling but could contribute to signaling to surrounding cells.

To identify the effects of the Notch pathway on UCB proliferation, cells were cultured for

three days in media with or without the Notch inhibitor L685,458. Cell number was determined

daily by visual inspection and manual counting. Inclusion of the Notch inhibitor had no

significant effect on cell number over the three day period (Figure 4-4). Furthermore, UCB

culture in the presence of the Notch inhibitor had no effect on the presence of Oct4, nanog, or

Sox2 as determined by RT-PCR (data not shown).

Notch signaling is mediated by the transcription factors hes and hey. The presence of hes

and hey in UCB was determined. 23A2 myoblasts were used as a positive control. No

transcript was amplified for hey, however hes transcripts were present in both cell types.

Furthermore, there appeared to be no difference in cells treated with L685,458 and control cells

of either type (Figure 4-5). To further verify this, real-time PCR was performed following three

days of treatment with the Notch inhibitor. Delta Ct values are reported in Table 4-4. No

difference in expression levels was apparent in either cell type.

To verify the efficacy of the Notch inhibitor, 23A2 myoblasts were stimulated to

differentiate. The presence of BMP6 abrogates differentiation however, this can be inhibited by

the inclusion ofL685,458. Control cells formed myotubes within 48 hours in differentiation

medium (Figure 4-6). At this time, 76% of nuclei were included in myosin heavy chain (MyHC)

expressing cells. The presence of BMP6 in differentiation media inhibited myotube formation,

with fewer than 10% of cells expressing MyHC. However, inhibition of the Notch pathway in

the BMP6 stimulated cells abrogated the effects of BMP6 and allowed a partial recovery of









BIOGRAPHICAL SKETCH

Sarah Reed was born in Bellefonte, Pennsylvania, to Lisa and Howard Grove, Jr. She

grew up riding and showing Quarter Horses and was a member of 4-H and the American Quarter

Horse Youth Association. Sarah graduated as valedictorian from Bellefonte Area High School in

Bellefonte, Pennsylvania in 2000. Following high school, Sarah pursued a bachelor's degree in

equine science at Delaware Valley College where she graduated summa cum laude in December

2003. While at DVC, Sarah was involved in the Equine Science Organization and the

agricultural honor society, Delta Tau Alpha. Following graduation, she worked as a laboratory

technician for Dr. Sally E. Johnson at the Pennsylvania State University. In July 2004, Sarah

married Jared Reed. Upon Dr. Johnson's move to the University of Florida, Sarah enrolled as a

master's student and completed her degree in Dr. Johnson's laboratory. The title of her master's

thesis was Identification ofDifferentially Expressed Proteins as a Result ofRafKinase Activity.

Sarah continued her education with Dr. Johnson, pursuing her doctoral degree in the Animal

Molecular and Cellular Biology program. While working on her degree, Sarah stayed involved

with the horse industry by working at Sundaram Farms in Newberry, Florida and taking show

jumping lessons from Beth Stelzleni and Ella Rukin. Sarah currently resides in Alachua, Florida,

with her husband, Jared, and their two dogs, Annie and Bella. She and Jared are active members

of Grace United Methodist Church at Fort Clarke. Sarah enjoys photography, horseback riding,

and reading fiction in her spare time.









Likewise, equine embryos have also yielded ES-like cells. Several groups have isolated

ES-like cells from frozen and freshly collected equine blastocysts (Saito et al., 2002; Li et al.,

2006; Guest and Allen, 2007). Equine ES-like cells exhibit morphology similar to mES cells

with underrun cell borders, a small cytoplasmic to nuclear ratio, and prominent nucleoli (Li et

al., 2006). These cells express Oct4, alkaline phosphatase, SSEA1, SSEA4, Tral-60 and Tral-81

(Saito et al., 2002; Li et al., 2006; Guest and Allen, 2007). No SSEA3 was apparent in ES-like

cells, although it was detected in equine blastocysts (Guest and Allen, 2007). Equine ES-like

cells were capable of differentiation into all three germ layers in vitro but did not form teratomas

when injected subcutaneously into SCID/beige immunoincompetent mice (Li et al., 2006).

Despite the similarities in marker expression, reports of the culture conditions required differ

among reports. In the absence of LIF, eES-like cells lost expression of all markers of

pluripotency and exhibited morphological properties identical to differentiated cells (Li et al.,

2006; Guest and Allen, 2007). However, other reports indicate that a feeder layer, but not LIF, is

necessary for the maintenance of eES-like cells (Saito et al., 2002). The difference between the

reports is unclear but may result from differences in isolation protocols or source of the equine

embryos.

Embryos from rats and dogs have also yielded ES-like cells. Rat ES-like cells cultured on

a feeder layer in the presence of LIF express Oct4, SSEA1, and alkaline phosphatase (Vassilieva

et al., 2000). Canine ES-like cells were isolated from inner cell masses collected from

blastocysts (Hatoya et al., 2006). Unlike mouse and human ES cells, this population is limited in

life span, entering replicative senescence after nine passages. Domed colonies with tightly

packed cells and an apparent border were present during early passages when cultured on a

feeder layer. Cells expressed alkaline phosphatase, SSEA1, and Oct4, but no SSEA4.









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CHAPTER 3
EQUINE UMBILICAL CORD BLOOD CONTAINS A POPULATION OF STEM CELLS
THAT EXPRESS OCT4 AND DIFFERENTIATE INTO MESODERMAL AND
ENDODERMAL CELL TYPES

Introduction

Bone marrow (BM) derived mesenchymal stem cells (MSCs) are the conventional model

of choice for adult stem cell based therapeutics in humans due to their multi-lineage

differentiation capabilities. Their relative ease of expansion in vitro without loss of plasticity

makes MSCs an attractive repair aid for damaged or diseased heart, bone and vascular tissues

[for review see (Giordano et al., 2007)]. However, enthusiasm for the use of MSCs as

cytotherapeutics is tempered by their age-dependent decline in absolute numbers and the

invasive nature of their harvest (Stenderup et al., 2003). To counter these problems, umbilical

cord blood (UCB) MSCs may represent a viable alternative. Several reports define a clonogenic

population of cells from the umbilicus that differentiate into both mesenchymal and non-

mesenchymal tissue derivatives (McGuckin et al., 2003; Aoki et al., 2004; Baal et al., 2004;

Bonanno et al., 2004; Peled et al., 2004; Ruzicka et al., 2004; He et al., 2005; Holm et al., 2006;

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like progenitors versus endothelial progenitors detached from the umbilicus remains debatable

(Kogler et al., 2004).

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pluripotent and adult MSCs are more limited in their differentiation capacity (Feinberg, 2007).

UCB stem cells likely fall in the area between the two. The three classes of stem cells

demonstrate variable stage specific embryonic antigen (SSEA) and tumor rejection antigen (Tra)

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Table 4-2. Effect of substrata and media on equine UCB1 derived stem cell doubling time
P-value
Con Gel Fib Col
Substrata 3 4 5 COn
2 Media3 PDT4 SEM5 GM+FG Gel GM GM+FG Fib GM GM+FG Col GM GM+FG
GM
F F F F
Con GM 30.07 1.59 --- <0.0001 0.0628 0.0079 0.0370 0.1055 0.0091 0.0029
GM+FG
Con GM+FG 41.74 3.01 --- --- <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
F
Gel GM 27.32 1.50 --- --- --- <0.0001 0.8187 0.0006 0.4463 <0.0001
GM+FG
Gel GM+FG 34.01 2.14 --- --- --- --- <0.0001 0.2908 <0.0001 0.7366
F
Fib GM 26.99 1.57 --- --- --- --- --- 0.0003 0.5941 <0.0001
GM+FG
Fib GM+FG 32.46 2.05 --- --- --- --- --- --- <0.0001 0.1643
F
Col GM 26.21 1.18 --- --- --- --- --- --- --- <0.0001
GM+FG
Col GMFG 34.50 2.38 --- --- --- --- --- ---
F


1 UCB=umbilical cord blood
2 Con= uncoated, Gel = gelatin, Fib = fibronectin, Col = collagen
3 GM = growth media, GM+FGF = growth media + FGF2
4 PDT= population doubling time in hours, N=No2(t/pdt) where N
population doubling time
SEM = Standard error of the mean


= final cell number, No = initial cell number, t=time, and pdt=









LIST OF FIGURES


Figure pae

3-1 Foal UCB cells express stem cell marker proteins ................................. ................72

3-2 Induction of chondrogenesis in foal UCB stem cells............................................. 73

3-3 U CB stem cells form osteocytes. .............................................. .............................. 74

3-4 Foal UCB stem cells form hepatocytes.......................................... 75

3-5 Incomplete initiation of adipogenesis and myogenesis in foal UCB stem cells and
A dM S C s ................................................................................................7 6

3-6 AdM SC fail to express embryonic stem cell markers ....................................... .......... 77

4-1 GM and GM+FGF support equine UCB stem cell propagation.................... ........ 91

4-2 Equine UCB stem cells express markers of embryonic stem cell pluripotency ...............94

4-3 UCB and AdMSC express a limited number of molecules in the Notch signaling
p ath w ay ........ ........ ...................................................................... 9 6

4-4 Inhibition of the Notch signaling pathway does not affect proliferation...........................97

4-5 UCB and 23A2 myoblasts express hes. ........................................ ........................ 98

4-6 BMP6 inhibits myoblast differentiation in a Notch dependent manner ........................100

5-1 AdM SC and UCB express markers of tenocytic cells.................................................... 118

5-2 Mouse scleraxis promoter with putative transcription factor binding sites .....................119

5-3 Scleraxis promoter activity is not increased by growth factor supplementation in
U C B stem cells. ..........................................................................122

5-4 UCB stem cells express Erm, Pea3, and Scleraxis........................................................ 123

5-5 UCB and AdMSC attach to various culture surfaces...........................................124

5-6 Culture in matrigel increases tenocyte gene expression ..............................................125

5-7 UCB and AdMSC respond uniquely to FGF stimulation.........................................126

5-8 Fibroblast growth factors stimulate proliferation of AdMSC and UCB stem cells in a
M A PK dependent m anner................ ......................... ........................... ............... 127

5-9 Culture conditions affect tenocyte gene expression in AdMSC and UCB .....................128









phase of G1 (White et al., 2005). By eliminating the requirement for cyclin D expression

(MAPK induced), ES cells uncouple cell cycle traverse from differentiation, allowing for self

renewal.

ES cells isolated from cynomolgus monkey require a feeder layer to maintain pluripotency

and cannot rely on LIF supplementation as do murine ES cells. LIF treatment induces

phosphorylation of tyrosine 705 but does not affect the phosphorylation status of serine 727 of

STAT3 (Sumi et al., 2004). Serine phosphorylation of STAT3 is required for full transcriptional

activity (Wen et al., 1995).

Interestingly, down-regulation of connexin 43, a protein responsible for formation of gap

junctions, results in the rapid differentiation of mES cells (Todorova et al., 2008). This included

loss of Oct4 expression, morphological changes, and up regulation of differentiation markers

such as glial fibrillar associate protein (GFAP). Formation of embroid bodies was hindered by

treatment with gap junction intercellular communication (GJIC) blockers. Recovery of GJIC

allowed the restoration of the differentiation program.

Research with ES cells is not limited to humans and mice. ES cells were successfully

isolated from the ICM of bovine embryos (created by IVF or nuclear transfer) and cultured on

mitomycin-c treated MEFs (Wang et al., 2005b). They assumed the typical morphology of ES

cells: multicellular colonies with a smooth surface and distinct colony boundary. Bovine ES

cells react positively with SSEA4 and Oct4 but not SSEA1 antibodies. No Tral expression was

found. In the absence of a feeder layer and LIF, bES cells spontaneously differentiated into EBs

and gave rise to cells from three germ layers. Ectodermal cells expressed the neurofilament

marker TUBB3, mesodermal cells expressed smooth muscle ACTA2, and cuboidal and net-like

epithelial structures expressed creatine kinase.









demonstrate that UCB stem cells are a source of osteocytes under appropriate in vitro cultivation

conditions.

Foal UCB Stem Cells can Differentiate into Endodermal-Derived Cell Types

A key feature of ES cells is their potential to contribute to any tissue type in the body.

Adult stem cells possess a more limited plasticity than their embryonic counterparts. The ability

of foal UCB stem cells to differentiate into hepatocytes, a cell type that originates from the

endoderm, was examined. In brief, UCB stem cells were cultured for 2 weeks in media that

supports hepatocyte formation in human UCB stem cells (Kang et al., 2005). Subsequently, cells

were fixed with paraformaldehyde or lysed for RNA isolation. As shown in Figure 3-4A, a

change from an elongated, spindle-shaped morphology to one exhibiting a larger cytoplasmic

volume with an elliptical shape occurs in response to the treatment media. These cells express

mRNA for both albumin and cytokeratin 18, definitive markers of hepatocytes (Figure 3-4B).

Equine UCBs maintained in the absence of induction media failed to express the liver marker

genes (data not shown). The ability to respond in a manner similar to ES cells and form

hepatocytes suggests that our UCB cell population may be more plastic than other adult MSC.

Inefficient Formation of Myocytes and Adipocytes by UCB Cells

Koerner et al. (Koerner et al., 2006) reported limited formation of adipocytes from adult

horse BM-derived MSCs. Thus, we compared the adipogenic differentiation capabilities of foal

UCB stem cells and adult horse adipocyte-derived MSCs (AdMSC). In brief, both cell types

were incubated for 21 days in adipocyte induction media. Cells were fixed and evaluated by Oil

Red O histology for the presence of lipid droplets. In our hands, neither UCB nor AdMSC

efficiently formed adipocytes. Sporadic fat cells containing limited amounts of lipid droplets

were evident in foal UCB cell cultures; no Oil Red O positive cells were found in the AD-MSCs.

This restricted differentiation profile by the two forms of stem cell was further exemplified









discovered that possesses stem cell-like markers and multilineage differentiation capabilities.

The isolation and characterization of these cells represent a first-step toward their application in

cytotherapeutic repair of sport horse injuries.

Materials and Methods

Umbilical Cord Blood (UCB) Collection and Stem Cell Isolation

Cord blood (n = 25) was collected from the intact umbilicus at foaling into a sterile 50 ml

centrifuge tube containing ethylenediamine tetraacetic acid (EDTA) as an anti-coagulant. Blood

was stored at 40C and further processed within 12 h of collection. Samples were incubated for 20

min with RosetteSep Human Cord Blood Progenitor Enrichment Cocktail (50 pl/ml blood; Stem

Cell Technologies, Seattle, WA), a commercially available product for negative selection of

human UCB stem cells. An equal volume of phosphate-buffered saline (PBS) containing 2%

fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) was added. The cell suspension was layered

atop a Ficoll-Paque Plus (Stem Cell Technologies) cushion and centrifuged at 1,200g for 20 min.

The cell interface was collected and cultured.

Equine UCB and Adipose-Derived (AD) Stem Cell Culture

MSCs isolated from equine adipose tissue were purchased from Sciencell Research

Laboratories (San Diego, CA). Cells were cultured in Mesenchymal Stem Cell Medium

(Sciencell Research Laboratories) on standard tissue plasticware, according to manufacturer's

recommendations. UCB stem cells were cultured in Dulbecco's modified Eagle media (DMEM,

Invitrogen) supplemented with 10% FBS and 5 tg/ml Plasmocin (Invitrogen). Culture medium

was exchanged every three days. Cells were passage at 70% confluency using 0.025% trypsin-

EDTA (Invitrogen).









differentiation. Greater than 30% of cells immunostained positive for MyHC. Supplementation

of the differentiation media with L685,458 alone had no effect on differentiation.

We conclude from these experiments that while the Notch pathway may play an important

role in differentiation of specific stem cell types, it is not crucial for the proliferation of UCB

stem cells. Furthermore, we have been unable to demonstrate that UCB stem cells have an active

Notch signaling network.

Discussion

Initial culture strategies for equine UCB stem cells involved growth on uncoated tissue

plasticware surfaces (Koch et al., 2007; Reed and Johnson, 2008) but no work has assessed the

effects of various substrata or media on the prolonged culture of these cells. Use of UCB stem

cells as injury repair aids likely will require an initial expansion in culture to provide sufficient

numbers of cells. Several media supplements were assessed for their ability to extend the

timeframe that UCB stem cells can be maintained in vitro. Expansion of human UCB stem cells

in media supplemented with thrombopoietin, Flt3 and c-kit ligand results in a population that

expresses ES-like surface markers, morphology and importantly, a delay in replicative

senescence (McGuckin et al., 2003). Passage of equine UCB stem cells in this media did not

duplicate the events and phenotypes reported for the human counterparts. Equine UCB stem

cells proliferated slowly and exhibited large nuclei with pronounced nucleoli, characteristics of

senescent fibroblasts. Close examination of the cultures indicated an absence of any cell clusters

with ES-like morphology. Because Tpo, Flt3 and c-kit were supplemented into a basal media

that supports equine UCB stem cell growth, we conclude that the growth factors are detrimental

to the routine culture and passage of these cells in vitro.

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3000- C2C12

2500-

2000-

1500

1000

PT 5 0 0 -

0
Scx-luc Scx-luc Scx-luc Scx-luc
+BMP6 +FGF4 +FGF5

Figure 5-3. Scleraxis promoter activity is not increased by growth factor supplementation in
UCB stem cells. C2C12 mesenchymal cells and UCB stem cells were transiently
transfected with Scx-luc and supplemented with 50 ng/ml BMP6, 10 ng/ml FGF4 or
10 ng/ml FGF5 for 24 hours. Luciferase activity was measured from cell lysates.
Relative luciferase units were calculated by dividing luciferase activity by renilla
expression.









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the experiment. Sox2 and nanog expression declined with time in culture. Finally, the effect of

culture conditions on expression of tendon markers was assessed. Initial stem cell populations

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did not affect scleraxis levels; however culture in 30% Matrigel significantly increased levels of

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possessed a flat, spindle-shaped, fibroblast-like morphology. A lesser population formed light-

refractile colonies that grew upward from the substratum surface in a manner consistent with

transformed fibroblast foci. These colonies of small cells with a high nuclear to cytoplasmic

volume were evident in our cultures of newborn foal UCB stem cells only after reaching

confluency. Our UCB stem cells were maintained as a monolayer and passage at approximately

60% confluency thereby, selecting against the development of these cell clusters that appear to

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times than other cultures. GM+Tpo/Flt/Kit and CM were discontinued due to morphological

changes and a prolonged PDT and cell death.

Protein Surface Matrixes Promote UCB Growth

UCB stem cells were seeded at equal cell densities on gelatin, fibronectin, collagen and

uncoated surfaces. Cell population doubling times were calculated following four days of

culture (Table 4-1). UCB stem cells on coated surfaces demonstrated faster PDTs than controls

maintained on uncoated tissue plasticware. Changes in PDT were independent of the type of

matrix used.

Additionally, PDT was increased in cells cultured in GM+FGF (35.681.25 hr) compared

to growth media controls (27.650.74 hr; p<0.0001). The interaction of media and substrata

types demonstrated that cells cultured on any type of matrix in control media have significantly

faster doubling times than their respective cultures in media supplemented with FGF2 (Table 4-

2). Cells cultured on collagen, fibronectin, and gelatin matrices in control media exhibit no

differences in growth kinetics.

Few stem cell populations are immortal in vitro, most enter replicative senescence over

time in culture. Thus, population doubling times during continuous culture were determined at

passages three, six, and nine. Time in culture significantly increases PDT from passage three

through passage nine (21.090. 3 hr, 34.841.28 hr, and 39.061.0 hr, respectively; p<0.0001 for

all interactions). While there was no significant effect of media at passage three (p=0.1361),

later passages showed significantly shorter doubling times when cultured in control growth

media compared to GM+FGF (p<0.0001, data not shown).

Oct4 is Maintained Throughout UCB Culture

Having determined appropriate culture conditions to maintain proliferation, markers of

stemness were measured by RT-PCR. Oct4 expression is maintained over time in culture









markers nanog and Sox2 were gradually lost with time in culture, however Oct4 expression

remained.

Unmanipulated UCB and AdMSC express the bHLH transcription factor scleraxis, which

is crucial to the development of flexor tendons. Culture on the complex protein matrix Matrigel

resulted in the upregulation of scleraxis in both AdMSC and UCB stem cells. In Matrigel cells

form tight colonies while they retain their fibroblast-like morphology when grown on gelatin or

collagen coated surfaces. Supplementation of FGF2 or FGF5 resulted in changes in proliferation

of both cell types, but had limited effects on scleraxis or tenascin c mRNA expression compared

to culture on matrigel alone.

Overall, this work highlights the potential of equine umbilical cord blood derived stem

cells as not only a therapeutic aid for horses but as a model system for human medicine. The

cells appear to be more naive than either bone marrow or adipose derived stem cells yet do not

retain the tumorigenicity of embryonic stem cells. The ability to form immature myocytes as

well as hepatocytes suggests that UCB have greater plasticity than other adult stem cells. This

may be a result of a less restrictive epigenetic status than adipose derived stem cells. This open

genome structure likely allows for greater plasticity and naivete. The apparent inability of UCB

and AdMSC to efficiently differentiate into adipose tissue was surprising but reflects other work

in the literature. In truth, the inefficiency of adipocyte formation is a positive aspect from a

clinical aspect as these cells may be less likely to precociously differentiate into that cell type if

used in vivo.

While initial populations express all of the common markers of a naive stem cell (Oct4,

nanog, Sox2, and Klf4), the majority of these markers are lost with time in culture. This may

reflect one of several possibilities: (1) UCB stem cells may be differentiating to a more mature









ACKNOWLEDGMENTS

There are so many people whom have helped me throughout this process that it would be

impossible to thank you all. First and foremost, I am thankful to my advisor, Dr. Sally Johnson.

Sally gave me the chance to work in her lab as an unproven technician and then invited me to

join her lab as a student. Over the past five years, she has coaxed, encouraged, motivated and

made me into a better scientist and a better person. She pushed me beyond what I thought were

my limits because, in the end, she knew I could do more.

My committee has been supportive throughout my tenure at UF. Dr. Moore was

instrumental in teaching me valuable ES culture techniques. Dr. Brown provided a wonderful

outside perspective of my project from a clinician's point of view. Dr. Ealy was a great

sounding board for ideas and was extremely helpful with statistical analysis.

My lab mates have been wonderful at providing friendship and support. Sophia, Dane, Juli,

Sara, John Michael, Lulu and Dillon Thank you. I am forever indebted to my friends who have

been there for me throughout the last five years. Sara, Beth, and Ella have all been the greatest

friends to have, whether I needed a shoulder to cry on or to just go out and have fun. John

Michael has been my lab best friend and the person that I could always gripe to when I had a

frustration with anything. I couldn't have gotten through all of the real-time without him.

My family has provided constant support and understanding, even when attending graduate

school meant moving 1100 miles away from home, missing Christmases, birthdays and all of the

little things in family life. My Mom and Dad, Lisa and Howard Grove, have provided endless

encouragement and faith that I could do this. My grandparents have always let me know how

much I am loved and that they believe I can do anything I set my mind to do. My in-laws, Don

and Barb Reed, have also provided much support and encouragement along the way, and I thank

them for accepting me into their family.































B 4 T

+ -+ +RT

1300 bp- [
603 bp -
310bp -






Figure 3-4. Foal UCB stem cells form hepatocytes. UCB stem cells were cultured for 14 days in
absence or presence of HGF and FGF4. Cells were fixed with 4% paraformaldehyde
and representative phase-contrast images were captured at 200x (A). Separate
cultures were analyzed by RT-PCR for cytokeratin 18 (CK18), albumin and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression (B). RT,
reverse transcriptase.









interstitial spaces. FGFs bind FGFR in a 2:2:2 fashion where each fully active complex consists

of two each ofFGF, FGFR, and heparin sulfate. FGF receptors contain two or three

immunoglobulin-like domains, a short, conserved area of acidic residues which contains separate

ligand and heparin binding domains, a transmembrane region and two intracellular tyrosine

kinase domains. To achieve downstream signaling, binding of the ligand to the FGFR and

heparin sulfate must occur. The resulting tyrosine phosphorylation of FRS2, a docking protein,

allows the recruitment of Grb2 molecules which, in turn, recruit the nucleotide exchange factor

SOS. The formation of the FRS2-Grb2-SOS complex activates the Ras-Raf-MAPK pathway,

resulting in changes in gene transcription. Phosphorylation of the FGFRs can also activate the

PLCy-PKC pathway. Alternatively, Grb2 can recruit Gab1 to activate PI3K/Akt signaling. This

diversity allows a single set of receptors to influence a wide variety of cellular events.

Receptor diversity is controlled by alternative splicing and results in differential ligand

binding characteristics as well as varying temporal and spatial expression patterns (Lee et al.,

1989; Johnson et al., 1990). Alternative splicing specifies the sequence of the immunoglobulin

domain III determining the receptor isoform (i.e. IIIb, IIIc). Splicing occurs in a tissue specific

manner and dramatically affects ligand specificity (Miki et al., 1992; Orr-Urtreger et al., 1993;

Scotet and Houssaint, 1995).

Identified as potent mitogens, FGFs play a major role in stem cell biology that ranges from

self-renewal to differentiation to cell attachment and migration. Importantly, FGF2 is required

by human ES cells to sustain self-renewal and plasticity (Amit et al., 2000; Xu et al., 2001).

Inhibition of FGFRs in hES cells suppressed activation of downstream signaling, led to the

downregulation of Oct4 and stimulated rapid differentiation (Dvorak et al., 2005).

Supplementation of FGF2 at levels greater than 5 ng/ml decreased the outgrowth of hES colonies









Table B-l Continued.
Size,
Primer Sequence bp Source
Jagged-1 F GCCTGGTGACAGCCTTCTAC 305 XM_001495238.2
R- GGGGCTTCTCCTCTCTGTCT
Jagged-2 F CATGATCAACCCCGAGGAC 169 XM_001494763.2
R- CGTACTGGTCGCAGGTGTAG
Notch-1 F GAGGACCTGGAGACCAAGAAGGTTC 297 XM 001498582.2
R AGATGAAGTCGGAGATGACGGC
Notch-2 F GCAGGAGCAGGAGGTGATAG 188 NM 010928.2
R GCGTTTCTTGGACTCTCCAG
Notch-3 F GTCCAGAGGCCAAGAGACTG 219 NM 008716.2
R CAGAAGGAGGCCAGCATAAG
Dll-1 F ACCTTCTTTCGCGTATGCCTCAAG 221 NM 007865.3
R AGAGTCTGTATGGAGGGCTTC
Dll-4 F CGAGAGCAGGGAAGCCATGA 378 NM 019454.2
R- CCTGCCTTATACCTCTGTGG









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Wang, J., Rao, S., Chu, J., Shen, X., Levasseur, D.N., Theunissen, T.W., and Orkin, S.H. (2006).
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B C


Figure A-1. Stages of mES colony differentiation. Naive mES colonies maintain a compact
shape with no discernable individual cells (A, day one). More differentiated colonies
often have a ring of epithelial cells surrounding a more densely packed core (B, day
two). Fully differentiating cells adopt the morphology of their mature cell type and
are commonly found in monolayers on the culture surface (C, day 5). Scale bar = 10
tm














GMI GM\I + FGF


GM +
Flt/Tpo/Kit


CM


Figure 4-1. GM and GM+FGF support equine UCB stem cell propagation. UCB stem cells
cultured in GM and GM+FGF have faster doubling times than those grown in GM
containing thrombopoietin(tpo), Flt3, and c-kit, or UCB conditioned media.
Population doubling times are presented below their respective phase photograph in
hours SEM. Scale bar = 10 tm. GM = growth media; GM + FGF = growth media
containing FGF2; GM + Tpo/Flt/kit = growth media containing thrombopoietin, Flt3,
and c-kit; CM = conditioned media.










Table 4-1. Effect of substrata on equine UCB1 derived stem cell population doubling time


Substrata2 PDT3 SEM4 Con Gel
Con 35.91 1.89 --- <0.000
Gel 30.67 1.38 -
Fib 29.72 1.34 -
Col 30.36 1.44 --- --
UCB = umbilical cord blood
2 Con= uncoated, Gel = gelatin, Fib = fibronectin, Col = collagen
3 PDT= population doubling time in hours, N=No2(t/pdt) where N
initial cell number, t=time, and pdt= population doubling time
4 SEM = Standard error of the mean


P-value
Fib Col
1 <0.0001 <0.0001
0.3633 0.7634
--- 0.5427
--- ---


final cell number, No









module both in vitro and in viable mouse and human ES cells. Expression of constitutively active

nanog maintains mES cells in the undifferentiated state in the absence of LIF (Mitsui et al.,

2003).

Recent work has suggested that Oct4, nanog and Sox2 cooperate in regulating self renewal

and plasticity. Numerous target genes have been identified to be bound by the three factors.

Target genes of Oct4 have a Sox binding element 0 or 3 base pairs from the octamer binding

element (Rodda et al., 2005). Significantly, a complex containing both Oct4 and Sox2 regulates

the expression of nanog (Kuroda et al., 2005; Rodda et al., 2005). Oct4 and Sox2 recognize and

bind elements in the regulatory regions of their own genes (Chew et al., 2005). Large numbers

of target genes of Oct4 are also bound by nanog in ES cells, suggesting that these pathways work

in cooperation rather than independently of one another (Boyer et al., 2005; Loh et al., 2006).

The gene targets identified represent a variety of products required for pluripotency but also

those required for differentiation, suggesting that these transcription factors not only promote the

maintenance of the naive state but also block the progression of differentiation. Klf4 binds the

regulatory regions of Pou5fl and nanog and is also found in many complexes of these proteins at

transcriptional regulatory sites on other genes. Nanog and Oct4 may interact directly with each

other and chromatin remodeling proteins (Liang et al., 2008). A transient reduction in Pou5fl

and nanog induced differentiation in mES cells (Hough et al., 2006).

Epigenetic Regulation of Plasticity

Another mechanism by which plasticity is maintained is the epigenetic regulation of

chromatin structure. Epigenetic marks (including methylated DNA and possibly modified

histones) are propagated at S phase, thus epigenetic information can be transmitted through

sequential rounds of cell division (Jaenisch and Bird, 2003; Henikoff et al., 2004). Chromatin is

subjected to various forms of epigenetic regulation that modulate the transcriptional activity of


































To Jake, who taught me what it meant to be a true horsewoman; Aja, who relit the fire; and Jared
who was there for it all








UCB
0 5 10 20 30 60
m ---


-


AdMSC
0 5 10 20 30 60 min
- a-pERKl/2

1 oa-ERKl/2

W1m a-tubulin

Sa-pERK1/2

-- ca-ERKl/2


[- c-tuAlbulin


IIiL


I l a-pERKl/2

I- MW I -ERK1/2


1 "-O OW W, *I .-tubulin


Figure 5-7. UCB and AdMSC respond uniquely to FGF stimulation. UCB and AdMSC were
stimulated with 10 ng/ml FGF2, FGF4, or FGF5 for the times shown. Protein
extracts were probed with antibodies specific to phosphorylated and total ERK1/2.
Tubulin antibodies were used as a loading control.









BMSC indicates that AdMSC are less efficient at osteogenic differentiation, despite considerable

similarity in gene expression throughout the differentiation process (Liu et al., 2007).

Adipogenic differentiation was observed following treatment with insulin and IBMX (Gronthos

et al., 2001; Katz et al., 2005; Guilak et al., 2006; Liu et al., 2007). Cells formed lipid vacuoles

and secreted leptin. Culture in chondrogenic differentiation media containing TGFP led to the

expression of proteoglycans and type II collagen (Guilak et al., 2006; Liu et al., 2007;

Yoshimura et al., 2007). Culture on a poly-lactide-co-glycolide scaffold in the presence of TGFP

resulted in the expression of Col2a] and a homogenous distribution of proteoglycans (Mehlhom

et al., 2009). However, the AdMSC are again less efficient at chondrogenesis producing less

Col2al, chondroitin sulfate, and hyaluronan (Yoshimura et al., 2007). Neural differentiation has

also been reported by several labs (Katz et al., 2005; Guilak et al., 2006). In instructive media,

AdMSC gain elongated cytoplasmic processes and express NeuN, GFAP, and p3-tubulin.

Injection of murine AdMSC into ischemia induced angiogenesis resulted in a greater degree of

perfusion in the implanted limbs (Kondo et al., 2009). Injection of mature adipocytes resulted in

weaker recovery of perfusion than saline injected controls. Additionally, AdMSC recruited

endothelial precursor cells and stimulated the secretion of SDF 1 and VEGF in ischemic hind

limbs. Human AdMSC co-cultured with neonatal rat cardiomyocytes resulted in elongation of

AdMSC and formation of myotube-like structures (Zhu et al., 2008). After two weeks of co-

culture, AdMSC stained positively for myosin heavy chain, troponin-I and connexin 43. At this

time, the AdMSC and cardiomyocytes contracted synchronously. Rarely, binucleate AdMSC

were found in co-cultures.

Therapeutic Uses of Stem Cells

Stem cells have been widely accepted as a potential therapeutic aid in disease and injury

states in both humans and animals. Diseases ranging from metabolic inefficiencies to









Table 4-3. Effects of horse, passage, media and substrata on mRNA expression
P-value
mRNA Horse Passage Media Substrata
Oct4 0.3535 0.6441 1.0000 0.4272
Nanog 0.0004 0.0001 0.3018 0.0001
Sox2 0.0823 <0.0001 0.5343 0.0574



































Figure 3-6. AdMSC fail to express embryonic stem cell markers. AD-MSCs were fixed and
incubated with antibodies directed against Oct4, SSEA-1, SSEA-4, Tral-60 or Tral-
81. Immunoreactivity was detected with goat anti-mouse AlexaFluor 488 or anti-
rabbit AlexaFluor 568 (Oct4). Nuclei were visualized with Hoechst 33245. SSEA-1
and SSEA-4 were not detected. Scale bar = 10 im.









enrichment of the UCB stem cell population by affinity purification with CD133 antibodies may

provide an alternative to both cell heterogeneity and cryopreservation issues.

The capacity of foal UCB stem cells to initiate hepatocyte-specific gene transcription

demonstrates an endodermal developmental potential. Reports exist demonstrating hepatocyte

formation from human UCB stem cells and MSC isolates from BM (Hong et al., 2005; Talens-

Visconti et al., 2006). However, this is the first report of hepatocyte formation using equine

multipotential cells. Putative stem cells from the inner cell mass of equine blastocysts undergo

spontaneous differentiation in vitro to yield cell derivatives of the three germ layers with

endoderm defined by RT-PCR detection of a-fetoprotein (Li et al., 2006). The ability of

newborn foal UCB stem cells to form liver cells is encouraging as it provides additional evidence

for a population with plasticity characteristics that more closely resemble an ES cell than an

adult stem cell. Additional endoderm-derived cell types of clinical importance include pancreatic

and cardiogenic. Human UCB stem cells can be induced to form heart cells following a two-step

differentiation protocol that involves 5-azacytidine treatment (Kadivar et al., 2006). Culture with

the hypomethylating agent suggests that UCB stem cells are more restricted in their

differentiation capabilities than ES cells and require chemical-induced reprogramming. In our

hands, treatment of foal UCB stem cells with 5-azacytidine did not induce the expression of

myosin immunopositive cells. Because our antibody (MF20) recognizes all forms of sarcomeric

myosin, this result provides indirect evidence that a full-fledged cardiocyte is not created in

response to epigenetic modification. However, a more comprehensive analysis of growth factor,

morphogen and substratum requirements for UCB stem cell differentiation into cardiocytes is

warranted.









following their incubation in myocyte induction media. UCB stem cells and AD-MSCs were

incubated for 7 days in media supplemented with galectin-1, a glycoprotein that promotes

myogenesis in human fetal MSCs (Chan et al., 2006). Subsequently, cells were fixed and

immunostained for the skeletal muscle marker protein, desmin. UCB stem cell cultures contained

several multinucleated, spindle-shaped cells that are reminiscent of myocytes. Anti-desmin

immunofluorescent detection reveals that these structures express the intermediate filament

protein (Figure 3-5B). No desmin expressing muscle cells were present in the AD-MCSs treated

in a similar manner (data not shown). The presence of organized actin filaments was examined

using Texas Red conjugated phalloidin. Equine UCB-derived myoblasts contained organized

actin structures throughout their cytoplasm (Figure 3-5C). By contrast, AdMSC cells contained

fewer phalloidin-reactive filaments. The cytoskeletal structures pointed to distinct differences in

overall cellular morphology between the differentiated AdMSC and UCB myoblasts. UCB

myoblasts were thin, elongated and cylindrical in shape whereas the AdMSC cells were

fibroblast-like with an enlarged cytoplasmic space. Our results demonstrate differences between

the two types of stem cells and suggest that foal UCB cells are more plastic than adult horse

MSCs.

AdMSC do not Express the Same Complement of Stem Cell Markers

The inability of AdMSC to form adipocytes was surprising given that they originate from

the fat depot. To ensure that the cells were naive and undifferentiated, subconfluent cultures of

AdMSC were immunostained for stem cell markers. Similar to UCB stem cells, AD-MSCs

express Oct4, Tral-60 and Tral-81 (Figure 3-6). However, SSEA-1 and SSEA-4 were

undetectable. These results indicate that AdMSC retain markers of adult stem cells but do not

express those more closely associated with ES cells.









Alternative UCB Differentiation Protocols

Following is a brief description of differentiation protocols that were tested on UCB stem

cells but were not reported in earlier work due to the lack of success.

Myogenic Differentiation

UCB stem cells were plated on gelatin coated plasticware and cultured in DMEM

containing 10% FBS and 10 [iM azacytidine for 24 hours. The media was subsequently replaced

with low glucose DMEM containing 5% FBS. Cells were continuously cultured in this media for

up to three weeks. No evidence of myogenic differentiation was found. RT-PCR for the

myogenic regulatory factors MyoD and Myf5 was negative. No immunostaining of desmin or

myosin heavy chain could be detected.

Neural Differentiation

To initiate neural differentiation, three protocols were attempted. The first two protocols

contain the same basic components but included either low or high amounts of FBS (2% and

10%, respectively). This medium was based on aMEM and contained 10 ng/ml PDGF, 10 ng/ml

bFGF and 10 ng/ml EGF in addition to serum. The third media was DMEM supplemented with

15% FBS, 20 ng/ml bFGF, 50 ng/ml neural growth factor (NGF) and 0.5 mM IBMX. Cells were

maintained in these media for up to three weeks. Following culture time, cells were

immunostained for nestin, glial fibrillary acidic protein (GFAP), and beta-3-tubulin. No

immunoreactivity was apparent at any time.

Adipogenic Differentiation

Three protocols for adipogenic differentiation were attempted during this study. The

previously described protocol gave the greatest level of success and is thus the protocol of choice

for UCB. However, it should be noted that the efficiency of differentiation remained extremely

low. A low glucose DMEM based media containing 10% FBS, 1 [iM dexamethasone, 0.5 mM









paraformaldehyde in PBS for 15 min on days 7, 14, and 21. Total RNA was isolated from

parallel plates.

Chondrogenic Differentiation

UCB stem cells were pelleted to a micromass, promoting chondrogenic differentiation in a

three-dimensional environment. Cells (4 x 105) were pelleted at 1,000g for 5 min. The medium

was removed and 0.5 ml chondrogenic medium was added (Worster et al., 2000; Tondreau et al.,

2005). Chondrogenic medium consisted of DMEM, 1.0 g/L insulin, 0.55 g/L transferring, 0.67

mg/L sodium selenite (ITS-X, Invitrogen), 10 ng/ml transforming growth factor beta-1 (TGF P1,

R&D Systems, Minneapolis, MN), 35 pg/ml ascorbic acid and 100 nM dexamethasone (Sigma,

St. Louis, MO). Media was changed twice weekly. After 7, 14, and 21 days, the micromass was

embedded and frozen in OCT freezing compound. Alternately, micromasses were washed with

PBS and used for RNA isolation.

Adipogenic Differentiation

UCB stem cells were plated at a density of 3,000 cells/cm2 in growth medium. Adipogenic

differentiation was induced with Iscove's modified Dulbecco's media (IMDM) supplemented

with 10% FBS, 1 iM dexamethasone, 10 pg/ml recombinant human insulin, 0.25 mM 3-

isobutyl-1-methylxanthine (IBMX) and 100 iM indomethacin (Wagner et al., 2005). Medium

was replaced every three days. Cells were fixed with 4% paraformaldehyde in PBS after 7, 14,

and 21 days in culture.

Hepatogenic Differentiation

UCB stem cells were plated at a density of 5,000 cells/cm2 and allowed to attach overnight

in normal growth medium. The following day, cells were washed twice with PBS and placed in

hepatogenic medium [1% FBS, 20 ng/ml recombinant human hepatocyte growth factor (HGF,

R&D Systems), 10 ng/ml recombinant human fibroblast growth factor 4 (FGF4, R&D Systems)









extracellular regulated kinase 1 and 2 (ERK1/2) may be the Ets transcription factor, PEA3 (Brent

and Tabin, 2004). Retroviral mis-expression of PEA3 in chick embryos causes transcription of

scleraxis; ectopic dominant inhibitory PEA3 represses scleraxis expression.

The objective of this experiment was to examine scleraxis and tenascin C mRNA

expression in equine umbilical cord blood (UCB) stem cells and equine adipose-derived

mesenchymal stem cells (AdMSCs) in response to FGF treatment. Results demonstrate that

UCB stem cells and AdMSCs express scleraxis and tenascin C. Culture in matrigel upregulated

expression of both transcripts. UCB stem cells treated with FGF2 or FGF5 causes an increase in

MEK-dependent phosphoERKl/2, although with different activation kinetics. Scleraxis mRNA

content was measured following 48 hours of treatment with either FGF2 or FGF5. Neither

caused an increase in sex over that seen on matrigel alone. However, TnC expression was

increased in AdMSC cultured with FGF2 or FGF5.

Materials and Methods

Stem Cell Culture

Umbilical cord blood stem cells (UCB) were cultured on plastic tissue culture plates coated

with 0.1% gelatin (Fisher Scientific, Pittsburgh, PA) in Dulbecco's Modified Eagle Medium

supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and 5 tg/ml

Plasmocin (InVivogen, San Diego, CA). Equine adipose derived cells (AdMSC) were purchased

from ScienCell (Carlsbad, CA). Cells were cultured on 0.1% gelatin coated tissue culture plates

in Mesenchymal Stem Cell Medium (ScienCell) according to manufacturer's recommendations.

Cells were passage at 70% confluency using 0.025% trypsin-EDTA. Three dimensional

cultures were achieved by growing cells on Cytodex3 collagen coated beads (Invitrogen) or

allowing cells to embed into 30% Matrigel (BD Biosciences, San Jose, CA).









0 min 10min 30 min
































Figure 5-10. FGF5 supplementation does not affect UCB actin structure. UCB stem cells were
serum starved for one hour prior to treatment with FGF5 or growth media. Cells were
fixed at 0, 10 and 30 minutes prior to visualizing actin structures with fluorescein
conjugated phalloidin. Nuclei were stained with Hoechst 33342. SF = serum free
media, FGF5 = FGF5 containing media, GM = growth media. Scale bar = 10 im









pluripotent cells, only trophectodermal cells which cannot be used to produce ES cells (Nichols

et al., 1998). Decreased expression of Oct4 by siRNA resulted in the downregulation of

pluripotency related genes such as nanog and Sox2, markers of undifferentiated stem cells such

as Lefty], Lefty2, and Thyl, and chromatin modifying factors such as DNMT3B (Babaie et al.,

2007). Relative amounts of Oct4 protein effects cell fate; overexpression of Oct4 results in

differentiation into primitive endoderm and mesoderm, while loss of the protein results in

spontaneous differentiation into trophectodermal cells (Yeom et al., 1996; Niwa et al., 2000;

Niwa, 2001). Thus, Pou5fl must be strictly regulated to maintain ES cell fate. The Oct4

pathway appears to be independent from LIF/STAT3 signaling, as disruptions to either pathway

have no direct effect on the other. However, the two transcription factors may cooperate to

regulate target genes, as many target genes contain both Oct and STAT binding sites (Tanaka et

al., 2002; Ginis et al., 2004).

Sox2.

Sox2 is a high mobility group (HMG) domain DNA binding protein that regulates

transcription and chromatin architecture (Pevny and Lovell-Badge, 1997). Sox2 forms a ternary

complex with Oct4 on the FGF4 enhancer as well as other genes involved in maintaining ES cell

pluripotency (Yuan et al., 1995; Boyer et al., 2005). Expression of Sox2 in the developing

embryo is similar to that of Oct4 (Avilion et al., 2003). Null embryos are incapable of giving

rise to pluripotent cells from the ICM, instead producing trophectoderm-like cells (Avilion et al.,

2003; Masui et al., 2007). Microarray screening indicates that Sox2 null cells express greater

levels of genes that negatively regulate Oct4 and downregulate factors that positively regulate

Oct4. Introduction of exogenous Oct4 into Sox2 null cells restored their proliferation and

pluripotency, indicating that Sox2 may upregulate the positive regulators of Oct4 while

downregulating factors that negatively affect Oct4 transcription (Masui et al., 2007). In human









musculoskeletal defects and neurological disorders could benefit from the use of a naive cell type

which promotes repair or production of healthy tissue. The potential of stem cells lies in not only

their ability to contribute healthy, differentiated cells to the "unhealthy" region, but in the

production of beneficial growth promoting factors and recruitment of additional reparative cells.

The precise effects of stem cells in each disease state remain to be determined as do the

mechanisms by which they may help.

Spinal Cord Injuries

A number of stem cell populations have been used to treat spinal cord injuries in hope of

improving neural regeneration and recovery of locomotion. Embryonic stem cells differentiated

into neural precursors were transplanted into a spinal cord injury (SCI) model in the rat

(McDonald et al., 1999). Transplanted cells were found at the injury site and at distances up to 8

mm from the injury. Engrafted ES cells expressed markers of oligodendrocytes, neurons, and

astrocytes with no evidence of tumor formation. Rats receiving UCB transplants demonstrated

partial hind limb weight bearing and improved coordination compared to a complete lack thereof

in sham operated rats. Engraftment of fetal neural stem cells into immunodeficient mice after a

traumatic spinal cord injury showed locomotory recovery (Cummings et al., 2005). Cells had

migrated extensively from the injection site at 17 weeks post injury. Transplantation resulted in a

higher degree of locomotor recovery with greater coordinated forelimb-hind limb function after

16 weeks. Ablation of the stem cells after transplantation by diphtheria toxin resulted in similar

coordination to animals with no stem cell transplantation, indicating that the improvement is due

to the presence of these cells. Engrafted cells differentiated into neurons and oligodendrocytes.

Transplantation of neurospheres into a contusion model of SCI by Ogawa et al resulted in

engrafted cells differentiating into neurons, oligodendrocytes, and astrocytes (Ogawa et al.,









protein scaffolds differentiated into more mature phenotypes connected by gap and tight

junctions (Jurga et al., 2009). Cells migrated away from aggregates and formed neural networks

that were capable of generating spontaneous electrical activity, indicating the presence of a

functional action potential.

Hepatogenic differentiation has also been shown in a number of studies. Treatment of

UCB with fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) stimulated the

expression of hepatogenic markers alpha-fetoprotein and cytokeratin 18 (Kang et al., 2005; Tang

et al., 2006). These cells were capable of producing urea, storing glycogen, and LDL

endocytosis, functional measures of liver cells (Hong et al., 2005; Kang et al., 2005; Tang et al.,

2006). Additionally, to determine if these cells were capable of producing insulin in an in vivo

environment, xenograft transplantation was performed by Zhao et al. Human UCB were

transplanted into Balb/c nude mice with induced diabetes. Mice were then evaluated for their

ability to correct hyperglycemia. Mice receiving the UCB transplantation displayed significantly

lower blood glucose levels than the untransplanted controls (Zhao et al., 2006).

Adult Stem Cells

Bone Marrow Derived Mesenchymal Stem Cells

Isolation of bone marrow derived mesenchymal stem cells (or mesenchymal stromal cells;

BMSC) has been occurring in research and medical practice for a number of years. In vivo these

cells support the formation of hematopoietic cells. BMSC derived from humans, mice and

horses attach to culture plates and exhibit a fibroblast like morphology when cultured in vitro.

They represent a very small portion of the total cell population of the bone marrow; reported

numbers vary from 0.001-0.01% (Pittenger et al., 1999). BMSC are positive for cell surface

markers SH2, SH3, CD29, CD44, CD71, CD106, CD120a, and CD124 but negative for CD14,

CD34 and CD45 (Pittenger et al., 1999). Common differentiation protocols can be used to elicit









Umbilical Cord Blood Derived Stem Cells

Identified as a population of cells from blood that are adherent in plastic cultureware,

umbilical cord blood derived stem cells (UCB) are further purified by centrifugation through a

density gradient such as Ficoll. These cells exhibit a fibroblast like appearance and proliferate,

though they are contact inhibited. UCB have been successfully isolated from humans, sheep,

pigs, dogs, and horses and appear to maintain the same basic characteristics across species

(Bieback et al., 2004; Lee et al., 2004b; Fuchs et al., 2005; Zhao et al., 2006; Koch et al., 2007;

Kumar et al., 2007).

While the explicit protein markers of UCB remain elusive, most reports agree that this

population is CD34, CD45 and major histocompatibility complex (MHC) class II negative and

only weakly positive for MHC class I (Lee et al., 2004a). The minor expression of MHC classes

I and II indicate the lack of immunogenicity of this cell type. Additionally, a mixed lymphocyte

reaction indicated that human UCB did not stimulate lymphocyte proliferation, consistent with

low levels of immunogenicity (Zhao et al., 2006). The absence of CD34 and CD45 suggests a

non-hematopoietic lineage. Additionally, cell surface markers commonly found on MSC, CD29,

CD44, CD73, and CD90 (Thy-1) are found in the majority of UCB (Bieback et al., 2004; Lee et

al., 2004b; Fuchs et al., 2005; Zhao et al., 2006; Kumar et al., 2007). The presence of Oct4,

nanog, SSEA3 and SSEA4 by some researchers suggests a naive phenotype for UCB (Baal et al.,

2004).

Collection and Processing

Aspiration of blood via syringe or through a canula into a sterile container containing anti-

coagulant (EDTA, citrate phosphate dextrose, or other citrate based anticoagulant) at the time of

parturition or caesarean section yields the highest number of UCB (Sparrow et al., 2002;

McGuckin et al., 2003; Bieback et al., 2004; Chang et al., 2006; Kern et al., 2006). Processing









cord blood derived stem cells for tenocytic markers, RT-PCR was performed using gene specific

primers for scleraxis and tenascin C. Messenger RNA for both transcripts were present in both

adipose and umbilical cord blood derived stem cells (Figure 5-1). Transcripts were sequenced to

verify identity.

Scleraxis Minimal Promoter Activity

The mouse minimal scleraxis promoter was sequence verified. Analysis of the sequence

by the Transcription Element Search System (TESS, http://www.cbil.upenn.edu/cgi-bin/tess)

revealed the presence of several putative transcription factor binding sites. The list of binding

sites with p-values less than or equal to 0.01 is presented in Table 5-3. Figure 5-2 shows a

schematic of the promoter highlighting potentially important regulatory sites. Of particular

interest is the presence of POU factor, Hes, SP1, and API binding sites. A Klf4 binding site is

present at base pair 35, but is not included on the list as the p-value was given as 0.011.

However, there were multiple sites strongly recognized in a small region lending strength to this

prediction and its inclusion on the figure.

UCB stem cells show much lower basal levels of scleraxis promoter activity than do

C2C12s (Figure 5-3). Supplementation of C2C12 cells with BMP6 reduced luciferase

expression driven by the scleraxis promoter. FGF4 and FGF5 had no effect on the scleraxis

promoter in these cells. Scleraxis promoter activity in UCB stem cells appears to be unaffected

by supplementation with BMP6, FGF4 or FGF5. This may be due to the low basal level of the

minimal promoter's activity. Other transcription factor binding sites may be required for full

activity of this promoter in this environment.

Downstream effectors of FGF signaling include the transcription factors Pea3 and Erm.

Activation of Pea3 and Erm is required for scleraxis expression in the developing somite (Brent

& Tabin). Thus, we examined the presence of these factors by RT-PCR in UCB stem cells. RT-









Maturation of UCB into bone and cartilage is routinely achieved. Both human and equine

UCB have been differentiated into cells capable of producing calcium deposits stained by von

Kossa and Alizarin Red (Kogler et al., 2004; Koch et al., 2007). Transcription of osteopontin,

osteocalcin, and type I collagen has been reported in these cells (Kogler et al., 2004). Culture of

human UCB in media containing dexamethasone and bone morphogenic protein 2 (BMP2)

resulted in morphological changes from spindle shaped cells to cuboidal cells in twenty days

coupled with increased alkaline phosphatase and type I collagen expression (Hildebrandt et al.,

2009). However, it has been noted by some researchers that UCB which differentiate into

osteoblasts do not take on the cuboidal appearance typical of bone marrow derived osteoblasts

(Goodwin et al., 2001).

Chondrogenesis is commonly achieved by culture in a three-dimensional micromass

environment in the presence of transforming growth factor 0 (TGFP). These masses react

positively with Alcian Blue and Safranin O, indicating the presence of glycosaminoglycans

typical of cartilage (Kogler et al., 2004; Koch et al., 2007). Further analysis of these cells

reveals transcription of cart-], Col2al and chondroadherin (Kogler et al., 2004). Ovine UCB

from blood collected at 80-120 days of gestation formed tissue reminiscent of hyaline cartilage

when placed on a construct of biodegradable polyglycolic acid polymer treated with poly-L-

lactic acid solution and coated with collagen. This was placed in a bioreactor in permissive

medium for 12 weeks. Marked chondrogenic differentiation was apparent, presenting

characteristics of hyaline cartilage and staining positively for Safranin O and Toluidine Blue.

Type II collagen was primarily expressed with little type I collagen present and no type X

collagen (Fuchs et al., 2005). Inclusion of insulin-like growth factor 1 (IGF-1) after the initial









Plasmids and Transfections

The 500 base pair minimal promoter of the mouse scleraxis gene was amplified from

C2C12 myoblasts with specific primers (F:CACACGGCCTGGCACAAAAGACCC

R:TTCGGACTGGAGTGGGGCCGCCAGC). Following sequencing to verify the identity of

the product, the promoter was cloned into the TOPO Zero Blunt vector prior to subcloning into

the pGL3 basic vector such that promoter activity would drive the expression of luciferase (Scx-

luc). C2C12 mesenchymal cells and UCB stem cells in 12 well plates were transiently

transfected with 250 ng Scx-luc and 25 ng pRL-tk, a Renilla luciferase plasmid, as a monitor of

transfection efficiency using FuGene 6.0. Media was replaced after 6 hours to include 50 ng/ml

BMP6, 10 ng/ml FGF4, or 10 ng/ml FGF5. Cells were passively lysed in luciferase lysis buffer

after 24 hours of culture and luciferase activities measured (Dual-Luciferase Reporter kit,

Promega, Madison, WI). Scx-luc activity was corrected for pRLtk activity. The mouse minimal

Scleraxis promoter was sequence verified. Analysis of the sequence by the Transcription

Element Search System (TESS, http://www.cbil.upenn.edu/cgi-bin/tess) revealed the presence of

several putative transcription factor binding sites.

Confocal Microscopy

UCB and AdMSC cultured on gelatin, matrigel, and collagen beads were fixed in 4%

paraformaldehyde for 15 minutes. Cells were permeabilized with 0.1% TritonX100 in phosphate

buffered saline (PBS) supplemented with 5% FBS. Actin filaments were visualized using

fluorescein conjugated phalloidin (Invitrogen) and nuclei were stained with Hoechst 33342. All

immunofluorescence work was completed on glass bottom plates to aid microscopy. Confocal

microscopy was performed on a Leica TCS SP5 Laser Scanning Confocal Microscope running

Leica LAS-AF software for instrument control and image analysis. Images were adjusted for

brightness and contrast in Adobe Photoshop CS.









the absence of LIF (Matsuda et al., 1999). These cells were capable of forming chimeric mice

when injected into blastocysts. However, LIF is not required for embryonic development null

embryos develop to a stage beyond that of ES cell isolation. It is important to remember that the

presence of the pluripotent ICM in the developing embryo is a transient state, thus the lack of

requirement of LIF is not surprising. A notable difference between hES and mES cells is the

lack of requirement for LIF signaling in hES cells. When maintained on a feeder layer in the

presence of serum, human ES cells do not require LIF signaling to prevent differentiation. Bone

morphogenic protein 4 (BMP4) is an anti-neurogenesis factor that has been shown to contribute

to LIF cascade, enhancing self renewal and pluripotency by activating SMADs which, in turn,

promote transcription of the Id gene family (inhibitors of differentiation, (Ying et al., 2003)). In

the absence of LIF, BMP4 promotes differentiation. Like LIF, this factor is not required for the

initial "creation" of the ES cell population null embryos develop past the stage of ES cell

derivation. However, ES cells can be derived in the absence of serum with media supplemented

with LIF and BMP4 (Ying et al., 2003). Wnt activation helps maintain the undifferentiated

phenotype in mouse and human ES cells by sustaining the expression of Oct4, Rex], and nanog

(Sato et al., 2004).

Oct4.

Oct4 is a Pou domain transcription factor expressed by all pluripotent cells during mouse

embryogenesis as well as undifferentiated mouse ES cells and embryonic carcinoma cell lines

(Scholer etal., 1989a; Scholer et al., 1989b; Okamoto etal., 1990; Rosner etal., 1990).

Upregulated during the 4 cell stage of embryo development, Oct4 later becomes restricted to

pluripotent stem cells. Expression of Oct4 is downregulated with differentiation (Brandenberger

et al., 2004). In addition, embryos lacking Oct4 expression develop to a stage resembling a

blastocyst and have a mass of cells designated to the location of the ICM, but do not contain









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number of differentiated or pre-cursor cell types. In permissive medium, human PBPC

underwent differentiation into neural precursor cells, evidenced by changes in morphology and

expression of nestin, 33 tubulin, and NeuN, a nuclear protein present in neurons (Porat et al.,

2006). These cells also underwent limited myogenic differentiation in vitro. PBPC cultured

with galectin 1 expressed desmin and subsequently formed multinucleated fibers over the

following week of culture (Chan et al., 2006). In vivo transplantation into an injury model (c-/y-

/RAG2- mouse) resulted in more muscle fibers expressing human spectrin in galectin 1

stimulated PBPCs than control PBPCs (
the rat striatum, where they migrated and maintained neurogenic morphology (Price et al., 2006).

Adipogenic, chondrogenic, and osteogenic conversions are also commonly completed with

PBPCs (Koerner et al., 2006; Price et al., 2006; Giovannini et al., 2008). Stem cells derived

from peripheral blood are easily obtained, but are more limited in plasticity and self-renewal than

other sources of adult stem cells.

Umbilical Cord Vein Derived Stem Cells

An alternative to umbilical cord blood, a population of stem cells has also been isolated

from the human, equine and porcine umbilical cord veins (UCV) (Kim et al., 2004; Kestendjieva

et al., 2008). The umbilicus itself is digested with collagenase; cells isolated from this procedure

form a confluent monolayer with cobblestone or spindle-shaped fibroblast like morphology

(Kestendjieva et al., 2008). These cells express oct4, sox2,and nanog as well as the cell surface

markers CD29, CD73, and CD90. However, they lacked expression of CD45, CD14, CD3,

CD19, CD16, CD34 and HLA-DR. UCV are capable of differentiation into adipogenic,

osteogenic, and endothelial cell types. Lipid droplets and Oil Red O staining were present in

UCV treated with adipogenic induction medium. Adipsin, lipoprotein lipase and PPARy2 mRNA

are also expressed in treated cells (Kim et al., 2004; Kestendjieva et al., 2008). Osteogenic










5 CULTURE OF EQUINE UMBILICAL CORD BLOOD AND ADIPOSE DERIVED
STEM CELLS TO PROMOTE TENOCYTIC DIFFERENTIATION ................................101

In tro d u ctio n ................... ...................1............................1
M materials and M methods ............................................................................. ......................103
S tem C ell C u ltu re ................................................................. .................................10 3
P lasm ids and T ransfections ............................................. ........................................ 104
Confocal Microscopy ............................... ................ ..............104
Protein Isolation and Evaluation ............................................................................105
A ssessm ent of Proliferation..................................... ......... ............... ............ 105
RNA Isolation, Reverse Transcription, and Polymerase Chain Reaction.....................106
Quantitative PCR....................... .................... ............ 106
Results ........................ ......... ........ ..... .................. .................. 106
AdMSC and UCB Express Markers of Tenocytic Cells ........................................106
Scleraxis M inim al Prom other A ctivity....................................................... ............... 107
AdMSC and UCB Survive on Various Matrices........................... ........................108
Culture in Matrigel Increases Tenocyte Gene Expression ...........................................108
Fibroblast Growth Factors Elicit Differing ERK1/2 Responses in UCB and AdMSC.109
Effect of FGF5 Supplementation on Actin Structure ................................................... 110
Response of the PI3K Pathway to FGF2 and FGF5 Supplementation......................111
D is c u s sio n ............................................................................................................................. 1 1 1

6 SUMMARY AND CONCLUSIONS ............ ..... ........ .... ............... 131

APPENDIX

A SUPPLEM EN TARY DATA ................................................................... ............... 136

Mouse Embryonic Stem Cell Culture and Differentiation ................................................136
Alternative UCB Differentiation Protocols ........................................ ....... ............... 137
M yogenic D differentiation ............................................................................ .. 137
N eural D differentiation .......................................... .. .. .... .......... ....... 137
A dipogenic D ifferentiation................................ ................................................... 137
Quantification of Oct4, nanog, and Sox2 Across Time in Culture................. .......... 138

B TABLE OF PRIMER SEQUENCES ............................................................................142

L IST O F R E FE R E N C E S ................................................................................................... 144

B IO G R A PH ICA L SK ETCH ........... ................................................................ ............... 171










8









differentiation is visualized by von Kossa staining of calcium deposits and osteopontin and

Runx2 expression (Kim et al., 2004; Kestendjieva et al., 2008).

Adipose Derived Stem Cells

A population of stem cells can be derived from adipose tissue by obtaining fat from

subcutaneous surgery. The tissue undergoes enzymatic digestion followed by filtration and

centrifugation. Adherence to plastic and subsequent expansion produces a relatively

homogenous population. Morphologically similar to bone marrow and umbilical cord blood

derived stem cells, adipose derived stem cells are spindle shaped. Adipose derived

mesenchymal stem cells (AdMSC) express similar cell surface markers to bone marrow derived

MSC, including CD13, CD44, CD73, CD90, CD105, CD106, CD166, CD29, CD49e, and HLA-

ABC (Gronthos et al., 2001; Katz et al., 2005; Wagner et al., 2005; Yanez et al., 2006). While

HLA-ABC surface markers are expressed on these cells, they lack expression of HLA-DR.

Additionally, AdMSC did not stimulate lymphocyte proliferation (Yanez et al., 2006). In fact,

AdMSC inhibited the proliferation of T cells stimulated with peripheral blood mononuclear cells.

When co-cultured in a transwell, soluble factors secreted by AdMSC exerted immunosuppressive

effects on responder T cells but only when AdMSC could interact with responder lymphocytes.

Furthermore, AdMSC infusion decreased the severity of graft v. host disease in mice when used

in the first two weeks.

Adipose derived stem cells have been induced to differentiate into osteogenic,

chondrogenic, adipogenic, cardiomyogenic and neurogenic cell types (Gronthos et al., 2001;

Katz et al., 2005; Wagner et al., 2005; Guilak et al., 2006; Oedayraj singh-Varma et al., 2006;

Liu et al., 2007; Yoshimura et al., 2007; Zhu et al., 2008; Mehlhom et al., 2009). Cells induced

to osteogenic differentiation express RunX2 and Collal after 7 days of culture, with

mineralization after 3 weeks (Oedayrajsingh-Varma et al., 2006). Comparison of AdMSC and
























500 bp
300 bp




Figure 5-4. UCB stem cells express Erm, Pea3, and Scleraxis. RT-PCR was performed on UCB
stem cells with gene specific primers as shown. 18S was included as an internal
control. Scx = scleraxis.


,~d




Full Text

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1 ISOLATION AND CHARACTERIZATION OF EQUINE UMBILICAL CORD BLOOD DERIVED STEM CELLS By SARAH ANN REED A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FO R THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Sarah Ann Reed

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3 To Jake, who taught me what i t meant to be a true horsewoman; Aja, who relit the fire; and Jared who was there for it all

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4 ACKNOWLEDGMENTS There are so many people whom have helped me throughout this process that it would be impossible to thank you all. First and foremost, I am thankful to my advisor, Dr. Sally Johnson. Sally gave me the chance to work in her lab as an unproven technician and then invited me to join her lab as a student. Over the past five years, she has coaxed, encouraged, motivated and made me into a better scientist and a better person. She pushed me beyond what I thought were my limits because, in the end, she knew I could do more. My committee has been supportive throughout my tenure at UF. Dr. Moore was instrumental in teaching me valuable ES culture techniques. Dr. Brown provided a wonderful outside perspective of my project from a clinician s point of view. Dr. Ealy was a great sounding board for ideas and was extremely helpful with statistical analysis. My lab mates have been wonderful at providing friendship and support. Sophia, Dane, Juli, Sara, John Michael, Lulu and Dillon Thank you. I am forever indebted to my friends who have been there for me throughout the last five years. Sara, Beth, and Ella have all been the greatest friends to have, whether I needed a shoulder to cry on or to just go out and have fun. John Michael has been my lab best friend and the person that I could always gripe to when I had a frustration with anything. I couldnt have gotten through all of the real time without him. My family has provided constant support and understanding, even when attending graduate school meant moving 1100 miles away from home, missing Christmas es, birthdays and all of the little things in family life. My Mom and Dad, Lisa and Howard Grove, have provided endless encouragement and faith that I could do this. My grandparents have always let me know how much I am loved and that they believe I can do anything I set my mind to do. My inlaws, Don and Barb Reed, have also provided much support and encouragement along the way, and I thank them for accepting me into their family.

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5 Fin ally, I am supremely thankful to my husband, Jared, who has been there through all of the tears, successes, late night blood collections and long work hours. He had faith that I would succeed even when I lost mine. Even when he didnt understand what, he understood why and stood behind me the whole way. His unfailing support is the only reason I stand where I do today. He is the love of my life, my partner, my other half, my husband and I am the luckiest girl in the world to have found him.

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6 TABLE OF C ONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................9 LIST OF FIGURES .......................................................................................................................10 ABSTRACT ...................................................................................................................................12 CHAPTER 1 INTRODUCTION ..................................................................................................................14 Self Renewal ...........................................................................................................................14 Stem Cell Plasticity .................................................................................................................17 Transcriptional Regulation of Plasticity ..........................................................................17 Oct4. .........................................................................................................................18 Sox2. .........................................................................................................................19 Nanog .......................................................................................................................20 Epigenetic Regulation of Plasticity .................................................................................21 Role of Fibroblast Growth Factors in Stem Cell Maintenance and Differentiation ...............23 Embryonic Stem Cells ............................................................................................................25 Characteristics .................................................................................................................25 Differentiation .................................................................................................................30 Umbilical Cord Blood Derived Stem Cells ............................................................................33 Collection and Processing ...............................................................................................33 Differentiation .................................................................................................................34 Adult Stem Cells .....................................................................................................................38 Bone Marrow Derived Mesenchymal Stem Cells ...........................................................38 Peripheral Blood Derived Progenitor Cells .....................................................................39 Umbilical Cord Vein Derived Stem Cells .......................................................................40 Adipose Derived Stem Cells ...........................................................................................41 Therapeutic Uses of Stem Cells ..............................................................................................42 Spinal Cord Injuries .........................................................................................................43 Vertebrae .........................................................................................................................44 Cardiovascular Repair .....................................................................................................45 Tendon and Ligament Injuries .........................................................................................47 Bone Injuries ...................................................................................................................49 Cartilage Injuries .............................................................................................................51 Muscular Dystrophies ......................................................................................................53 Conclusion ..............................................................................................................................54 2 CENTRAL HYPOTHESIS ....................................................................................................55

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7 3 E QUINE UMBILICAL CORD BLOOD CONTAINS A POPULATION OF STEM CELLS THAT EXPRESS OCT4 AND DIFFERENTIATE INTO MESODERMAL AND ENDODERMAL CELL TYPES ..................................................................................57 Introduction .............................................................................................................................57 Materials and Methods ...........................................................................................................59 Umbilical Cord Blood (UCB) Coll ection and Stem Cell Isolation .................................59 Equine UCB and Adipose Derived (AD) Stem Cell Culture ..........................................59 RNA Isolation, Reverse Transcription (RT), and Polymerase Chain Reaction (PCR) ...60 Osteogenic Differentiation ..............................................................................................60 Chondrogenic Differentiation ..........................................................................................61 Adipogenic Differentiation ..............................................................................................61 Hepatogenic Differentiation ............................................................................................61 Myogenic Differentiation ................................................................................................62 Histology .........................................................................................................................62 Immunocytochemistry .....................................................................................................62 Results .....................................................................................................................................63 Foal Umbilical Cord Blood Contains an Oct4Expressing Cell Population ...................63 UCB Stem Cells Form Chondrocytes ..............................................................................64 Differentiation of UCB Stem Cells into Osteocytes ........................................................64 Foal UCB Stem Cells can Differentiate into EndodermalDerived Cell Ty pes ..............65 Inefficient Formation of Myocytes and Adipocytes by UCB Cells ................................65 AdMSC do not Express the Same Complement of Stem Cell Markers ..........................66 Discussion ...............................................................................................................................67 4 REFINEMENT OF CULTURE CONDITIONS TO PROMOTE THE MAINTENANCE OF EQUINE UMBILICAL CORD BLOOD DERIVED STEM CEL LS ....................................................................................................................................78 Introduction .............................................................................................................................78 Materials and Methods ...........................................................................................................79 UCB Collection and Stem Cell I solation .........................................................................79 Stem Cell Culture ............................................................................................................80 RNA Isolation, Reverse Transcription (RT) and Polymerase Chain Reaction (PCR) ....81 Statistical Analysis ..........................................................................................................82 Results .....................................................................................................................................83 UCB Express Markers of Pluripotent Stem Cells ...........................................................83 GM and GM+FGF Maintain UCB Proliferation .............................................................83 Protein Surface Matrixes Promote UCB Growth ............................................................84 Oct4 is Maintained Throughout UCB Culture ................................................................84 Notch Signa ling in UCB Stem Cells ...............................................................................85 Discussion ...............................................................................................................................87

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8 5 CULTURE OF EQUINE UMBILICAL CORD BLOOD AND ADIPOSE DERIVED STEM CELLS TO PROMOTE TENOCYTIC DIFFERENTIATION ................................101 Introduction ...........................................................................................................................101 Materials and Methods .........................................................................................................103 Stem Cell Culture ..........................................................................................................103 Plasmids and Transfections ...........................................................................................104 Confocal Microscopy ....................................................................................................104 Protein Isolation and Evaluation ...................................................................................105 Assessment of Prolife ration ...........................................................................................105 RNA Isolation, Reverse Transcription, and Polymerase Chain Reaction .....................106 Quantitative PCR ...........................................................................................................106 Results ...................................................................................................................................106 AdMSC and UCB Express Markers of Tenocytic Ce lls ...............................................106 Scleraxis Minimal Promoter Activity ............................................................................107 AdMSC and UCB Survive on Various Matrices ...........................................................108 Culture in Matrigel Increases Tenocyte Gene Expression ............................................108 Fibroblast Growth Factors Elicit Differing ERK1/2 Responses in UCB and AdMSC .109 Effect of FGF5 Supplementation on Actin Structure ....................................................110 Response of the PI3K Pathway to FGF2 and FGF5 Supplementation ..........................111 Discussion .............................................................................................................................111 6 SUMMARY AND CONCLUSIONS ...................................................................................131 APPENDIX A SUPPLEMENTARY DATA ................................................................................................136 Mouse Embryonic Stem Cell Culture and Differentiation ...................................................136 Alternative UCB Differentiation Protocols ..........................................................................137 Myogenic Differentiation ..............................................................................................137 Neural Differentiation ...................................................................................................137 Adipogenic Differentiation ............................................................................................137 Quantification of Oct4, nanog, and Sox2 Across Time in Culture .......................................138 B TABLE OF PRIMER SEQUENCES ...................................................................................142 LIST OF REFERENCES .............................................................................................................144 BIOGRAPHICAL SKETCH .......................................................................................................171

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9 LIST OF TABLES Table page 41 Effect of substrata on equine UCB1 derived stem cell population doubling time .............92 42 Effect of substrata and media on equine UCB1 derived s tem cell doubling time ..............93 43 Effects of horse, passage, media and substrata on mRNA expression ..............................95 44 Delta Ct values for hes realtime PCR. ...............................................................................99 51 Real time PCR primers ....................................................................................................116 52 Cycle threshold ranges .....................................................................................................117 53 Putative transcription factor binding sites on the mouse scleraxis minimal promoter1. ..120 B 1 Primer sequences and sources. .........................................................................................142

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10 LIST OF FIGURES Figure page 31 Foal UCB cells ex press stem cell marker proteins ............................................................72 32 Induction of chondr ogenesis in foal UCB stem cells .........................................................73 33 UCB stem cells form osteocytes .......................................................................................74 34 Foal UCB stem cells form hepatocytes ..............................................................................75 35 Incomplete initiation of adipogenesis and myogenesis in foal UCB stem cells and AdMSCs .............................................................................................................................76 36 AdMSC fail to expr ess embryonic stem cell markers .......................................................77 41 GM and GM+FGF support e quine UCB stem cell propagation ........................................91 42 Equine UCB stem cells express markers of em bryonic stem cell pl uripotency ................94 43 UCB and AdMSC express a limited number of molecules in the Notch signaling pathway. .............................................................................................................................96 44 Inhibition of the Notch signaling pathwa y does not affect proliferation ...........................97 45 UCB and 23A2 myoblasts express hes .............................................................................98 46 BMP6 inhibits myoblas t differentiatio n in a Notch dependent manner ..........................100 51 AdMSC and UCB express markers of tenocytic cells ....................................................118 52 Mouse scleraxi s promoter with putative tra nscription factor binding sites .....................119 53 Scleraxis promoter activity is not increased by growth factor supplementation in UCB stem cells. ...............................................................................................................122 54 UCB stem cells express Erm Pea3 and Scleraxis .........................................................123 55 UCB and AdMSC att ach to various culture surfaces .......................................................124 56 Culture in matrigel inc reases tenocyte gene expression ..................................................125 57 UCB and AdMSC respond uniquely to FGF stimulation. ...............................................126 58 Fibroblast growth factors stimulate proliferation of AdMSC and UCB stem cel ls in a MAPK dependent manner ................................................................................................127 59 Culture conditions affect tenocyte gene expr ession in AdMSC and UCB. .....................128

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11 510 FGF5 supplementation does not affect UCB actin structure ...........................................129 511 FGF2 and FGF5 do no activat e Akt in UCB (A) or AdMSC (B). ...................................130 A 1 Stages of mES colony differentiation. .............................................................................140 A 2 mES embroid bodies differentiate into a variety of cell types.. .......................................141

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ISOLATION AND CHARACT ERIZATION OF EQUINE UMBILICAL CORD BLOOD DERIVED STEM CELLS By Sarah Ann Reed August 2009 Chair: Sally E Johnson Major: Animal Molecular and Cellular Biology Musculoskeletal injuries are responsible for a large portion of wastage in sport horses. Mesenc hymal stem cells (MSCs) offer promise as therapeutic aids in the repair of tendon, ligament, and bone damage suffered by these horses. The objective of thes e studies was to identify and characterize stem like cells from newborn foal umbilical cord blood (U CB). UCB was collected and MSC isolated using human reagents. The cells exhibit a fibroblast like morphology and express the stem cell markers Oct4, SSEA 1, Tra160 and Tra181. UCB express transcripts implicated in embryonic stem (ES) cell pluripotency, namely Oct4, nanog, Sox2, Klf4 and c myc Culture of the cells in tissue specific differentiation media leads to the formation of cell types characteristic of mesodermal and endodermal origins including chondrocytes, osteocytes, and hepatocytes Limited a dipogenic and myogenic differentiation occurred. Population doubling time and the presence of Oct4, nanog and Sox2 transcripts were used to determine culture conditions that promoted the proliferation of a stem cell population. Culture on a protein matrix (gelatin, collagen or fibronectin) shortened population doubling time compared to growth on uncoated plasticware. Inclusion of fibroblast growth factor 2 (FGF2) in the growth media slowed proliferation. The persistence of Oct4 nanog and Sox2 expression was monitored over time in culture in UCB stem cells. Oct4 was detected throughout the duration of

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13 the experiment. Sox2 and nanog expression declined with time in culture. Finally, the effect of culture conditions on expression of tendon markers was as sessed. Initial stem cell populations express scleraxis an early marker of tenocytic differentiation. Culture on collagen coated beads did not affect scleraxis levels; however culture in 30% Matrigel significantly increased levels of the transcript. This change was accompanied by differences in morphology. Cells grown in Matrigel formed tight colonies reminiscent of ES cell colonies while those on collagen coated beads maintained a fibroblast like morphology. In conclusion, we have isolated a population of stem cells from equine umbilical cord blood that possess a number of stem cell markers, can be expanded in culture, and can differentiate into a variety of potentially useful cell types.

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14 CHAPTER 1 INTRODUCTION The millions of cells that compose an o rganism stem from a small population of cells of the inner cell mass (ICM) in the developing embryo. During development, these cells give rise to more committed cells which form the three germ layers: endoderm mesoderm, and ectoderm. With increasing num bers of divisions, the cells become more committed until the majority of the organism is composed of fully differentiated adult cell types. However, in most tissues a population of cells remains that is capable of recapitulating at least some of the cell types specific to that tissue. These stem cells maintain homeostasis throughout aging and insult. Stem cells have several characteristic properties that define the global stem cell population self renewal and differentiation into a variety of more matur e cell types. Self renewal is an essential property that allows repopulation of the stem cell community. One daughter cell is destined to become a more committed cell type, while the other remains a malleable stem cell. Plasticity, or the ability to dif ferentiate, is a property that is unique to the type of stem cell. Different populations have varied amounts of plasticity thus, while one cell type may be capable of differentiation into two committed cell types, another such as an embryonic stem cell can contribute to every tissue of the adult organism. Self Renewal The process of self renewal allows stem cells to contribute to a population of committed cells while maintaining a stable population of stem cells. This can occur through a variety of me chanisms including asymmetric cell division, polarization initiated by external factors, fate determination by cell:cell contact, and stochastic regulation of intrinsic processes. It is most likely a complex combination of all four mechanisms that creates a permissive and instructive environment for stem cell self renewal. During development, the axis of polarity is established

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15 and coordinated with the body plan. Cell fate determinants are localized asymmetrically along this axis. During mitosis, the mitotic spindle is oriented along the axis such that cytokinesis creates two daughter cells containing different concentrations of these determinants. In Drosophila germ line stem cells, differential positioning of the mother and daughter centrosomes during mitosis determines the fate of each resulting cell. The mother centrosome is anchored to the niche by astral microtubules. The cell containing the daughter centrosome migrates away from the niche following cytokinesis and differentiates (Yamashita et al. 2003; Spradling and Zheng, 2007; Yamashita et al. 2007) In neural stem cells, asymmetrical localization of cellular proteins influences cell fate. Numb, a negative regulator of Notch signaling is localized asymmetrically at one pole of mitotic spindle in the sensory organ precursor cell of the Drosophila peripheral nervous system such that only one daughter cell inherits the protein (Rhyu et al. 1994) In this system, Numb influences cell fate by repressing Notch signalin g. The primary function appears to be the creation of two daughter cells that can respond differently to external cues rather than to specify a specific cell fate (Rhyu et al. 1994) Using videomicroscopy, Li et al report that 3040% of mouse neuroepithelial cells segregate Numb to one daughter cell during division (Li et al. 2003) This is correlated wi th asymmetric division to one neuroepithelial cell and one neuron. In addition, 18% of cortical precursor cells divided and expressed Notch1 asymmetrically. Notch1 was associated with the basal cell of the dividing pair (Chenn and McConnell, 1995) Hematopoietic stem cells (HSC) provided the first indication that stem cells were capable of s elf renewal through asymmetric division. Colony formation assays of HSC demonstrated that approximately 20% of progenitors divide asymmetrically ( Suda et al. 1984a; Suda et al. 1984b) In a population of CD34+ HSC, one daughter remained quiescent or divided very

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16 slowly while the other multiplied quickly and yielded committed progenitors (Huang et al. 1999) Slow dividing fractions of HSC are associated with primitive function and self renewal while the fast dividing fraction proceeds to differentiation (Huang et al. 1999; Cheng et al. 2000; Punzel et al. 2002) Using time lapse microscopy, Wu et al showed that hematopoietic precursor cells are capable of symmetric commitment, symmetric self renewal, and asymmetric division (Wu et al. 2007c) Additionally, overexpression of Numb in these cells resulted in fewer lineage ne gative cells. In addition, HSC self renewal is mediated at least in part by the stem cell niche. Long term repopulating cells (LTRC; CD34+CD38) are located in the HSC niche (the endosteal region associated with bone lining osteoblasts or endothelial cel ls) in a quiescent state (Yahata et al. 2008) Approx 75% of the most primitive long term repopulating hematopoietic stem cells are resting in G0 (Cheshier et al. 1999) Following isolation, these cells were capable of successful engraftment and reconstitution of hematopoiesis (Yahata et al. 2008) Clonally distinct LTRC controlled hematopoietic homeostasis and created a stem cell pool hierarchy by asymmetric self renewing division that produced both lineage restricted, short term repopulating cells and LTRC s (Yahata et a l. 2008) Quiescent LTRC clones can expand to reconstitute the hematopoiesis of the secondary recipient (Yahata et al. 2008) More restricted adult stem cells are also capable of self renewal. Muscle satellite cells that are Pax7+/Myf5 can give rise to Myf5+ cel ls when dividing in the basal/apical orientation (Kuang et al. 2007) Myf5 expressing cells lay next to fiber in a position to differentiate and fuse while null cells remain next to basal lamina in a position to remain as a precursor cell. Committed cells expressed Delta1 while the more nave progenitor cells did not. Conversely, Notch3 was highly expressed in the progenitor but not committed cells. Notch 1, 2 and Numb levels were equal in both populations. Using videomic roscopy, Shinin et al examined the

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17 segregation of NumbGFP in dividing satellite cells (Shinin et al. 2006) Both symmetric and asymmetric segregation was observed; approximately 34% of primary m yogenic cells in culture displayed asymmetric Numb localization following division. Numb segregated to the mother cell. Slow dividing, label retaining satellite cells contain the template DNA strand during division and maintain Pax7 expression. In cells in which both the DNA and Numb were segregated asymmetrically, 90% retained the Numb and template DNA in the same (mother) cell. Furthermore, these cells expressed Pax7 and did not differentiate. Stem Cell Plasticity As mentioned above, different populat ions of stem cells have different levels of plasticity. Totipotent cells are those capable of recapitulating the entire organism including the extraembryonic materials. Pluripotent cells, such as ES cells, contribute to the three germ layers. Cells with limited abilities that can only differentiate into a few cell types (generally of the same germ layer) are termed multipotent. Pluripotency of ES cells has only been shown conclusively in the mouse, where ES cells completely integrated into a developing embryo and produced a highly chimeric fetus (Evans and Kaufman, 1981) The mechanisms that regulate plasticity must be pliable enough to allow differentiation under appropriate stimuli but rigorous enough to override the developmental program when needed. Transcriptional Regulation of Plasticity In ES cells, there are several well characterized pathways of transcriptional regulation of pluripotency. Leukemia inhibitory factor (LIF) is essential for the maintenance of mES cells in vitro LIF binds to the LIF receptor gp130 heterodimer to activate STAT3 signaling in mES cells (Niwa et al. 1998) Inhibition of STAT3 phosphorylation results in loss of DNA binding ability and morphological changes to ES cells resembling differentiation (Boeuf et al. 1997) In contrast, activation of STAT3 is sufficient to maintain mES cells in an undifferentiated state in

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18 the absence of LIF (Matsuda et al. 1999) These cells were capable of forming chimeric mice when injected into blastocysts. However, LIF is not required for embryonic development null embryos develop to a stage beyond that of ES cell isolation. It is important to remember that the presence of the pluripotent ICM in the developing embr yo is a transient state, thus the lack of requirement of LIF is not surprising. A notable difference between hES and mES cells is the lack of requirement for LIF signaling in hES cells. When maintained on a feeder layer in the presence of serum, human ES cells do not require LIF signaling to prevent differentiation. Bone morphogenic protein 4 (BMP4) is an anti neurogenesis factor that has been shown to contribute to LIF cascade, enhancing self renewal and pluripotency by activating SMADs which, in turn, pr omote transcription of the Id gene family (inhibitors of differentiation, (Ying et al. 2003) ). In the absence of LIF, BMP4 promotes differentiation. Like LIF, this factor is not required for the initial creation of the ES cell population null embryos develop past the stage of ES cell derivation. However, ES c ells can be derived in the absence of serum with media supplemented with LIF and BMP4 (Ying et al. 2003) Wnt activation helps maintain the undifferentiated phenotype in mouse and human ES cells by sustaining the expression of Oct4, Rex1 and nanog (Sato et al. 2004) Oct4. Oct4 is a Pou domain trans cription factor expressed by all pluripotent cells during mouse embryogenesis as well as undifferentiated mouse ES cells and embryonic carcinoma cell lines (Scholer et al. 1989a; Scholer et al. 1989b; Okamoto et al. 1990; Rosner et al. 1990) Up regulated during the 4 cell stage of embryo development, Oct4 later becomes restricted to pluripotent stem cells. Expression of Oct4 is downregulated with differentiation (Brandenberger e t al. 2004) In addition, embryos lacking Oct4 expression develop to a stage resembling a blastocyst and have a mass of cells designated to the location of the ICM, but do not contain

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19 pluripotent cells, only trophectodermal cells which cannot be used to produce ES cells (Nichols et al. 1998) Decreased expression of Oct4 by si RNA resulted in the downregulation of pluripotency related genes such as nanog and Sox2, markers of undifferentiated stem cells such as Lefty1, Lefty2, and Thy1, and chromatin modifying factors such as DNMT3B (Babaie et al. 2007) Relativ e amounts of Oct4 protein effects cell fate; overexpression of Oct4 results in differentiation into primitive endoderm and mesoderm, while loss of the protein results in spontaneous differentiation into trophectodermal cells (Yeom et al., 1996; Niwa et al. 2000; Niwa, 2001) Thus, Pou5f1 must be strictly regulated to maintain ES cell fate. The Oct4 pathway appears to be independent from LIF/STAT3 signaling, as disruptions to either pathway have no direct effect on the other. Howeve r, the two transcription factors may cooperate to regulate target genes, as many target genes contain both Oct and STAT binding sites (Tanak a et al., 2002; Ginis et al. 2004) Sox2. Sox2 is a high mobility group (HMG) domain DNA binding protein that regulates transcription and chromatin architecture (Pevny and Lovell Badge, 1997) Sox2 forms a ternary complex with Oct4 on the FGF4 enhancer as well as other genes involved in maintaining ES cell pluripotency (Yuan et al. 1995; Boyer et al. 2005) Expression of Sox2 in the developing embryo is similar to that of Oct4 (Avilion et al. 2003) Null embryos are incapable of giving rise to pluripotent cells from the ICM, instead producing trophectoderm like cells (Av ilion et al. 2003; Masui et al. 2007) Microarray screening indicates that Sox2 null cells express greater levels of genes that negatively regulate Oct4 and downregulate factors that positively regulate Oct4. Introduction of exogenous Oct4 into Sox2 null cells restored their proliferation and pluripotency, indicating that Sox2 may upregulate the positive regulators of Oct4 while downregulating factors that negatively affect Oct4 transcription (Masui et al. 2007) In human

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20 ES cells, treatment with Sox2 siRNA significantly reduced Sox2 Oct4 and nanog expression levels (Fong et al. 2008) Decreased Sox2 expression also resulted in significantly reduced populations of SSEA3, SSEA4, Tra160 and Tra1 81 expressing cells. Expression levels of chromatin remodeling factors and transcript ion factors known to regulate pluripotency were also downregulated in Sox2 knockdown cells. Nanog The homeobox domain containing transcription factor Nanog is expressed by undifferentiated ES cells, however it is not capable of preventing differentiatio n after the withdrawal of LIF (Chambers et al. 2003; Mitsui et al. 2003) Additionally, Oct4 is required for Nanog mediated self renewal in ES cells (Chambers et al. 2003) Null embryos lack a primitive ect oderm and consist almost entirely of disorganized extraembryonic tissues (Mitsui et al. 2003; Hyslop et al. 2005) ES cells lacking nanog differentiate slowly into extra embryonic lineages (Mitsui et al. 2003) Nanog transcriptionally represses gene s involved in differentiation. Overexpression of nanog in ES cells allows the cells to remain pluripotent in the absence of LIF, although the ability to self renew is reduced (Chambers et al. 2003; Mitsui et al. 2003) This ability is abrogated if nanog is mutated to be dimerization incompetent (Wang et al. 2008) In cooperation with Sall4, a spalt like zinc finger protein, nanog occupies target genes important for pluripotency, including Pou5f1, Sox2, and nanog (Wu et al. 2006; Zhang et al. 2006) Transcription of Nanog itself is suppressed by p53 binding of the nanog promoter (Lin et al. 2005) Oct4, Sox2 and Nanog map to promoters of a large population of genes that are both active in defining ES cell identity (the undifferentiated phenotype) and r epressing developmental/differentiation genes. These factors also participate in autoregulatory and feed forward loops (Boyer et al. 2005; Loh et al. 2006) Nanog contains a 15 base pair Oct Sox composite element in the proximal promoter (Rodda et al. 2005) Sox2 and Oct4 bind this

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21 module both in vitro and in viable mouse and human ES cells. Expression of constitutively active nanog maintains mES cells in the undif ferentiated state in the absence of LIF (Mitsui et al. 2003) Recent work has suggested that Oct4, nanog and Sox2 cooperate in regulating self renewal and plasticity. Numerous target genes have been identified to be bound by the three factors. Target genes of Oct4 have a Sox binding element 0 or 3 base pairs from the octamer binding element (Rodda et al. 2005) Significantly, a complex containing both Oct4 and Sox2 regulates the expression of nanog (Kuroda et al. 2005; Rodda et al. 2005) Oct4 and Sox2 recognize and bind elements in the regulatory regions of their own genes (Chew et al. 2005) Large n umbers of target genes of Oct4 are also bound by nanog in ES cells, suggesting that these pathways work in cooperation rather than independently of one another (Boyer et al. 2005; Loh et al. 2006) The gene targets identified represent a variety of products required for pluripotency but also those required for differentiation, suggesting that these transcription factors not only promote the maintenance of the nave state but also block the progression of differentiation. Klf4 binds the regulatory regions of Pou5f1 and nanog and is also found in many complexes of these proteins at transcriptional regulatory sites on ot her genes. Nanog and Oct4 may interact directly with each other and chromatin remodeling proteins (Liang et al. 2008) A transient reduction in Pou5f1 and nanog induced differentiation in mES cells (Hough et al. 2006) Epigenetic R egulation of P lasticity Another mechanism by which plasticity is maintained is the epigenetic regulation of chromatin structure. Epig enetic marks (including methylated DNA and possibly modified histones) are propagated at S phase, thus epigenetic information can be transmitted through sequential rounds of cell division (Jaenisch and Bird, 2003; Henikoff et al. 2004) Chromatin is subjected to various forms of epigenetic regulation that modulat e the transcriptional activity of

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22 specific genomic regions including chromatin remodeling, histone modifications, histone variants, and DNA methylation. For example, trimethylation of lysine 9 and lysine 27 on histone 3 (H3K9 and H2K27, respectively) corr elate with inactive regions of chromatin. However, trimethylation of H3K4 and acetylation of histones three and four are associated with active areas of transcription (Jenuwein and Allis, 2001) Generally, methylation is considered a repressive mark (Santos and Dean, 2004) Polycomb group proteins (PcG) also silence developmental regulators, aiding in the maintenance of a plastic state (Boyer et al. 2006; Lee et al., 2006) PcG proteins form two repre ssor complexes, PRC1 and PRC2. PRC2 inhibits initiation of transcription, while PRC1 maintains the repressed state (Boyer et al. 2006) Genes that are co occupied by PRC1 and PRC2 also exhibit H3K27 trimethylation which is catalyzed by PcG proteins (Cao et al. 2002; Ringrose et al. 2004) Recently, a configuration of epigenetic modification has been described on target genes poised for transcription but not yet active. These bivalent histones contain both repressive and active histone markers, with large regions of H3K27 trimethylation interrupted by a smaller region of H3K4 trimethylation. This configuration is frequently associated with developmentally regulated factors that may be expressed at low levels in stem cells. Upo n differentiation, most bivalent domains become H3K4 or H3K27 methylated (Azuara et al. 2006; Bernstein et al. 2006) BAF, a member of the SWI/SNF family of ATP dependent chromatin remodeling complexes, is expressed abundantly in ES cells. BAF250a is a member of the BAF complex which can target and antagonize PcG proteins. Absence of BAF250a results in embryos that fail to undergo gastrulation and proper germ layer development (Gao et al. 2008) Null ES cells differentiate into cells with endoderm like morp hology. These cells express a marked reduction

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23 in Oct4 and Sox2. Loss of BAF250a resulted in the inability to specify some lineage specific differentiation. Overall, the stem cell must maintain a highly dynamic and transcriptionally permissive state to b e poised to respond to appropriate stimuli. These cells exhibit fewer heterochromatin foci that are more diffuse than in differentiated cells. Using fluorescent recovery after photobleaching, Meshorer et al demonstrated the presence of an increased fract ion of loosely bound or soluble architectural chromatin proteins (Meshorer et al. 2006) With this method, higher recove ry rates reflect loose binding of these proteins to chromatin, rendering it more accessible to transcription factors and chromatin modifiers. Overall, the chromatin of stem cells exists in a more permissive transcriptional state than that of differentiated cells. Role of Fibroblast Growth Factors in Stem Cell Maintenance and Differentiation The fibroblast growth factors (FGF) were first discovered as a family of growth factors that promoted proliferation. There are now over twenty FGFs identified in the human with different temporal and spatial expression patterns. FGFs function in proliferation, differentiation and migration during development and adult life. Most FGFs share a conserved internal region of residues responsible for receptor binding (Baird et al. 1988; Plotnikov et al. 1999) Two of the twelve B strands in the core region of the protein are thoug ht to contain the heparin binding domain which is separate from the domain which binds the receptor. Most FGFs have aminoterminal signal peptides and are readily secreted. However, FGF1 and FGF2 lack this sequence and may be released via exocytosis (Mignatti et al. 1992) Extracellular FGFs bind four high affinity transmembrane r eceptor tyrosine kinases which propagate signal transduction intracellularly. Stable interaction between the FGF and FGF receptor (FGFR) requires the presence of heparin sulfate proteoglycans which protect the complex from proteolysis and thermal degradat ion. Heparin sulfate may also limit the amount of FGF diffusion into the

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24 interstitial spaces. FGFs bind FGFR in a 2:2:2 fashion where each fully active complex consists of two each of FGF, FGFR, and heparin sulfate. FGF receptors contain two or three immunoglobulinlike domains, a short, conserved area of acidic residues which contains separate ligand and heparin binding domains, a transmembrane region and two intracellular tyrosine kinase domains. To achieve downstream signaling, binding of the ligand to the FGFR and heparin sulfate must occur. The resulting tyrosine phosphorylation of FRS2, a docking protein, allows the recruitment of Grb2 molecules which, in turn, recruit the nucleotide exchange factor SOS. The formation of the FRS2Grb2 SOS complex activates the Ras Raf MAPK pathway, resulting in changes in gene transcription. Phosphorylation of the FGFRs can also activate the PKC pathway. Alternatively, Grb2 can recruit Gab1 to activate PI3K/Akt signaling. This diversity allows a single set of receptors to influence a wide variety of cellular events. Receptor diversity is controlled by alternative splicing and results in differential ligand binding characteristics as well as varying temporal and spatial expression patterns (Lee et al. 1989; Johnson et al. 1990) Alternative splicing specifies the sequence of the immunoglobulin domain III determining the rec eptor isoform (i.e. IIIb, IIIc). Splicing occurs in a tissue specific manner and dramatically affects ligand specificity (Miki et al. 1992; Orr Urtreger et al. 1993; Scotet and Houssaint, 1995) Identified as potent mitogens, FGFs play a major role in ste m cell biology that ranges from self renewal to differentiation to cell attachment and migration. Importantly, FGF2 is required by human ES cells to sustain self renewal and plasticity (Amit et al. 2000; Xu et al. 2001) Inhibition of FGFRs in hES cells suppressed activation of downstream signaling, led to the downregulation of Oct4 and stimulated rapid differentiation (Dvorak et al. 2005) Supplementation of FGF2 at levels greater than 5 ng/ml decreased the outgrowth of hES colonies

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25 while not affecting proliferation suggesting a role in cell attachment and spreading. Exogenous FGF2 enhances growth of bone marrow mesenchymal stem cells (Solchaga et al. 2005) It prolongs the life span of bone marrow stromal cells by stimulating telomere elongation (Bianchi et al. 2003) Murine bone marrow derived Sca1positive stem cells proliferated in response to FGF4 and FGF2 under low serum conditions and exhibited a simultaneous phosphorylation of ERK1/2 and Akt. Changes in proliferation were abrogated by the inclusion of MEK and PI3K inhibitors. The upregulation of the immediate early gene c jun was also apparent following ERK1/2 activity (Choi et al. 2008) FGF4 is secreted from undifferentiated hES cells and promoted self renewal. Not only did expression of FGF4 decline with differentiation, but knockdown of the growth factor with siRNA resulted in differentiation, indicating a role for FGF4 in the maintenance o f the undifferentiated state in human ES cells (Mayshar et al. 2008) Accordingly, Sox2 and Oct4 can transactivate the FGF4 enhancer in F9 embryonal carcinoma cells suggesting a circular feedback mechanism (Yuan et al. 1995) Alternatively, Fgf4 / mES cells do not undergo differentiation upon removal of LIF, but will differ entiate along the neural lineage if further supplemented with exogenous FGF4. Cells null for FGF4 exhibit a reduced ability to enter neuronal and mesodermal lineages which does not stem from a lack of proliferation or death of precursor cells. Null cells d o not select alternative commitment programs, rather they remain in an undifferentiated state (Kunath et al. 2007) Embryonic Stem Cells Characteristics Embryonic stem cells were first derived from the i nner cell mass of a mouse blastocyst in 1981 (Evans and Kaufman, 1981) Established human lines followed in the late 1990s (Thomson et al. 1998) The immortal cells derived from the inner cell mass are an in vitro

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26 phenomenon, existing only transiently in vivo Much work has been done to characterize these cells in hopes of obtaining a population of stem cells us eful for regeneration and repair of human tissue. The chromatin of ES cells exists in an open state, allowing transcription of genes for maintenance and eventual differentiation. Epigenetic modifications of chromatin structure or DNA methylation may lead to more restricted lineage specific gene transcription (Arney and Fisher, 2004) High levels of telomerase activity are associated with ES cells and their ability to be cultured indefinitely (Zeng and Rao, 2007) While murine and human ES cells exhibit similar morphologies, there are several distinct characteristics of each species. Embryonic stem cells derived from the mouse can be cultured in feeder free culture sy stems in the presence of leukemia inhibitory factor (LIF). Conversely, human ES cells do not require LIF and can be cultured off of feeder layers if supplemented with fibroblast growth factor 2 (Xu et al. 2005) Cells from both species grow in tightly packed colonies and appear to be inherently heterogeneous, as a portion of the outer layer of cells will differentiate. Whether this is truly a heterogeneous population or an artifact of inefficient culture is unknown at this point in time. The deletion of both copies of lif in mES did not completely abolish the ability of these cells to self renew. Instead, these cells produced a soluble factor that allowed the propagation of a small number of colonies despite the presence of a differentiation permissive environment. This factor, termed ES cell renewal factor (ESRF), works independently of the STAT3 signaling pathway and can sustain the undifferentiated phenotype of ES cells in vitro (Dani et al. 1998) In culture, ES cells possess a very short cell cycle (1116 hr) mainly due to a reduction in G1 phase. Early G1 is omitted by the constitutive presence of cyclin E CDK2 activity (Savatier et al., 1994; White et al. 2005) LIF withdrawal from the culture med ia brings cyclin E expression under control of pocket proteins, requiring cyclin D complexes and the obtainment of the early

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27 phase of G1 (White et al. 2005) By eliminating the requirement for cyclin D expression (MAPK induced), ES cells uncouple cell cycle traverse from differentiation, allowi ng for self renewal. ES cells isolated from cynomolgus monkey require a feeder layer to maintain pluripotency and cannot rely on LIF supplementation as do murine ES cells. LIF treatment induces phosphorylation of tyrosine 705 but does not affect the phosphorylation status of serine 727 of STAT3 (Sumi et al. 2004) Serine phosphorylation of STAT3 is required for full transcriptional activity (Wen et al. 1995) Interestingly, downregulation of connexin 43, a protein responsible for formation of gap junctions, results in the rapid differentiation of mES cells (Todorova et al. 2008) This included loss of Oct4 expressio n, morphological changes, and up regulation of differentiation markers such as glial fibrillar associate protein (GFAP). Formation of embroid bodies was hindered by treatment with gap junction intercellular communication (GJIC) blockers. Recovery of GJIC allowed the restoration of the differentiation program. Research with ES cells is not limited to humans and mice. ES cells were successfully isolated from the ICM of bovine embryos (created by IVF or nuclear transfer) and cultured on mitomycin c treated MEFs (Wang et al. 2005b) They assumed the typical morphology of ES cells: multicellular colonies with a smooth surface and distinct colony boundary. Bovine ES cells react positively with SSEA4 and Oct4 but not SSEA1 antibodies. No Tra1 expression was found. In the absence of a feeder layer and LIF, bES cells spontaneously differentiated into EBs and gave rise to cells from three germ layers. Ectodermal cells expressed the neurofilament marker TUBB3, mesoderm al cells expressed smooth muscle ACTA2, and cuboidal and net like epithelial structures expressed creatine kinase.

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28 Likewise, equine embryos have also yielded ES like cells. Several groups have isolated ES like cells from frozen and freshly collected equi ne blastocysts (Saito et al. 2002; Li et al. 2006; Guest and Allen, 2007) Equine ES like cells exhibit morphology similar to mES cells with underrun cell borders, a small cytoplasmic to nuclear ratio, and prominent nucleoli (Li et al., 2006) These cells express Oct4, alkaline phosphatase, SSEA1, SSEA4, Tra160 and Tra1 81 (Saito et al. 2002; Li et al. 2006; Guest and Allen, 2007) No SSEA3 was apparent in ES like cells, although it was detected in equine blastocysts (Guest and Allen, 2007) Equine ES like cells were capable of differentiation into all three germ layers in vitro but did not form teratomas when injected subcutaneously into SCID/beige immunoincompetent mice (Li et al. 2006) Despite the similarities in marker expression, reports of the culture conditions required differ among reports. In the absence of LIF, eES like cells lost expression of all markers of pluripotency and exhibited morphological properties identical to differentiated cells (Li et al. 2006; Guest and Allen, 2007) However, other report s indicate that a feed er layer, but not LIF, is necessary for the maintenance of eES like cells (Saito et al. 2002) The difference between the reports is unclear but may result from differences in isolation protocols or source of the equine embryos. Embryos from rats and dogs have also yielded ES like cells. Rat ES like cells cultured on a feeder layer in the presence of LIF express Oct4, SSEA1, and alkaline ph osphatase (Vassilieva et al. 2000) Canine ES like cells were isolated from inner cell masses collected from blastocysts (Hatoya et al. 2006) Unlike mouse and human ES cells, this population i s limited in life span, entering replicative senescence after nine passages. Domed colonies with tightly packed cells and an apparent border were present during early passages when cultured on a feeder layer. Cells expressed alkaline phosphatase, SSEA1, and Oct4, but no SSEA4.

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29 Aggregation into EBs produced cells with morphologies that represented cells from all three germ layers. In an effort to avoid the ethical controversies surrounding embryonic stem cells and to make pluripotent stem cells that matche d the unique identity of each individual, Takahashi and Yamanaka sought to find a set of factors that would induce a pluripotent state in a somatic cell (Takahashi and Yamanaka, 2006) They identified four factors which, when transduced into mouse embryonic fibroblasts, elicited a nave state with ES cell morphology and the ability to produce teratoma s in nude mice that contained cell types from all three germ layers. Oct4, Sox2, Klf4 and c Myc induced the expression of nanog and other pluripotency factors. The induced pluripotent stem cells (iPS) showed increased acetylation of histone H3 of the Po u5f1 and nanog promoters as well as decreased methylation of lysine 9 of histone H3, indicating a permissive state for Pou5f1 and nanog transcription. However, the CpG dinucleotides remained partially methylated. Additionally, iPS cells exhibited higher telomerase activity at levels similar to ES cells. Human embryonic fibroblasts respond similarly to induction with Oct4, Sox2, Klf4 and c Myc, expressing ES cell markers at both the protein and mRNA level (Park et al. 2008) However, recovery of iPS from postnatal human cells was successful only when hTERT and SV40 large T were included in the retroviral cocktail. The authors speculate that these factors are required to act on supportive cells in the culture to enhance the efficiency with which the reprogra mmed colonies can be selected (Park et al. 2008) Further work has indicated that only two factors, Oct4 and Sox2 may be necessary for induction of iPS cells from human fibroblasts when coupled with valproic acid (Huangfu et al. 2008) These cells are morphologically similar to hES cells and express nanog, Oct4, Sox2, SSEA4, Tra160 and Tra181. iPS cells have been postulated to be functionally equivalent to mouse ES cells in that they express the same markers,

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30 possess similar gene expression profiles, form teratomas, and contribute to cells of chimeric animals, including the germ line (Takahashi and Yamanaka, 2006; Maherali et al. 2007; Okita et al., 2007; Wernig et al. 2007) Differentiation Differentiation of ES cells can be obtained by removing LIF from the culture media and allowing the formation of embroid bodies (EBs). These masses of cells will differentiate and, upon dispersal onto a culture pl ate, produce cells of the three germ layers. Differentiating EBs mimic the genetic changes of early development the downregulation of pluripotency and self renewal genes coupled with the upregulation of genes responsible for the development of the three germ layers making this system useful for studying the mechanisms of early development (Gerecht Nir et al. 2005) Directed differentiation of ES cells is much more difficult than the spontaneous differentiation found in EBs. Zheng et al were successful in creating a pre myoblast line from hES cells but the differentiated cells did not fully differentiate in vi tro (Zheng et al. 2006) However, placing these preconditioned cells into an injured muscle resulted in moderate incorporation into host fibers (approximately 7%). Irradiati on following cardiotoxin treatment resulted in a higher degree of incorporation (approximately 29%). Transducing CGR8 ES cells with Ptf1a and Mist1 prior to differentiation resulted in increased pancreatic differentiation (Rovira et al. 2008) Acinar genes including CPA and Ela1 were upregulated in a manner similar to early exocrine cells. Pancreatic signaling mediators and gap junction protein mRNAs were expressed in transduced cells. Additionally, differentiated cells contained zymogen granule like vesicles filled with Amyl which was released in response to carbachol, indicating functional properties similar to exocrine cells. Phillips et al cultured hES cells in a three dimensional environment followed by stimulation by a series of growth factors to

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31 induce pancreatic differentiation (Phillips et al. 2007) Embroid bodies were cultured with activin A and BMP4 to induce endodermal cell types. Pancreatic progenitor cells were formed following supplementation with HGF, exendin4, and cellulin. These cells expressed Pdx 1 as well as the early pancreatic epithelial marker Ptf1a and endocrine progenitor marker Ngn3. Following further differentiation, cells expressed insulin and released cpeptide when stimulated with forskolin and glucose. When transplanted into the intraperitoneal cavity of SCID mice, differentiated cells maintain the endocrine identity and show modest glucose responsiveness. Multiple ES cells lines were tested for their ability to differentiate into mature cartilage (Kramer et al. 2005) Embroid bodies contained regions deeply stained with Alcian blue representing cartilage nodules. Nodules consisted of dense groups of rounded cells surrounded by a rigid membranous structure of extracellular matrix proteins containing type II collage n. In particular, the BLC6 line of ES cells had markedly high numbers of cartilage nodules (60 fold that of other lines tested) and a distinct increase in the expression of the adult splice variant B of type II collagen, a marker of mature chondrocytes. Osteogenic differentiation was achieved in hES cells when cultured as embroid bodies in glycerophosphate (Sottile et al., 2003) Culture in osteogenic induction media generated mineralized cultures that stained positive for calci um deposits with Alizarin Red and von Kossa. Expression of osteogenic genes such as osteocalcin, type I collagen, Cbfa1, and osteopontin were increased while Pou5f1 expression decreased during differentiation. The x ray diffraction pattern of mineralized nodules was characteristic of hydroxyapatite, the primary component of mineralized bone. Osteogenesis glycerophosphate by NA) (Yamashita et al. 2006)

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32 es the proportion of ES cells that differentiate into osteogenic cells. Osteogenic transcripts Cbfa1, type I collagen, and osteocalcin were expressed later in siR NA treated cells than those treated with osteogenic induction medium. positively with Alizarin Red and produced an equivalent amount of calcium as cells induced wit h osteogenic medium. Neural inductive signals provided by coculture of mES cells on the PA6 stromal cell line generated Sox1/TuJ1 expressing neurons (Wichterle et al. 2002) Reti noic acid was also capable of generating post mitotic neurons expressing Sox1, NeuN and TuJ1. Combination of retinoic acid and an agonist of the Sonic hedge hog signaling pathway generated spinal motor neurons. Similar to the gradient produced during embr yogenesis, a small variation in the level of sonic hedge hog signaling led to the generation of ventral interneurons rather than spinal motor neurons. Pre differentiated cells introduced into the embryonic chick spinal cord engrafted and differentiated in to motor neurons in vivo Post mitotic neurons also survived engraftment. ES cells are capable of undergoing hematopoiesis when cultured in serum free media and activated sequentially with a number of growth factors (Pearson et al. 2008) Addition of bone morphogenic protein 4 (BMP4) promoted efficient formation of mesodermal cells. Subsequent stimulation with activin A and fibroblast growth factor 2 (FGF2) induced the formation of hemangioblast precursors. Finally, supplementing the media with VEGF was required for the progression to a committed hematopoietic precursor. Sequential addition of these factors to mES cells resulted in a significant increase of CD41 expressing hemat opoietic precursor cells.

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33 Umbilical Cord Blood Derived Stem Cells Identified as a population of cells from blood that are adherent in plastic cultureware, umbilical cord blood derived stem cells (UCB) are further purified by centrifugation through a densit y gradient such as Ficoll. These cells exhibit a fibroblast like appearance and proliferate, though they are contact inhibited. UCB have been successfully isolated from humans, sheep, pigs, dogs, and horses and appear to maintain the same basic characteri stics across species (Bieback et al. 2004; Lee et al. 2004b; Fuchs et al. 2005; Zhao et al. 2006; Koch et al. 2007; Kumar et al. 2007) While the explicit protein markers of UCB remain elusive, most reports agree that this population is CD34, CD45 and major histocompatibility complex (MHC) class II negative and only weakly positive for MHC class I (Lee et al. 2004a) The minor expression of MHC classes I and II indicate the lack of immunogenicity of this cell type. Additionally, a mixed lymphocyte reaction indicated that h uman UCB did not stimulate lymphocyte proliferation, consistent with low levels of immunogenicity (Zhao et al. 2006) The absence of CD34 and CD45 suggests a nonhematopoietic lineage. Additionally, cell surface markers commonly found on MSC, CD29, CD44, CD73, and CD90 (Thy 1) are found in the majority of UCB (Bieback et al. 2004; Lee et al., 2004b; Fuchs et al. 2005; Zhao et al. 2006; Kumar et al. 2007) The presence of Oct4, nanog, SSEA3 and SSEA4 by some researchers suggests a nave phenotype for UCB (Baal et al. 2004) Collection and Processing Aspiration of blood via syringe or through a canula into a sterile container containing anti coagulant (EDTA, citrate phosphate dextrose, or other citrate based anticoagulant) at the time of parturition or caesarean section yields the highest number of UCB (Sparrow et al. 2002; McGuckin et al. 2003; Bieback et al. 2004; Chang et al. 2006; Kern et al. 2006) Proc essing

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34 is performed as soon as possible after collection, generally within 15 hours (Bieback et al. 2004) Prolonged time from collection to process ing may decrease the number of cells obtained. Blood is diluted in a buffered saline solution prior to separation of the mononuclear layer by density gradient centrifugation (Sparrow et al. 2002; McGuckin et al. 2003; Romanov et al. 2003; Bieback et al. 2004; Gang et al. 2004a; Lee et al. 2004a; Chang et al. 2006; Kern et al. 2006) The buffy coat, containing the mononuclear cells, is collected and subjected to at least two further washes in saline solution or medium. Lysing of the red blood cells may be performed at this time. Cells are then plated in expansion medium which is typically composed of Dulbeccos modified eagle medium (DMEM), 1020% fetal bovine serum (FBS), and penicillin/streptomycin. Some researchers suggest the addition of various growth factors and supplements (Sparrow et al. 2002; Romanov et al. 2003; Bieback et al. 2004; Gang et al. 2004a; Lee et al. 2004a; Kang et al. 2005; Wagner et al. 2005; Chang et al. 2006; Kern et al. 2006) Fibroblast like colonies can be seen two to four weeks after plating (Bieback et al ., 2004; Kern et al. 2006) Upon confluency, UCB can be detached with trypsinEDTA and subcultured. In contrast to stem cells derived from bone marrow, most UCB populations show an increased (but not indefinite) potential for proliferation (Bieback et al. 2004) Differentiation In an effort to prove the plasticity of umbilical cord blood derived stem cells in relation to other stem cell population s, in vitro and in vivo differentiation protocols have been performed. UCB have been successfully differentiated into cells from all three germ layers. Mesodermal cell types, such as osteoblasts, chondrocytes, and adipocytes, are most commonly used to id entify a stem cells ability to differentiate. However, differentiation into cells of the ectodermal or endodermal layer tends to be more difficult.

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35 Maturation of UCB into bone and cartilage is routinely achieved. Both human and equine UCB have been dif ferentiated into cells capable of producing calcium deposits stained by von Kossa and Alizarin Red (Kogler et al. 2004; Koch et al. 2007) Transcription of osteopontin, osteocalcin, and type I collagen has been reported in these cells (Kogler et al. 2004) Culture of human UCB in media containing dexamethasone and bone m orphogenic protein 2 (BMP2) resulted in morphological changes from spindle shaped cells to cuboidal cells in twenty days coupled with increased alkaline phosphatase and type I collagen expression (Hildebrandt et al. 2009) However, it has been noted by some researchers that UCB which differentiate into osteoblasts do not take on the cuboidal appearance typical of bone marrow derived osteoblasts (Goodwin et al. 2001) Chondrogenesis is commonly achieved by culture in a threedimensional micromass positively with Alcian Blue and Safranin O, indicating the presence of glyco s aminoglycans typical of cartilage (Kogler et al. 2004; Koch et al. 2007) Further analysis of these c ells reveals transcription of cart 1, Col2a1 and chondroadherin (Kogler et al. 2004) Ovine UCB from blood collected at 80120 days of gestation formed tissue reminiscent of hyaline cartilage when placed on a construct of biodegradable polyglycolic acid polymer treat ed with poly L lactic acid solution and coated with collagen. This was placed in a bioreactor in permissive medium for 12 weeks. Marked chondrogenic differentiation was apparent, presenting characteristics of hyaline cartilage and staining positively for S afranin O and Toluidine Blue. Type II collagen was primarily expressed with little type I collagen present and no type X collagen (Fuchs et al. 2005) Inclusion of insulinlike growth factor 1 (IGF 1) after the initial

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36 (Wang and Detamore, 2009) UCB treated with insulin and 3isobutyl 1 methyl xanthine, a phosphodiesterase inhibitor, will obtain lipid vacuoles identified by Oil Red O (Goodwin et al., 2001; Kogler et al. 2004; Koch et al. 2007) It has been noted that UCB present much less obvious adipogenic differentiation than do bone marrow or adipose derived MSC (Wagner et al. 2005; Rebelatto et al., 2008) Other researchers have struggled to form adipocytes from UCB. Biebeck et al could not obtain adipocytes after culture of UCB in induction medium containing dexamethasone, IBMX, insulin, indomethacin and fetal calf serum (Bieback et al. 2004) No lipid vacuoles were formed in UCB despite the appearance of lipid vacuoles in BM MSC treated in a parallel culture. However, continuous culture in induction medium for 5 weeks did result in some adipocy tic differentiation. Kern et al also reported a failure to induce differentiation into adipocytes, even following 5 weeks of culture (Kern et al. 2006) Lee et al achieved adipocytic differentiation but only after the addition of rabbit serum to the induction medium (Lee et al. 2004b) Less frequently, UCB have been shown capable of limited myogenic conversion in vitro Human UCB cultured in myogenic medium resulted in an increase in MyoD and myogenin transcription. These cells also expressed myosin, but no fusion of cells was reported (Gang et al. 2004b) When cocultured on murine fetal cardiomyocytes, human UCB began synchronized beating in culture after seven days (Nishiyama et al. 2007) Immunocytochemistry indicated actinin, and connexin 43 on these ce lls. Transcripts for GATA4, cardiac actin, cardiac troponin T, cardiac troponin I, and Nkx 2.5 were also amplified. Cells successfully engrafted into the atria, ventricles and septum when transplanted into fetal sheep hearts (Kogler et al. 2004) No cell fusion was apparent between host and donor

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37 cells. Myosin heavy chain, dystrophin, and ryanodine receptor were all identified in engrafted UCB. Treatment of human UCB with VEGF stimulated the expression of endothelial markers Flt1, Flk1, von Willebrand Factor, and t he transmembrane glycoprotein CD146 as well as a change in morphology to broad endothelial like cells with spontaneous formation of chain like structures (Zhao et al. 2006) Elongated or branched morphologies forming neuronal like networks were formed following treatment of human UCB with neural growth factor. These cells expressed the neuronal markers microtubule associated protein 1B, synaptophysin, neuronal transmitter gamma aminobutyric acid (GABA), and glutamic acid decarboxylase (GAD) (Zhao et al. 2006) Formation of neural pre cursors in vitro was achieved in approximately 90% of human UCB as evidenced by the presence of neurofilament, synaptophysin and GABA protein after 4 weeks of culture. When these cells were labeled and transplanted into the hippocampus region of adult ra t brain, three months after grafting cells were detected throughout the brain with a neuronal like highly differentiated morphology (Kogler et al. 2004) Additionally, Jeong et al report that human UCB exhibit morphological changes including a narrowing and thickenin g of the area around the nucleus while remainder of the cytoplasm elongated to give rise to multiple cellular processes (Jeong et al. 2004) These cells further progressed to yield network like structures and express glial fibrillar acidic protein (GFAP), Tuj1, TrkA and CNPase, markers of mature neuroglia. Neural differentiation can also be obtai ned following cryopreservation of UCB. Under permissive conditions, previously cryopreserved human UCB differentiated into neuronal cell types and expressed neurofilament, GAD, acety l cholinesterase, and GFAP (Lee et al. 2007) Neural stem cells derived from hUCB cultured on biodegradable human keratinassociated

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38 protein scaffolds differentiated into more mature phenotypes connected by gap and tight junctions (Jurga et al. 2009) Cells migrated away from aggregates and formed neural networks that were capable of generating spontaneous electrical activity, indicating the presence of a functional action potential. Hepatogenic differentiation has also been shown in a number of studies. Treatment of UCB with fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) stimulated the expression of hepatogenic markers alphafetoprotein and cytokeratin 18 (Kang et al. 2005; Tang et al. 2006) These cells were capable of producing urea, storing glycogen, and LDL endocytosis, functional measures of liver cells (Hong et al. 2005; Kang et al. 2005; Tang et al. 2006) Additionally to determine if these cells were capable of producing insulin in an in vivo environment, xenograft transplantation was performed by Zhao et al. Human UCB were transplanted into Balb/c nude mice with induced diabetes. Mice were then evaluated for their ability to correct hyperglycemia. Mice receiving the UCB transplantation displayed significantly lower blood glucose levels than the untransplanted controls (Zhao et al. 2006) Adult Stem Cells Bone Marrow Derived Mesenchymal Stem Cells Isolation of bone marrow derived mesenchymal stem cells (or mesenchymal stromal cells; BMSC) has been occurring in research and medical practice for a number of years. In vivo these cells support the formation of hematopoietic cells. BMSC derived from humans, mice and horses attach to culture plates and exhibit a fibroblast like morphology when cultured in vitro They represent a very small portion of the total cell population of the bone marrow; reported numbers vary from 0.001 0.01% (Pittenger et al. 1999) BMSC are positive for cell surface markers SH2, SH3, CD29, CD44, CD71, CD106, CD120a, and CD124 but negative f or CD14, CD34 and CD45 (Pittenger et al. 1999) Common differentiation protocols can be used to elicit

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39 osteogenic, adipogenic and chondrogenic differentiation of BMSC. Adipogenic differentiation resulted in the (Pittenger et al. 1999; Meirelles Lda and Nardi, 2003; Tropel et al. 2004; Vidal et al. 2006) expression of type II collagen, aggrecan, and the formation of a proteoglycan rich extracellular matrix (Pittenger et al. 1999; Tropel et al. 2004) Furthermore, culture in osteogenic induction me dium resulted in rapid mineralization and nodule formation as well as runx2, type I collagen, osteopontin, and osteonectin expression (Pittenger et al. 1999; Meirelles Lda and Nardi, 2003; Tropel et al. 2004; Vidal et al. 2006) Mouse, rat, and human BMSC differentiated into hepatocyte like cells in presence of HGF and/or FGF4, expressing markers HNF3B, Gata4, HNF1a, albumin and CK18 by day 21. These cells produced urea and albumin at levels similar to monolayer cultures of primary rat hepatocytes and were also capable of endocytosing LDL and storing glycogen (Schwartz et al. 2002) Culture of hBMSC in enhanced DMEM further (Gong et al. 2008) 4, Nkx2.5, troponinT, troponinI, and connexin43 in murine BMSC (Li et al., 2008) Cells exhibited morphological but not functional differentiation no spontaneous beating was observed. Peripheral B lood D erived P rogenitor C ells In addition to cells derived from bone marrow, other sources of blood related stem cells have be en explored. Peripheral blood derived progenitor cells (PBPC) have been isolated from human, swine, and equine with limited success. Reports range from 3575% of collections that give rise to an adherent cell population (Koerner et al. 2006; Giovannini et al. 2008) These mononuclear cells exhibit fibroblast like morphology and proliferate rapidly (Chan et al. 2006; Koerner et al. 2006; Porat et al. 2006; Giovannini et al. 2008) PBPC ca n give rise to a

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40 number of differentiated or pre cursor cell types. In permissive medium, human PBPC underwent differentiati on into neural precursor cells, evidenced by changes in morphology and (Porat et al. 2006) These cells also underwent limited myogenic differentiation in vitro PBPC cultured with galectin 1 expressed desmin and subsequently formed multinucleated fibers over the following w eek of culture (Chan et al. 2006) In vivo transplantation into an injury model (c /y /RAG2 mouse) resulted in more muscle fibers expressing human spectrin in galectin 1 stimulated PBPCs than control PBPCs (
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41 differentiation is visualized by von Kossa staining of calcium deposits and osteopontin and Runx2 expression (Kim et al. 2004; Kestendjieva et al. 2008) Adipose Derived Stem Cells A population of stem cells can be derived from adipose tissue by obtaining fat from subcutaneous surgery. The tissue undergoes enzymatic digestion followed by filtration and centrifugation. Adherence to plastic and subsequent expansion produces a relatively homogenous population. Morphologically similar to bone marrow and umbilical cord blood derived stem cells, adipose derived stem cells are spindle shaped. Adipose derived mesenchymal stem cells (AdMSC) express similar cell surface markers to bone marrow derived MSC, including CD13, CD44, CD73, CD90, CD105, CD106, CD166, CD29, CD49e, and HLA ABC (Gronthos et al. 2001; Katz et al. 2005; Wagner et al. 2005; Yanez et al. 2006) While HLA ABC surface markers are expressed on these cells, they lack expression of HLA DR. Additionally, AdMSC did not stimulate lymphocyte proliferation (Yanez et al. 2006) In fact, AdMSC inhibited the proliferation of T cells stimulated with peripheral blood mononuclear cells. When co cultured in a transwell, soluble factors secreted by AdMSC exerted immunosuppressive effects on responder T cells but only when AdMSC could interact with responder lymphocytes. Furthermo re, AdMSC infusion decreased the severity of graft v. host disease in mice when used in the first two weeks. Adipose derived stem cells have been induced to differentiate into osteogenic, chondrogenic, adipogenic, cardiomyogenic and neurogenic cell types (Gronthos et al. 2001; Katz et al. 2005; Wagner et al. 2005; Guilak et al. 2006; OedayrajsinghVarma et al. 2006; Liu et al. 2007; Yoshimura et al ., 2007; Zhu et al. 2008; Mehlhorn et al. 2009) Cells induced to osteogenic differentiation express RunX2 and Col1a1 after 7 days of culture, with mineralization after 3 weeks (Oedayrajsingh Varma et al. 2006) Comparison of AdMSC and

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42 BMSC indicates that AdMSC are less efficient at osteogenic differentiation, despite considerable similarity in gene expression throughout the differentiation process (Liu et al. 2007) Adipogenic differentiation was observed following treatment with insulin and IBMX (Gronthos et al. 2001; Katz et al. 2005; Guilak et al. 2006; Liu et al. 2007) Cells formed lipid vacuoles expression of proteoglycans and type II collagen (Guilak et al. 2006; Liu et al. 2007; Yoshimura et al. 2007) Culture on a poly lactide co glycolide scaffold resulted in the expression of Col2a1 and a homogenous distribution of proteoglycans (Mehlhorn et al. 2009) However, the AdMSC are again less efficient at chondrogenesis producing less Col2a1, chondroitin sulfate, and hyaluronan (Yoshimura et al. 2007) Neural differentiation has also been reported by several labs (Katz et al. 2005; Guilak et al. 2006) In instructive media, AdMSC gain elongated cytoplasmic processes and express NeuN, GFAP, and tubulin. Injection of murine AdMSC into ischemia induced angiogenesis resulted in a greater degree of perfusion in the implanted limbs (Kondo et al. 2009) Injection of mature adipocytes resulted in weaker recover y of perfusion than saline injected controls. Additionally, AdMSC recruited endothelial precursor cells and stimulated the secretion of SDF1 and VEGF in ischemic hind limbs. Human AdMSC cocultured with neonatal rat cardiomyocytes resulted in elongation of AdMSC and formation of myotube like structures (Zhu et al. 2008) After two weeks of coculture, AdMSC stained positively for myosin heavy chain, troponinI and connexin 43. At this time, the AdMSC and cardiomyocytes contracted synchronously. Rarely, binucleate AdMSC were found in cocultures. Therapeutic Uses of Stem Cells Stem cells have been widely accepted as a potential therapeutic aid in disease and injury states in both humans and animals. Diseases ranging from metabolic inefficiencies to

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43 musculoskeletal defects and neurological disorders could benefit from the use of a nave cell type which promotes repair or production of healthy tissue. The potential of stem cells lies in not only their ability to contribute healthy, differentiated cells to the unhealthy region, but in the production of beneficial growth promoting factors and recruitment of additional reparative cells. The precise effects of stem cells in each diseas e state remain to be determined as do the mechanisms by which they may help. Spinal Cord Injuries A number of stem cell populations have been used to treat spinal cord injuries in hope of improving neural regeneration and recovery of locomotion. Embryonic stem cells differentiated into neural precursors were transplanted into a spinal cord injury (SCI) model in the rat (McDonald et al. 1999) Transplanted cells were found at the injury site and at distances up to 8 mm from the injury. Engrafte d ES cells expressed markers of oligodendrocytes, neurons, and astrocytes with no evidence of tumor formation. Rats receiving UCB transplants demonstrated partial hind limb weight bearing and improved coordination compared to a complete lack thereof in sha m operated rats. Engraftment of fetal neural stem cells into immun o deficient mice after a traumatic spinal cord injury showed locomotory recovery (Cummings et al. 2005) Cells had migrated extensively from the injection site at 17 weeks post injury. Transplantation resulted in a higher degree of locomotor recovery with greater coordinated forelimb hind limb function after 16 weeks. Ablation of the stem cells after transplantation by diphtheria toxin resulted in similar coordination to animals with no stem cell transplantation, indicatin g that the improvement is due to the presence of these cells. Engrafted cells differentiated into neurons and oligodendrocytes. Transplantation of neurospheres into a contusion model of SCI by Ogawa et al resulted in engrafted cells differentiating into n eurons, oligodendrocytes, and astrocytes (Ogawa et al.

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44 2002) Cells underwent mitotic neurogenesis. Additionally, some donor axons were myelinated and formed pre synaptic structures. Infusion of UCB into a rat model of stroke ameliorated many of the physical and behavioral deficits associated with the injury (Chen et al. 2001) hUCB engrafted into the brain of rats that suffered a stroke with the majority of cells localized to the boundary of the ischemic region. Engrafted cells were reactive with neuronal and astrocyte markers. Treatment within 24 hours of injury significantly improved functional recovery measured by the motor rotarod test and neurological se verity scores (NSS), however later treatment resulted only in improved NSS. Transplantation of hUCB into either the femoral artery or the brain of induced stroke rats resulted in higher levels of spontaneous activity compared to nontransplanted controls (Willing et al. 2003) The recovery of motor asymmetry was shown to be dose dependent in animals treated with varying numbers of UCB (Vendrame et al. 2004) Transplantation also decreased the area of infarction, ischemic volume, and inflammatory cytokines TNF 6 compared to nontransplanted rats in a stroke model (Vendrame et al. 2004; Vendrame et al. 2005; Vendrame et al. 2006) When hUCB were injected into rats via the saphenous vein 24 hours after transient middle cerebral artery occlusion, an improvement in neurological severity scores was apparent in transplanted rats by day 14 (Xiao et al. 2005) Transplanted rats s howed improvement in the stepping test measuring asymmetric movement. Smaller lesion sizes were measured in transplanted animals, although very few cells engrafted into the brain. However, implants of hUCB into the cortex resulted in neuronal outgrowth fr om the contralateral side and improvement in NSS scores. Vertebrae In addition to examining the effect of stem cells on neurogenesis, stem cells have also been used to aid the repair of the vertebrae surrounding the spinal cord which is also likely to be

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45 marrow derived MSC and autografts from the iliac crest fused the L4 and L5 vertebrae similarly in the Cynomolgus monkey (Orii et al. 2005) However, bone formation assessed by micro computed tomography showed grea ter new bone formation at 12 weeks in BMSC engrafted vertebrae than those receiving autografts. Likewise, in an ovine model of vertebrae injury, higher fusion was apparent in implants of BMSC on a TCP scaffold than autografts (Gupta et al., 2007) Newly formed woven and lamellar bone trabeculae in a cancellous organization with evidence of remodeling was observed in BMSC/ TCP grafts. Formation of hematopoietic and fatty marrow tissue was also apparent. Using a canine cancellous bone matrix transplanted with BMSC, superior fusion volume and fusion area for mineralized and demineralized matrix was observed over graft alone (Muschler et al. 2003) In a clinical trial, bone grafts saturated with uncultured autologous BMSC were implanted into acute thoracolumbar fractures (Faundez et al. 2006) The resorbable matrix was replaced with new bone containing several active foci of membranous and/or endochondral ossification. Cardiovascular Repair In rabbits with surgical myocardial infarctions, hBMSC were injected into the border area of myocardial ischemia (Wang et al. 2005a) Lower mortality was reported in the cel l transplant group compared to controls. Surviving patients had higher ejection fractions. Engrafted cells differentiated into cardiac troponin I expressing cells. In a canine model of cardiovascular disease, labeled canine BMSC were injected into a chron ically infarcted myocardium due to a permanently ligated coronary (Bartunek et al. 2007) Labeled cells expressed cardiac specific myosin or troponin I by four and twelve weeks after injection. Newly differentiated cardiomyocyte like cells were observed within the fibrotic area of infarction. Transplanted BMSC expressed connexin43, consistent with the integration into the host tissue. No

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46 calcification or osteogenic formation was noted. Pre differentiated BMSC injected into heart resulted in increased shortening and regional wall thickening suggesting a higher degree of functional recovery. Eight weeks after transplantation of hBMSC into rats with acute myocardial infarcts, engrafted cells expressed connexin43 and cardiac troponin T (Chang et al. 2008b) Functional aspects of the heart were improved, with reduced left ventricular end diastolic diameter and end systolic dimension as well as improvement of fractional shortening. Comparing hBMSC and hUCB in a mouse model of cryoinjur y to the left ventricle, Ma et al determined that the presence of both cell types increased capillary density (Ma et al. 2006) How ever, hUCB had no effect on the shortening fraction while BMSC alleviated the decrease in contractility caused by cryoinjury. Additionally, while both BMSC and UCB could be identified in the myocardium, no neocardiomyocyte like cells were found. Stem cell s from umbilical cord blood have also been used as a therapeutic tool in the treatment of cardiovascular diseases. hUCB implantation into Sprague Dawley rats with acute myocardial infarction present engrafted cells that express cardiac troponin T, von Wil lebrand factor, and smooth muscle actin indicating that these cells contributed to cardiac, endothelial and smooth muscle cell types (Wu et al. 2007b) Left ventricular ejection fraction was increased within two weeks of transplantation. Rats transplanted with hU CB also demonstrated an increase in arteriole vessels and capillary density. hUCB implantation into a hind limb model of ischemia in nude mice resulted in marked improvement of perfusion as well as a time dependent increase in blood flow following the inj ection of hUCB (Wu et al. 2007a) Genetically modified hUCB were used by Chen et al to treat a murine model of acute myocardial infarction (Chen et al., 2005) hUCB transfected with vascular endothelial growth factor (VEGF) or Angiopoietin1 were imp lanted into the damaged myocardium. Transplanted cells incorporated into the

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47 myocardium and expressed VEGF or Ang1. Treatment with untransfected hUCB decreased the infarct size, however cells expressing VEGF reduced infarct size further. Transplantation of cells expressing VEGF or Ang1 also increased capillary density and improved fractional shortening and ventricular ejection fraction. Tendon and Ligament Injuries Current treatments for tendon injury are inefficient at best, including immobilization or s urgery to reattach partially severed tendons. Re injury is a common problem due to the replacement of tendon tissue with scar tissue. These changes in the tendons capacity to store energy and recoil affect the ability of the tendon to adapt and equally di sperse the load, potentially creating microdamage leading to re injury. In some animals, bone marrow derived stem cells (BMSC) have been used to treat tendon lesions with some success. Several reports suggest improved results using BMSC injections compare d to traditional treatment programs (Smith et al. 2003; Crovace et al. 2007; Pacini et al. 2007; Guest et al. 2008) No negative immune response was reported using autologous or allogeneic BMSC injections into core superficial digital flexor tendon (SDFT) lesions (Smith et al. 2003; Guest et al. 2008) The labeled cells integrated into tendon, assuming tenocyte like morphology (Guest et al. 2008) However, a low efficiency (0.001%) of engraftment was reported. Autologous BMSC transplanted into collagenase induced SDFT lesions decreased the lesion size as a percent of total tendon cross sectional area (Crovace et al. 2007) Racehorses with typical core SDFT lesions receiving stem cells suffered no adverse reactions (Pacini et al. 2007) One month following injection, greater tendon density was apparent in horses injected with BMSC compared to uninjected control horses. Tendons appeared almost completely repaired after six months. All returned to racing with no further reinjury more than two years after diagnosis. Control horses showed fibrosis during the healing process and all were reinjured within twelve months after the

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48 initial diagnosis. Autologous rat BMSC successfully eng rafted into injured MCL (Watanabe et al., 2002) These cells became spindle shaped with elongated nuclei and aligned with parallel collagen bundles within 14 days of treatment. Biomechanical properties including stiffness, maximum force, maximum stress, and modulus were all improved in tendons receiving a gel sponge seeded with autologous BMSC (Juncosa Melvin et al. 2006) In a patellar tendon defect, BMSC seeded collagen composite implants str engthened tendons over natural repair (Awad et al., 2003) However, 28% of these grafts showed formation of ectopic bone. Bone free tendons exhibited improved biomechanics, with increased maximum force, stiffness and strain energy after 26 weeks of recovery. Values reported for cell seeded grafts were interm ediate to naturally repaired tissue and normal, healthy tissue indicating an improvement but not return to completely native state. Twenty days after implantation of hBMSC into a patellar tendon defect in rats treated tendons exhibited spindle shaped cells among collagen fibers aligned in parallel (Hankemeier et al. 2007) The improvement in biomechanical properties was also reported by Young et al, who implanted autologous BMSC into a gap defect model in the gastrocnemius tendon of the rabbit (Young et al. 1998) Treated repairs were stiffer, withstood more force and energy than control repairs but were still weaker than normal tissue. The area of the treated repair declined at a significantly fast er rate than control repairs. An alternate source of autologous stem cells derived from adipose tissue (adipose derived stem cells; ADSC) has recently become commercially available for use in the horse (VetStem, CA). This putative population of cells ha s been used to treat tendon, ligament and joint injuries. However, no data is available regarding the true identity of these cells or their capability for differentiation into various types of tissue. A recent report indicates that use of adipose derived mononuclear cell fractions for repair of collagenase induced lesions in the horse results in

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49 improvements in overall tendon fiber architecture after six weeks of recovery (Nixon et al. 2008) These cells may be more mature in nature than BMSC, as Kisiday et al report indicates they are less efficient at chondrogenesis than BMSC, secreting and accumulating less extracellular matrix (Kisiday et al. 2008) Bon e Injuries Union at the host implant interface was apparent by eight weeks in dogs implanted with allogeneic BMSC on a hydroxyapatitetricalcium phosphate scaffold (Arinzeh et al. 2003) A callus was also present around the periphery of the implant and host bone at this time. Newly formed bone and connective tissue were apparent; in some cases a marrow cavity was reestablished. Similar results were obtained with autologous BMSC, however cell free matrices formed little to no callus or new bone. No adverse host response to allogenic BMSC was observed. On hydroxyapatite ceramic (HAC), culture expanded BMSC osteo progenitor cells were implanted into a critical sized defect in ewes and allowed to recover for sixty days (Kon et al., 2000) Callus formation was observed between the bone and scaffold, regardless of the presence of cells. Transplants including cells had more substantial callus formation and earlier presence of bone than cell free trans plants. In another study using sheep, Viateau et al transplanted BMSC on coral constructs into a bone defect (Viateau et al. 2007) In ewes implanted with scaffold only grafts, no union formed but some bone deposition was present. Grafts with BMSC exhibited significant new bone formation and complete resorption of the scaffold as early as one month after implant. Notably, the same amount of new bone formation was present in the coral/BMSC grafts as autogenic corticocancellous grafts. In bone m arrow derived osteogenic progenitor cells cultured on a matrix consisting of demineralized bone and cancellous chips and subsequently transplanted into a critical sized defect in the canine femur, autografted and stem cell grafts resulted in 100% healing c ompared to 50% healing in graft only

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50 transplants and 67% in bone marrow grafts (Brodke et al. 2006) Goats with tibial bone defects that received autologous BMSC on a beta tricalcium phosphate scaf fold achieved full union by 32 weeks post surgery (Liu et al., 2008) At this point, the ceramic scaffold was almost completely resorbed and the engrafted bone had reached similar biomechanical properties as a normal tibia. Little callus was observed at 16 or 32 weeks in acellular grafts, which were not resor bed and did not heal during the 32 week trial. hBMSC implanted into a rat femur defect on (Bruder et al. 1998) By eight weeks, the BMSC seeded graft had significant bone formation within the pores of the scaffo ld and were integrating into the ends of the host bone. Union was complete by twelve weeks. New woven and lamellar bone were detected in close contact with the edges of the defect and were directly contiguous. Defects corrected with cell seeded grafts exhi bited increased strength and stiffness in biomechanical testing compared to grafts alone. Pores of the acellular grafts were primarily filled with fibrous tissue at twelve weeks. Following lunate arthroplasty, autologous rabbit BMSC were seeded on a scaff old of hyaluronan and gelatin prior to transplantation into the surgical defect (Huang et al. 2006) Removal of the entire lunate resulted in carpal collapse. However, rabbits receiving the implant presented evidence of new bone formation twelve weeks after surgery. Scaffold only implants showed no bone f ormation. Repaired tissue contained intensely stained metachromatic matrix and islands of ossicles within the lunate space. Endochondral ossification had occurred in central areas of the scaffold seeded with BMSC by twelve weeks with chondrocytes being replaced by woven bone. In a clinical trial, Marcacci et al report that patients injected with autologous BMSC into bone defects resulting from trauma exhibit callus formation between the host bone and hydroxyapatite cylinder implant after one to two months (Marcacci et al. 2007) Complete resorption occurred

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51 five to seven months after surgery; this integration was maintained in three patients six to seven years after surgery (the longest follow up available). Cartilage Injuries Swine BMSC label ed with green fluorescent protein were seeded on polyglycolic acid fibers (PGA) and polylactic acid (PLA) scaffolds prior to implantation into an osteochondral defect in the femur trochlea of both knees (Zhou et al. 2006) Transplantation of scaffolds with cells predifferentiated to cartilage resulted in relatively regular surfaces and newly formed cartilage like tissue a fter three months. Noninduced BMSC on scaffolds had inferior results, with irregular surfaces and visible tissue deficits with defects filled mostly with fibrotic tissue. Six months post surgery, nearly normal osteochondral tissue and relatively smooth a rticular surfaces were apparent in induced BMSC seeded grafts with strong metachromatic matrix production. Green fluorescent protein positive cells remained present in the graft site up to seven months post operation. Mrugala et al transplanted ovine BMSC with chitosan in fibrin glue in the patella (Mrugala et al. 2008) BMSC implanted in the absence of chitosan (fibrin glue only) formed poorly integrated tissue with no glyco s aminoglycan proteoglycan, or type II collagen expression. However, when chitosan was included, tissue resembling cartilage with lacunae and associated cells was observe d but integration remained poor. The combination of chitosan, presence of chondrocyte like cells surrounded by a matrix that stained positive for Safranin O, type II collagen, and aggrecan. However, some fibrous tissue remained and overlapped the neocartilage in some areas. Autologous uncultured rabbit BMSC transplanted into a full thickness cartilage defect on a fibrin gel carrier resulted in the regeneration of cartilage that stained intensely with Safranin O but remained inferior to normal cartilage (Chang et al. 2008a)

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52 Implants containing gel alone had much less area of regeneration coupled with the presence of fibrous tissue and less intense staining of extracellular matrix for Safranin O. BMSC injected directly into a full thickness defect of the articular cartilage of the left distal femur in 16 New Zealand white rabbits showed smooth, consistent white tissue resembling articular cartilage in the regenerating area (Im et al. 2001) In contrast, control animals exhibited red, irregular tissue with the margin of the defect sharply differentiated from the surrounding normal cartilage. Defects in the group receiving BMSC had relatively normal surfaces with adjacent normal cartilage showing little degen erative change. Control animals showed degenerative changes in adjacent cartilage, thin and undifferentiated defects, and decreased cellularity. Collagen matrices containing type I, type II and type III collagen seeded with ovine BMSC were used to fill s urgical defects in the medial condyle of the femur (Dorotka et al. 2005) Defects treated with cell seeded matrices appeared more filled than control de fects and had the largest quantity of hyaline cartilage. Autologous BMSC were implanted into full thickness cartilage lesions in the femoropatellar articulation in mature horses (Wilke et al. 2007) After thirty days, defects repaired with BMSC appeared more completely filled with more homogenous tissue. Arthroscopic scores were significantly higher in the BMSC seeded def ects. No consistent differences between control and cell seeded scaffolds could be determined eight months after surgery. The authors concluded that early repair was improved by the presence of BMSC but no long term benefits were apparent. Yan and Yu compared the effects of chondrocytes, BMSC, fibroblasts and hUCB seeded on a polylactic acid scaffold on a full thickness cartilage defect in rabbits (Yan and Yu, 2007) While no apparent visible difference in the defects were found after twelve weeks, the fibroblast and control scaffolds were filled with noncartilaginous tissue while hUCB and BMSC seeded scaffolds housed tissue indistinguishable from host hyaline cartilage.

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53 Areas of union between the host and graft were indiscernible in defects implanted with hUCB seeded PLA scaffolds. However, the junction between the repaired tissue and subchondral bone was irregular in grafts seeded with BMSC. Fibroblast seeded scaffolds exhibited irregular borders with some degeneration of surrounding cartilage. Poly lactide co glycolide scaffolds seeded with hum subcutaneously into scid mice (Mehlhorn et al. 2009) After eight weeks, histological examination demonstrated the presence of proteoglycans throughout the implant. Cells were round in shape and distributed homogenously throughout the scaff old. The presence of type II collagen was determined by immunohistochemistry, however some implants also expressed type control implants. In a clinical study, autolo gous BMSC were injected into patella femoral lesions (Wakitani et al. 2007) A cartilaginous matrix formed at the site of injection. Patients reported clinical improvement in lessening pain and range of motion wit h no adverse reactions. Muscular Dystrophies The use of stem cells in muscle disease generally involves muscular dystrophies. When human UCB were transplanted into the sjl mouse, a model of limb girdle muscular dystrophies, the cells engrafted into host muscle (Kong et al. 2004) A small number of fibers were capable of expressing dysferlin, the missing protein, twelve weeks after transplantation. Addit ionally, when injected into the adductor muscle of an ischemic mouse limb, human UCB engrafted into the injured muscle (Pesce et al. 2003) Some donor cells expressed desmin, a marker of myogenic differentiation. Transplantation increased the number of regenerating cont rols at an earlier time point, suggesting that the presence of UCB initiated an earlier healing process. Transplantation of human circulating AC133+ cells into scid/mdx mouse tibialis anterior resulted in cells engrafting into the regenerating muscle (Torrente et al. 2004) Some cells took up

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54 residence in the satellite cell position while others produced human dystrophin and myosin heavy chain. Additionally, muscle injected with ACC133+ cells possessed fewer regenerating fibers with less central nuclei than controls. Human fetal BMSC were transplanted intraperitone ally into mdx murine fetuses (Chan et al. 2007) Engraftment of human cells wa s apparent in a number of tissues including the brain, lung, liver and spleen. Skeletal, diaphragmatic and cardiac muscle expressed human specific myosin heavy chain transcripts. Human nuclei were present in regenerating muscle of transplanted animals an d some human dystrophin was expressed. Conclusion The scientific community has access to a widely varied population of stem cells with which to investigate the biology and potential application of the stem cell. Undoubtedly, certain cell types are more su ited for use in different situations. ES cells likely recapitulate early embryonic development; while adult bone marrow derived cells are more suited for the study of hematopoiesis in the adult animal. Investigations focus not only the stem cells themselves, but the environment in which they exist in the organism. The effects of the stem cell niche on not only the stem cell but the surrounding cells and tissues provide a huge area of potential research for therapeutic application.

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55 CHAPTER 2 CENTRAL HYP OTHESIS Stem cells exist in a wide array of plasticity and self renewing abilities. From embryonic stem cells which can divide indefinitely and recapitulate the entire organism to adult stem cells which are limited to a few cell divisions and differentia tion into a small number of cell types, each stem cell has its niche both in the life of the animal and in the world of therapeutic medicine. Umbilical cord blood is a widely available, often discarded source of stem cells. This population of stem cells has the potential to be an intermediate population between the nave embryonic stem cell and the less plastic adult stem cell. Owing to its immature source, umbilical cord blood derived stem cells may be a more malleable population of stem cells with less of the inherent risk of tumor formation found in embryonic stem cells. Using the horse as a model animal, we sought to identify stem cells from umbilical cord blood that may be useful in treating tendon injuries. Horses provide a large animal model tha t is easily accessible, easy to work with, and provides large volumes of umbilical cord blood at birth. Most foalings are attended by farm hands who can collect the blood with minimal training. Moreover, large animal models may be more appropriate to evaluate possible human therapies owing to greater similarities in anatomy and physiology than available in rodent models. Our hypothesis is that equine umbilical cord blood contains a population of stem cells that can be isolated, cultured, and differentiat ed into a variety of cell types. Specifically, the ability of these cells to differentiate into an early tendon precursor cell was examined. The following objectives were designed to test the hypothesis:

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56 Aim 1: Stem cells can be isolated from equine umb ilical cord blood and cultured using methods currently used in human medicine and research. Equine UCB stem cells express markers and morphology similar to other mese n chymal stem cells. Aim 2: Equine UCB have the potential to contribute to tendon injury and repair. Stem cells were examined for expression of early tenocytes. Tenocytic differentiation was promoted by culture on a variety of protein matrices and in growth factor containing media.

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57 CHAPTER 3 EQUINE UMBILICAL COR D BLOOD CONTAINS A P OPULA TION OF STEM CELLS THAT EXPRESS OCT4 AND DIFFERENTIATE INTO MESODERMAL AND ENDODERMAL CELL TYPE S Introduction Bone marrow (BM) derived mesenchymal stem cells (MSCs) are the conventional model of choice for adult stem cell based therapeutics in humans due t o their mu l tilineage differentiation capabilities. Their relative ease of expansion in vitro without loss of plasticity makes MSCs an attractive repair aid for damaged or diseased heart, bone and vascular tissues [for review see (Giordano et al. 2007) ]. However, enthusiasm for the use of MSCs as cytotherapeutics is tempered by their age dependent decline in absolute numbers and the invasive nature of their harvest (Stend erup et al. 2003) To counter these problems, umbilical cord blood (UCB) MSCs may represent a viable alternative. Several reports define a clonogenic population of cells from the umbilicus that differentiate into both mesenchymal and non mesenchymal tiss ue derivatives (McGuckin et al. 2003; Aoki et al. 2004; Baal et al. 2004; Bonanno et al. 2004; Peled et al. 2004; Ruzicka et al. 2004; He et al. 2005; Holm et al. 2006; Martin Rendon et al. 2007; Guest et al ., 2008) The identity of these cells as circulating stemlike progenitors versus endothelial progenitors detached from the umbilicus remains debatable (Kogler et al. 2004) A hierarchy in stem cell plasticity exists such that embryonic stem (ES) cells are pluripotent and adult MSCs are more limited in their differentiation capacity (Feinberg, 2007) UCB stem cells likely fall in the area between the two. The three classes of stem cells demonstrate variable stage specific embryonic antigen (SSEA) and tumor re jection antigen (Tra) surface marker protein expression patterns as well as differences in transcriptional circuitry. SSEA 3 and SSEA 4 are prevalent on the surface of human ES cells; these undifferentiated cells

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58 do not express SSEA 1 (Thomson et al. 1998; Reubinoff et al. 2000; Henderson et al. 2002) By contrast, mouse blastocyst inner cell mass cells and ES cells express SSEA 1 but not SSEA 3 or SSEA 4 (Henderson et al. 2002; Tielens et al. 2006) The keratan sulfate proteoglycan markers, Tra1 60 and Tra181, are localized within the extracellular matrix of human ES cells (Henderson et al. 2002; Stojkovic et al. 2004) Key to the establishment and maintenance of the undifferen tiated state of ES cells are the coordinated activities of Oct4, nanog, and Sox2 (Boyer et al. 2005) This combination of surface markers and plasticity genes represent the minimal defining components of a nave ES cell. By comparison, human BM derived MSCs are more limited in their expression of the central ES indicators likely owing to the heterogeneity of the population. SSEA4 is present on the surface of BM MSC; the cells lack Oct4 but can be induced to form multiple lineages (Gang et al. 2007) Culture of BM MSC i n FGF2 supplemented media results in Oct4 and nanog transcription suggesting that a premature phenotype reminiscent of ES cells can be established (Battula et al. 2007) UCB stem cells are unique in that they possess an intermediate phenotype that more clos ely resembles ES cells. SSEA 3, SSEA 4, Tra1 60, Tra181, Oct4, and Nanog are present in this population (McGuckin et al. 2003; Zhao et al. 2006; Markov et al. 2007; Sun et al. 2007) BM MSC isolated from adult horses differentiate along the chondrogenic and osteogenic lineages comparable to their human counterparts (Fortier et al. 1998; Worster et al. 2001; Koerner et al. 2006) However, a reduced level of success exists for the formation of adipocytes from BM aspirates (Koe rner et al. 2006; Vidal et al. 2006) Because human UCB stem cells exhibit a heightened degree of plasticity, we chose to identify a comparable cell entity in newborn foal cord blood as an alternative to BM MSC. Using conventional human purification met hods, culture conditions and differentiation protocols, an equine UCB cell population was

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59 discovered that possesses stem cell like markers and multilineage differentiation capabilities. The isolation and characterization of these cells represent a first st ep toward their application in cytotherapeutic repair of sport horse injuries. Materials and Methods U mbilical C ord B lood (UCB) C ollection and S tem C ell I solation Cord blood (n = 25) was collected from the intact umbilicus at foaling into a sterile 50 ml c entrifuge tube containing ethylenediamine tetraacetic acid (EDTA) as an anti coagulant. Blood was stored at 4C and further processed within 12 h of collection. Samples were incubated for 20 min with RosetteSep Human Cord Blood Progenitor Enrichment Cockta il (50 l/ml blood; Stem Cell Technologies, Seattle, WA), a commercially available product for negative selection of human UCB stem cells. An equal volume of phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) w as added. The cell suspension was layered atop a Ficoll Paque Plus (Stem Cell Technologies) cushion and centrifuged at 1,200g for 20 min. The cell interface was collected and cultured. Equine UCB and Adipose D erived (AD) Stem C ell C ulture MSCs isolated fro m equine adipose tissue were purchased from Sciencell Research Laboratories (San Diego, CA). Cells were cultured in Mesenchymal Stem Cell Medium (Sciencell Research Laboratories) on standard tissue plasticware, according to manufacturer's recommendations. UCB stem cells were cultured in Dulbecco's modified Eagle media (DMEM, Invitrogen) supplemented with 10% FBS and 5 g/ml Plasmocin (Invitrogen). Culture medium was exchanged every three days. Cells were passaged at 70% confluency using 0.025% trypsinEDTA (Invitrogen).

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60 RNA Isolation, R everse T ranscription (RT), and P olymerase C hain R eaction (PCR) Total RNA was isolated by lysis in STAT60 (Iso Tex Diagnostics, Friendswood, TX) and ethanol precipitation. The RNA was digested with DNase (Ambion, Austin, TX) to remove genomic DNA contaminants. One microgram of total RNA was reverse transcribed (Superscript III, Invitrogen) in 20 l reaction volume. Two microliters of first strand cDNA was amplified with gene specific primers and AccuPrime DNA polymerase (Invitrogen). Primer sequences included glyceraldehyde 3 phosphate dehydrogenase (F GATTCCACCCATGGCAAGTTCCATGGCAC, R GCATCGAAGGTGGAAGAGTGGGTGTCACT), collagen 2a1 (F CAGCTATGGAGATGACAACCTGGC, R CGTGCAGCCATCCTTCAGGACAG), Sox9 (F GCTCCCAGCCCCACCATGTCCG, R CGCCTGCGCCCACACCATGAAG), osteonectin (F CCCATCAATGGGGTGCTGGTCC, R GTGAAAAAGATGCACGAGAATGAG), Runx2 (F CGTGCTGCCATTCGAGGTGGTGG, R CCTCAGAACTGGGCCCTTTTTCAG), albumin (F AACTCTTCGTGCAACCTACGGTGA, R AATTTCTGGCTCAGGCGAGCTACT) and cytokeratin18 (F GGATGCCCCCAAATCTCAGGACC, R GGGCCAGCTCAGACTCCAGGTGC). PCR products were visualized following electrophoresis through 2% agarose gels containing ethidium bromide. Representative images were captured with a Kodak ImageDoc system and inverted in Adobe Photoshop CS. Osteogenic D ifferen tiation Cells were plated at a density 1,300 cells/cm2 and allowed to attach overnight in normal growth medium. The following day, cells were washed twice with PBS and placed in an osteogenic differentiation medium composed of alpha modified Eagle medium ( MEM), 10 mM glycerophosphate, 0.1 M dexamethasone, 0.1 mM ascorbic acid (Tondreau et al. 2005; Wagner et al. 2005) Media was changed twice weekly. Cells were fixed in 4%

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61 paraformaldehyde in PBS for 15 min on days 7, 14, and 21. Total RNA was isolated from parallel plates. Chondrogenic D ifferentiation UCB stem cells were pelleted to a micromass, promoting chondrogenic differentiation in a threedimensional environment. Cells (4 105) were pelleted at 1,000 g for 5 min. T he medium was removed and 0.5 ml chondrogenic medium was added (Worster et al. 2000; Tondreau et al. 2005) Chondrogenic medium consisted of DMEM, 1.0 g/L insulin, 0.55 g/L transferrin, 0.67 mg/L sodium selenite (ITS X, Invitrogen), 10 ng/ml transforming growth factor beta 1 (TGF 1, R&D Systems, Minneapoli s, MN), 35 g/ml ascorbic acid and 100 nM dexamethasone (Sigma, St. Louis, MO). Media was changed twice weekly. After 7, 14, and 21 days, the micromass was embedded and frozen in OCT freezing compound. Alternately, micromasses were washed with PBS and used for RNA isolation. Adipogenic D ifferentiation UCB stem cells were plated at a density of 3,000 cells/cm2 in growth medium. Adipogenic differentiation was induced with Iscove's modified Dulbecco's media (IMDM) supplemented with 10% FBS, 1 M dexamethasone, 10 g/ml recombinant human insulin, 0.25 mM 3isobutyl 1methylxanthine (IBMX) and 100 M indomethacin (Wagner et al. 2005) Medium was replaced every three days. Cells were fixed with 4% paraformaldehyde in PBS after 7, 14, and 21 days in culture. Hepatogenic D ifferentia tion UCB stem cells were plated at a density of 5,000 cells/cm2 and allowed to attach overnight in normal growth medium. The following day, cells were washed twice with PBS and placed in hepatogenic medium [1% FBS, 20 ng/ml recombinant human hepatocyte gro wth factor (HGF, R&D Systems), 10 ng/ml recombinant human fibroblast growth factor 4 (FGF4, R&D Systems)

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62 in IMDM], as described (Kang et al. 2005) Medium was replaced twice weekly. On days 7 and 14 cells were fixed in 4% paraformaldehyde in PBS for 15 min. Total RNA was isolated from parallel plates. Myogenic D ifferentiation UCB stem cells were plated at 3,000 cell/cm2 on gelatincoated tissue cultureware. Differentiation was initiated by incubation in low glucose DMEM supplemented with 200 g/ml galectin 1 (R&D Systems) essentially as described (Chan et al. 2006) After 14 days, cells were fixed with 4% paraformaldehyde or lysed for RNA isolation. Histology Alkaline phosphatase enzymatic activity was detected colorimetrically using Nitro Blue Tetrazolium Chloride (NBT) and 5Bromo 4Chloro3' Indolyphosphate p Toluidine (BCIP; Pierce, Rockford, IL) following fixation with 4% paraformaldehyde. Oil Red O (0.1% in 60% isopropanol) was used to visualize lipid droplets. Alcian Blue (1% in 3% acetic acid) staining was used to detect glycosaminoglycans. Safranin O (0.1% in water) was used for the visualization of proteoglycans and cartilage. Alizarin Red (2% in water, pH 4.2) was used for the detection of mineral deposits. Calcium deposits were detected by the method of von Kossa using 1% silver nitrate and 5% sodium thiosulfate. Immunocytochemistry Cells wer e fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.1% Triton X 100 in PBS. Nonspecific antigen sites were blocked with 5% horse serum. For the detection of stem cell markers, anti Oct4 (1:50, Santa Cruz, Santa Cruz, CA) anti SSEA 1, anti SSEA 3, anti SSEA 4 (1:50, R&D Systems), anti Tra1 60 (1:50, Abcam, Cambridge, MA) and anti Tra1 81 (1:50, Abcam) were used. Myogenic cells were incubated with anti desmin (1:200, DE U 10, Sigma Aldrich, St. Louis, MO) and Texas Red

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63 con jugated Phalloidin (Invitrogen). All antibodies were diluted in PBS containing 1% horse serum and incubated with fixed cells for 1 h at room temperature. Immune complexes were visualized with goat antimouse AlexaFluor488 (1:200), goat anti rabbit AlexaFlu or488 (1:200), or goat anti rabbit AlexaFluor568 (1:200) on a Nikon T200 microscope equipped with epifluorescence. Images were captured with NIS Elements (Nikon Instruments, Melville, NY) software and compiled with Adobe PhotoShop CS. Results Foal U mbilica l C ord Blood Contains an Oct4 E xpressing Cell P opulation Multipotent stem cells are routinely isolated from fresh cord blood at birth from humans by density gradient centrifugation. Initial Ficoll gradient separation of equine UCBs yielded a heterogeneous population with poor recovery of adherent cells. As such, a negative selection procedure (RosetteSep) was employed with the potential to remove extraneous natural killer cells, macrophages, lymphocytes, and B cells. Significantly fewer cells were present i n the buffy coat following centrifugation through Ficoll that attached readily to plastic cultureware. To ascertain their identity, cells were fixed and evaluated by immunocytochemistry for the stem cell markers, SSEA 1, Oct4, Tra160 and Tra1 81. Results demonstrated the presence of Oct4 in the nuclei of greater than 90% of the cells (Fig ure 3 1). A similar percentage of the cells contained Tra1 60 and Tra1 81, as well as SSEA 1. In data not shown, alkaline phosphatase enzymatic activity was readily detect ed and minor amounts of SSEA 4 were noted by immunocytochemistry. Thus, newborn foal cord blood contains a cell population that can be isolated with conventional human reagents and protocols and that express characteristic ES cell marker proteins.

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64 UCB S tem C ells F orm C hondrocytes The plasticity of UCB stem cells was evaluated by differentiation into cartilage precursors. Adherent stem cells were pelleted and cultured as a micromass in a defined media supplemented with ascorbic acid, dexamethasone and TGF 1. At the end of 3 weeks in chondrogenic media, the cell pellet was cryopreserved or lysed for RNA isolation. Alcian blue histology revealed the presence of proteoglycans, a matrix component of mature chondrocytes (Fig ure 3 2A ). The extracellular constituen ts of the micromass were rich in glycosaminoglycans, as indicated by Safranin staining. Sox9 is a transcription factor that positively regulates the expression of collagen and extracellular matrix genes in chondrocytes (Ng et al. 1997; Bi et al. 1999; Lefebvre et al. 2001) Expression levels of Sox9 and collagen 2a1 were evaluated by RT PCR after 7 and 21 days in chondrogenic differentiation media. Gene transcripts for Sox9 are evident after 7 days but absent by 21 days in differentiation permissive media (Figure 3 2 B). Abundant amounts of collagen 2a1 mRNA was evident at both time frames. T hese results demonstrate temporal and specific activation of the chondrogenic gene program. Differentiation of UCB S tem C ells into O steocytes Young thoroughbred racehorses are prone to debilitating bone fractures whose repair may be aided by stem cell based therapeutics. Therefore, UCB stem cells were cultured on plasticware in a defined media capable of inducing osteocytes from human UCB stem cells (Tondreau et al. 2005) After 3 weeks, cells were fixed with paraformaldehyde or harvested for RNA isolation. Alizarin Red histology indicated that the putative bone cells were capable of calcium deposition (Fig ure 3 3A). In a similar manner, Von Kossa staining detected calcium aggregates. RT PCR confirmed the osteogenic program in these cells. Primers specific for osteonectin and Runx2 amplified products of the correct size (Fig ure 3 3B). These results

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65 demonstrate that UCB stem cells are a source of osteocytes under appropriate in vitro cultivation conditions. Foal UCB S tem C ells can Differentiate into Endodermal D erived C ell T ypes A key feature of ES cells is their potential to contribute to any tissue type in the body. Adult stem cells possess a more limited plasticity than th eir embryonic counterparts. The ability of foal UCB stem cells to differentiate into hepatocytes, a cell type that originates from the endoderm, was examined. In brief, UCB stem cells were cultured for 2 weeks in media that supports hepatocyte formation in human UCB stem cells (Kang et al. 2005) Subsequently, cells were fixed with paraformaldehyde or lysed for RNA isolation. As shown in Figure 34 A, a change from an elongated, spindle shaped morphology to one exhibiting a larger cytoplasmic volume with an elliptical shape occurs in response to the treatment media. These cells express mRNA fo r both albumin and cytokeratin 18, definiti ve markers of hepatocytes (Figure 3 4 B). Equine UCBs maintained in the absence of induction media failed to express the liver marker genes (data not shown). The ability to respond in a manner similar to ES cells a nd form hepatocytes suggests that our UCB cell population may be more plastic than other adult MSC. Inefficient Formation of M yocytes and A dipocytes by UCB C ells Koerner et al. (Koerner et al. 2006) reported limited formation of adip ocytes from adult horse BM derived MSCs. Thus, we compared the adipogenic differentiation capabilities of foal UCB stem cells and adult horse adipocyte derived MSCs (A dMSC). In brief, both cell types were incubated for 21 days in adipocyte induction media. Cells were fixed and evaluated by Oil Red O histology for the presence of lipid droplets. In our hands, neither UCB nor AdMSC efficiently formed adipocytes. Sporadic fat cells containing limited amounts of lipid droplets were evident in foal UCB cell cult ures; no Oil Red O positive cells were found in the AD MSCs. This restricted differentiation profile by the two forms of stem cell was further exemplified

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66 following their incubation in myocyte induction media. UCB stem cells and AD MSCs were incubated for 7 days in media supplemented with galectin 1, a glycoprotein that promotes myogenesis in human fetal MSCs (Chan et al. 2006) Subsequently, cells were fixed and immunostained for the skeletal muscle marker protein, desmin. UCB stem cell cultures contained several m ultinucleated, spindle shaped cells that are reminiscent of myocytes. Anti desmin immunofluorescent detection reveals that these structures express the intermediate filament protein (Fig ure 3 5 B). No desmin expressing muscle cells were present in the AD MC Ss treated in a similar manner (data not shown). The presence of organized actin filaments was examined using Texas Red conjugated phalloidin. Equine UCB derived myoblasts contained organized actin structures throughout their cytoplasm (Fig ure 3 5C). By co ntrast, AdMSC cells contained fewer phalloidinreactive filaments. The cytoskeletal structures pointed to distinct differences in overall cellular morphology between the differentiated AdMSC and UCB myoblasts. UCB myoblasts were thin, elongated and cylindr ical in shape whereas the AdMSC cells were fibroblast like with an enlarged cytoplasmic space. Our results demonstrate differences between the two types of stem cells and suggest that foal UCB cells are more plastic than adult horse MSCs. AdMSC do not Expr ess the S ame C omplement of Stem C ell M arkers The inability of AdMSC to form adipocytes was surprising given that they originate from the fat depot. To ensure that the cells were nave and undifferentiated, subconfluent cultures of AdMSC were immunostained for stem cell markers. Similar to UCB stem cells, AD MSCs express Oct4, Tra1 60 and Tra181 (Fig ure 3 6). However, SSEA 1 and SSEA 4 were undetectable. These results indicate that AdMSC retain markers of adult stem cells but do not express those more close ly associated with ES cells.

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67 Discussion There is widespread interest in tendon, ligament and cartilage repair in horses through the use of directed stem cell transplantation methods. To date, published reports of multipotent cells isolated from BM, periphe ral blood, and UCB exist (Fortier et al. 1998; Saito et al. 2002; Koerner et al. 2006; Li et al. 2006) Cells from each of these sources display limited differentia tion into mesodermal cell types with predominant induction of chondrogenic and osteogenic precursors. Beyond these two cell types, vast differences in differentiation efficiencies and alternate cellular identities exist. The disparities may be attributed t o tissue source or suboptimal culture conditions; both possibilities necessitate further study. Alternatively, the transcriptional regulators that govern pluripotency may be absent or inactive thereby, limiting plasticity. Key to the ES celllike nature is expression of Oct4, Sox2, nanog, c myc, and Klf4 (Takahashi and Yamanaka, 2006) Foal UCB stem cells maintained in a growth factor rich medium expressed Oct4, SSEA 1, Tra1 60 and Tra1 81, all stem cell marker proteins. However, repeated attempts to detect nanog and Sox2 mRNA were unsuccessful. The absence of these transcription factors may contribute to the restricted types of cells generated and their incomplete differentiation (myoblasts). Interestingly, the ability of UCB stem cells to express these embryonic markers sets them apart from adult MSCs. Surface expression of SSEA 1 and SSEA 4 were not evident in equine AD MSCs. The lack of SSEA markers points to a hierarchy in plasticity that may account for some of the differences in differentiation capabilities. Efforts to define culture media that support nanog, Sox2, and Klf4 expression may lead to an increased range of differentiated lineages from UCB stem cells. Stem cells isolated from the umbilical cord matrix of pigs develop a morphology that resembles that reported by others for equine UCB stem cells (Carlin et al. 2006; Koch et al. 2007) In both examples, the majority of the cells attached to the cultureware surface and

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68 possessed a flat, spindle shaped, fibroblast like morphology. A lesser population formed light refractile colonies that grew upward from the substratum surface in a manner consistent with transformed fibroblas t foci. These colonies of small cells with a high nuclear to cytoplasmic volume were evident in our cultures of newborn foal UCB stem cells only after reaching confluency. Our UCB stem cells were maintained as a monolayer and passaged at approximately 60% confluency thereby, selecting against the development of these cell clusters that appear to grow independent of contact inhibition. While the identity of this cell population remains less clear, it is possible that these colonies represent a more primitive progenitor cell. Indeed, these cell clusters resemble those found in cultures of mouse ES cells. As such, one would predict that confluent equine UCB cultures that contain both the fibroblast like and light refractile cell colonies would express the plast icity genes, nanog and Sox2. However, expression of SSEA 1, Tra1 60, Tra181 and alkaline phosphatase, in a manner consistent with equine inner cell mass derived ES cells, provides encouraging evidence that our monolayer cells are naive and undifferentiate d (Takahashi and Yamanaka, 2006) Equine UCB stem cells, in our hands, are not direct equivalents to human UCB stem cells but do possess many similarities. Human UCB stem cells can be isolated directly from the blood and frozen without expansion (Lee et al. 2005) This aspect of enrichment and storage remains elusive in our equine UCB cells. Partial purification by negative immunoselection and density gradient centrifugation produces a cell population that survives immediate cryopreservation very poorly. This may be due to the small numbers of stem cells and/or heightened sensitivity of these cells to plasma membrane perturbation. Culture of the fresh isolates for 3 5 days allows for the removal of contaminating lymphocytes and cellular debris and expansion of the putative stem cell population, which can be stored in liquid nitrogen and subsequently recovered. Direct

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69 enrichment of the UCB stem cell population by affinity purification with CD133 antibodies may provide an alterna tive to both cell heterogeneity and cryopreservation issues. The capacity of foal UCB stem cells to initiate hepatocyte specific gene transcription demonstrates an endodermal developmental potential. Reports exist demonstrating hepatocyte formation from human UCB stem cells and MSC isolates from BM (Hong et al. 2005; Talens Visconti et al. 2006) However, this is the first report of hepatocyte formation using equine multipotential cells. Putative stem cells fro m the inner cell mass of equine blastocysts undergo spontaneous differentiation in vitro to yield cell derivatives of the three germ layers with endoderm defined by RT PCR detection of fetoprotein (Li et al. 2006) The ability of newborn foal UCB stem cells to form liver cells is encouraging as it provides additional evidence for a population with plasticity characteristics that more closely resemble an ES cell than an adult stem cell. A dditional endoderm derived cell types of clinical importance include pancreatic and cardiogenic. Human UCB stem cells can be induced to form heart cells following a two step differentiation protocol that involves 5azacytidine treatment (Kadivar et al. 2006) Culture with the hypomethylating agent suggests that UCB stem cells are more restricted in their differentiation capabilities than ES cells and requ ire chemical induced reprogramming. In our hands, treatment of foal UCB stem cells with 5 azacytidine did not induce the expression of myosin immunopositive cells. Because our antibody (MF20) recognizes all forms of sarcomeric myosin, this result provides indirect evidence that a full fledged cardiocyte is not created in response to epigenetic modification. However, a more comprehensive analysis of growth factor, morphogen and substratum requirements for UCB stem cell differentiation into cardiocytes is war ranted.

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70 Induction of the myogenic gene program has proven difficult in MSC originating from multiple animal and tissue sources. Exposure of rat BM derived MSCs to 5azacytidine caused differentiation into elongated, multinucleated myofibers (Wakitani et al. 1995) However, reprogramming the equine UCB transcriptome with this chemical did not induce the myogenic gene program (data not shown). A similar result was noted by others (Chan et al. 2006) Others reported that human UCB stem cells formed limited numbers of desmin immunopositive cells following in vitro differentiation (Nunes et al. 2007) These cells were devoid of the myogenic regulatory factors (MRFs) as measured by RT PCR. Interestingly, injection of the putative stem cells into mdx mice resulted in engraftment suggesting that compon ents within the muscle niche are essential for myogenic progression. One of those proteins is likely galectin 1, a glycoprotein of the basal lamina. Chan et al. (Chan et al. 2006) demonstrated that culture of human fetal MSC in media containing galectin 1 initiated both biochemical and morphological differentiation into myocytes. These cells formed large, multinucleated fibers that expressed contractile proteins and the MRFs. We used a similar approach with some degree of success. Supplementation of foal UCB cell cu lture medium with purified galectin 1 caused myogenic lineage establishment as determined by desmin immunocytochemistry. However, a large percentage of the myoblasts were fusion defective. In addition, we were unable to detect gene transcripts for MyoD, an early MRF, or myogenin, an MRF required for fusion and contractile gene expression. In accordance with our failure to amplify members of the MRFs, we did not detect myosin heavy chain or troponin T by immunocytochemical methods. The constraints to full ac tivation of the myogenic program may be attributed to the absence of complementary soluble proteins. The source of galectin 1 used by Chan was spent media from COS cells that produce and secrete the glycoprotein. Thus, additional proteins within the galect in 1 supplement may have aided

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71 induction of myogenesis. Alternatively, specie specific differences may underlie the discrepant results. Given the relative ease of adipocyte formation by human and rodent MSC, the inefficiency of adipogenesis in equine UCB s tem cells was surprising. Less than 1% of cells contained Oil Red O reactive lipid droplets following application of conventional adipogenic induction protocols. Koerner reported a similar result using BM derived and peripheral bloodderived MSC isolated f rom adult horses (Koerner et al. 2006) A very small number of adipocytes were found and the cytoplasmic lipid droplets within said cells were miniscule. By contrast, robust lipid formation is evident by Oil Red O histology in equine UBCs cultured in a similar adipocyte induction media (Koch et al. 2007) The discrepancy between these various reports may be attributed to the heterogeneity of the starting population and/or culture conditions. Koch reported the presence of dome like, clusters o f small cells as well as a fibroblastlike cell type (Koch et al., 2007) While we observe the same morphologies, care was taken to maintain the adherent monolayer exclusive of the foci like colonies. Future efforts will concentrate on resolving the identity of th ese divergent cellular phenotypes and their contribution to plasticity.

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72 Figure 3 1. Foal UCB cells express stem cell marker proteins. UCB stem cells (passage 5) were fixed and incubated with antibodies directed against Oct4, SSEA 1, Tra160 or T ra1 81. Immunoreactivity was detected with goat anti mouse AlexaFluor 488 or anti rabbit AlexaFluor 568 (Oct4). Nuclei were counterstained with Hoechst 33245. Scale

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73 Figure 3 2. Induction of chondrogenesis in foal UCB stem cells. Micr omass cultures were established as described. Cell pellets were embedded in OCT freezing medium and 10 um cryosections were collected. Alcian Blue and Safranin O histology indicated the presence of glycosaminoglycans and proteoglycans (A). Total RNA was i solated from cells treated in an analogous manner. RT PCR using gene specific primers indicated Sox9 and collagen 2A1 expression after 7 days in chondrogenic medium (B). Sox 9 mRNA was not detected at d21. RT, reverse transcriptase.

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74 Figure 3 3. U CB stem cells form osteocytes. UCB stem cells were maintained for 21 days in media containing glycerophosphate, dexamethasone, and ascorbic acid. Cells were fixed and stained for calcium and mineral deposition with Alizarin Red and von Kossa. Representat ive brightfield images (left part) and corresponding phase contrast fields (right part) at 200 are shown (A). Total RNA was isolated after 21 days in osteogenic induction medium. RT PCR using gene specific primers for osteonectin and RunX2 was performed. Products were separated through agarose gels and visualized with ethidium bromide (B). RT, reverse transcriptase.

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75 Figure 3 4. Foal UCB stem cells form hepatocytes. UCB stem cells were cultured for 14 days in absence or presence of HGF and FGF 4. Cells were fixed with 4% paraformaldehyde and representative phasecontrast images were captured at 200 (A). Separate cultures were analyzed by RT PCR for cytokeratin 18 ( CK18 ), albumin and glyceraldehyde 3 phosphate dehydrogenase ( GAPDH) gene expressi on (B). RT, reverse transcriptase.

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76 Figure 35. Incomplete initiation of adipogenesis and myogenesi s in foal UCB stem cells and Ad MSCs. Foal UCB and AdMSC cells were cultured in adipogenic induction media for 21 days prior to fixation. Oil Red O histol ogy demonstrates very few adipocytes in foal UCB cells (A). UCB stem cells were cultured for 7 days in myogenic induction media prior to fixation and desmin immunostaining. Arrow indicates multinucleated cell (B). Actin filaments were detected by incubatio n with Texas Red phalloidin (C). Total nuclei were visualized with Hoechst 33245. Scale bar = 25 m.

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77 Figure 36. AdMSC fail to express embryonic stem cell markers. AD MSCs were fixed and incubated with antibodies directed against Oct4, SSEA 1, SSEA 4, Tra1 60 or Tra1 81. Immunoreactivity was detected with goat anti mouse AlexaFluor 488 or anti rabbit AlexaFluor 568 (Oct4). Nuclei were visualized with Hoechst 33245. SSEA 1 and SSEA 4 were not detected. Scale bar = 10 m.

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78 CHAPTER 4 REFINEMENT OF CULTUR E CONDITIONS TO PROMOT E THE MAINTENANCE OF EQUINE UMBILICAL COR D BLOOD DERIVED STEM CELLS Introduction Cytotherapeutic repair in horses is a proposed means of decreasing the prescribed stall confinement period. Current strategies involve isolation of mes enchymal stem cells (MSCs) from bone marrow (BM) aspirates or adipose tissue coupled with autologous engraftment into the site of damage (Richardson et al. 2007; Taylor et al. 2007) Tendon lesions in horses treated with BM MSC retained a portion of the cells at the lesion site, exhibited properly oriented collagen fibrils and returned to exercise sooner (Pacini et al. 2007) Injection of BM MSCs into damaged superficial digital flexor tendons (SDFT) demonstrates that a portion of the cells is retained at the lesion site indicating that the improved healing is likely a product of the engrafted MSCs (Guest et al. 2008) MSC from equine adipose, bone marrow and umbilical cord blood efficiently differentiate into chondrocytes and osteocytes in vitro (Fortier et al. 1998; Arnhold et al. 2007; Koch et a l. 2007; Stewart et al. 2007; Kisiday et al. 2008; Reed and Johnson, 2008) Their ability to transform into cell types of additional lineages is limited. Under the appropriate conditions, UCB stem cells differentiate into adipocytes (Koch et al. 2007) A limited set of hepatogenic and myogenic markers was reported following UCB stem cell differentiation (Reed and Johns on, 2008) The reason for the restricted plasticity is unknown but may be associated with suboptimal culture conditions. Implicit to the pluripotent nature of human and rodent embryonic stem (ES) cells is expression of Oct4 and nanog (Chambers et al. 2003; Yates and Chambers, 2005; Wang et al. 2006; Babaie et al. 2007) Loss of either factor is associated with differentiation of the pluripotent cell Recent evidence demonstrates that e ctopic expression of Oct4 Sox2, Klf4 and c -

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79 myc is sufficient to reprogram somatic cells into ES like cells (Takahashi and Yamanaka, 2006; Takahashi et al. 2007; Aoi et al. 2008; Lowry et al. 2008) Oct4 mRNA is present in MSCs isolated from multiple tissue sources sugges ting that the transcription factor participates in the global inhibition of stem cell differentiation (Tondreau et al. 2005; Ren et al. 2006; Greco et al., 2007) Undifferentiated equine UCB stem cells express Oct4 in the nucleus, which is lost upon introduction of lineage decisions (Reed and Johnson, 2008) The objective of the experiment was to refine culture conditions of equine UCB stem cells and examine the effects of continuous culture on genetic markers of ES cell identity. Results indicate i mproved UCB stem cell population doubling times (PDTs) with cultivation on matrix associated protein surfaces. UCB stem cells express Oct4 nanog and Sox2 immediately upon establishment in vitro Serial passage is associated with decreased expression of both nanog and Sox2. Materials and Methods UCB Collection and Stem Cell I solation Cord blood was collected from the intact umbilicus of Thoroughbred foals (N= 4) at foaling into a sterile 50 ml centrifuge tube containing EDTA (1 mg/ml) as an anticoagulan t. UCB was stored at 4 C and putative stem cells isolated within 12 hours of collection. Samples were incubated for 20 minutes with RosetteSep Human Cord Blood Progenitor Enrichment Cocktail (50 l/ml blood; Stem Cell Technologies, Seattle, WA). An equ al volume of phosphate buffered saline (PBS) containing 2% FBS was added and the mixture was layered on a bed of Ficoll Paque ( Sigma, St. Louis, MO ). Cell aggregates were sedimented through the density gradient by centrifugation at 1,200 X G for 20 minute s. Mononuclear cells at the gradient interface were collected, washed with PBS and placed into culture.

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80 Stem Cell C ulture All media, serum and supplements were purchased from Invitrogen (Carlsbad, CA) unless otherwise noted. Murine ES cells (E14TG2a; (Thompson et al. 1989) ) were cultured using a modified procedure of that previously described (Piedrahita et al. 1992) and served as positive controls. Briefly, ES cells were cultured in Dulbeccos modified Eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 1000 units/ml leukemia inhibitory fact or (LIF), 2mM L glutamine, 0.1 mM 2mercaptoethanol on 0.1% gelatin coated plasticware. Cells were passaged at 70% confluence using 0.025% trypsinethylenediamine tetraacetic acid (EDTA). UCB stem cells were cultured on 0.1% gelatin (Fisher Scientific, Pittsburgh, PA), 0.01% fibronectin (Sigma Aldrich, St. Louis, MO), rat tail collagen I (5 g/ cm2) or uncoated tissue cultureware. Conventional UCB stem cell growth media (GM) is DMEM supplemented with 10% FBS and 5 g/ml plasmocin (InVivogen, San Diego, C A). Test media included GM supplemented with10 ng/ml FGF2 (R&D Systems, Minneapolis, MN), 1% nonessential amino acids, 1% insulintransferrin selenium (GM+FGF), Iscoves modified Dulbeccos media (IMDM) supplemented with 10% FBS, 50 ng/ml Flt3, 10 ng/ml thrombopoietin and 20 ng/ml c kit [GM+Flt/Tpo/Kit; (McGuckin et al. 2003) ] or conditioned media (CM). CM was coll ected from confluent UCB stem cells cultured in growth medium and centrifuged at 1500 x g for 10 minutes. The supernatant was retained and supplemented with 3% FBS and 5 g/ml plasmocin. Culture medium was exchanged every three days and cells were passaged at 70% confluency using 0.025% trypsinEDTA. Additionally, c ell number was determined daily for 4 days on subpopulations of UCB for the calculation of PDT according to the formula N=N02(t/pdt) where N = final cell number, N0 = initial cell number, t=time, and pdt= population doubling time. Phase photographs were captured by a Nikon T200 microscope with NIS Elements software (Nikon

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81 Instruments, Melville, NY) and converted to grayscale in Adobe Photoshop CS. Cell number was determined by manually counting the number of attached cells in six random photographed fields. Manual counting was employed to minimize the disruption of cell growth. For extended passaging, UCB cells at passages three, six, and nine were plated at a density of 2000 cells/cm2 on the a ppropriate substrata in either FBS or Modified FBS media. Cell number was determined on days 0 through 4 and population doubling time was determined as above. Cells were harvested for RNA isolation on day 4 for subsequent RT PCR analysis. All samples were evaluated in duplicate. RNA Isolation, Reverse Transcription (RT) and P olymerase C hain R eaction (PCR) Total RNA was isolated by lysis in STAT60 (Iso Tex Diagnostics, Friendswood, TX) and ethanol precipitation. The RNA was digested with DNase (Ambion, A ustin, TX) to remove genomic DNA contaminants. One microgram of total RNA was reverse transcribed (Superscript III, Invitrogen, Carlsbad, CA) in 20 l reaction volume. Two microliters of first strand cDNA was amplified with gene specific primers and AccuPrime DNA polymerase (Invitrogen, Carlsbad, CA). Primer sequences are glyceraldehyde 3phosphate dehydrogenase (F GAGATCCCGCCAACATC, R CTGACAATCTT CAGGGAATTGTC), Oct4 (F GCTGCAGAAGTGGGTGGAGGAAGC, R GCCTGGGGTACCAAAATGGGGCCC), Nanog (F GTCTCTCCTCTGCCTTCCTCCATGG, R CCTGTTTGTAGCTAAGGTTCAGGATG), Sox2 (F AACGGCAGCTACAGCATGA, R TGGAGTGGGAGGAAGAGGTA), Klf4 (F TGGGCAAGTTTGTGTTGAAG, R TGACAGTCCCTGTTGCT CAG), c myc (F GACGGTAGCTCGCCCAAG, R ACCCCGATTCTGACCTTTTG) Jagged 1 ( F GCCTGGTGACAGCCTTCTAC R GGGGCTTCTCCTCTCTGTCT), Jagged2 ( F CATGATCAACCCCGAGGAC R CGTACTGGTCGCAGGTGTAG ), Notch 1 ( F -

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82 GAGGACCTGGAGACCAAGAAGGTTC R AGATGAAGTCGGAGATGACGGC ), Notch 2 (F GCAGGAGCAGGAGGTGATAG, R GCGTTTCTTGGACTCTCCAG), Notch 3 (F GTCCAGAGGCCAAGAGACTG, R C AGAAGGAGGCCAGCATAAG ), Dll 1 ( F ACCTTCTTTCGCGTATGCCTCAAG R AGAGTCTGTATGGAGGGCTTC ), and Dll 4 ( F CGAGAGCAGGGAAGCCATGA, R CCTGCCTTATACCTCTGTGG ) cDNA was amplified with the gene specific primers listed below using the following protocol: 5 minutes at 94C for the initial denaturation followed by 40 cycles of 94C for 30 seconds, 53C for 30 seconds, and 68C for 30 seconds. Amplicons then underwent a final elongation period a t 68C for 10 minutes. PCR products were visualized following electrophoresis through 2% agarose TAE (40 mM Tris, pH 8.0, 2 mM EDTA) gels impregnated with ethidium bromide. Representative images were captured with a Kodak ImageDoc system and inverted in Adobe Photoshop CS. All products were verified by sequencing. Statistical Analysis Transcript expression was analyzed by logistic regression using the LOGISTIC procedure in SAS (SAS Institute, Inc., Cary NC) Reference values for substrata, media, and p assage were set at uncoated, growth media, and passage three, respectively. Probability values from the logistics procedure were obtained using WALD Chi square statistics derived from type III analyses of effects. Initial models included all main effects and interactions. Data were reanalyzed after removing nonsignificant effects from the model. Chi square probability is reported. Doubling time data was analyzed by ANOVA using the GLM procedure of SAS. Initial models included all main effects and interac tions. Subsequent reanalysis removed all nonsignificant effects from the model. Passage, substrata, media and horse were tested as main

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83 effects. Comparisons of least squares means were examined by the PDIFF option of the LSMEANS statement. Statistical significance for all experiments was set at p<0.05. Results UCB Express Markers of Pluripotent Stem Cells Equine UCB stem cells are cultured routinely in basal media supplemented with 10% fetal bovine serum [GM; (Koch et al. 2007; Reed and Johnson, 2008) ]. Under these conditions, the majority of cells grew in a monolayer with fibroblast like morphology (Figure 41A). However, a small portion of cells formed colonies reminiscent of embryonic stem cell colonies (Fig ure 1B). Oct4 nanog, Sox2, Klf4 and c myc are involved in somatic cell reprogramming to ES like cells and the gene products are implicated in pluripotency (Takahashi and Yamanaka, 2006; Takahashi et al. 2007) Total RNA was isolated from mouse ES cells and equine UCB stem cells and analyzed by RT PCR for expression of the aforementioned gene products. As expected, transcripts for the reprogramming genes were detected in mo use ES cells (Fig ure 4 1C ). In a similar manner, Oct4, nanog, Sox2, Klf4 and c myc amplicons were present in UCB stem cell RNA isolates. No DNA products were evident in RT PCR reactions in the absence of reverse transcriptase. GM and GM+FGF Maintain UCB Proliferation To determine the effects of culture conditions on UCB stem cell proliferation and stemness, cells were supplemented with a number of growth factor combinations. Supplementation of GM with FGF2 did not disrupt morphology (Figure 42 ). How ever, a combination of Tpo, Flt3 and c kit, or CM resulted in a morphology resembling cells in replicative senescence. Cell number change over a four day culture period was measured and population doubling times (PDT) determined. GM and GM+FGF maintained shorter doubling

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84 times than other cultures GM+Tpo/Flt/Kit and CM were discontinued due to morphological changes and a prolonged PDT and cell death. Protein Surface Matrixes Promote UCB Growth UCB stem cells were seeded at equal cell densities on gelati n, fibronectin, collagen and uncoated surfaces. Cell population doubling times were calculated following four days of culture (Table 41). UCB stem cells on coated surfaces demonstrated fas ter PDTs than controls maintained on uncoated tissue plasticware Changes in PDT were independent of the type of matrix used. Additionally, PDT was increased in cells cultured in GM+FGF (35.681.25 hr) compared to growth media controls (27.650.74 hr; p<0.0001). The interaction of media and substrata types demonstra ted that cells cultured on any type of matrix in control media have significantly faster doubling times than their respective cultures in media supplemented with FGF2 (Table 4 2). Cells cultured on collagen, fibronectin, and gelatin matrices in control me dia exhibit no differences in growth kinetics. Few stem cell populations are immortal in vitro most enter replicative senescence over time in culture. Thus, population doubling times during continuous culture were determined at passages three, six, and ni ne. Time in culture significantly increases PDT from passage three through passage nine (21.090. 3 hr, 34.841.28 hr, and 39.061.0 hr, respectively; p<0.0001 for all interactions). While there was no significant effect of media at passage three (p=0.136 1), later passages showed significantly shorter doubling times when cultured in control growth media compared to GM+FGF (p<0.0001, data not shown). Oct4 is Maintained Throughout UCB Culture Having determined appropriate culture conditions to maintain prol iferation, markers of stemness were measured by RT PCR. Oct4 expression is maintained over time in culture

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85 regardless of substrata or media (Table 4 3). However, nanog and Sox2 mRNA decline with serial passage. Expression of both genes is affected by substrata, maintaining expression longer on a protein matrix (particularly collagen) than on uncoated plasticware. Media containing FGF2 does not affect the maintenance of either nanog or Sox2 mRNA. The presence of nanog transcripts was significantly differ ent among horses, suggesting an innate heterogeneity among animals. Notch Signaling in UCB Stem Cells Notch signaling plays a crucial role in cell:cell communication among mature and naive cells. In human ES cells, constitutive Notch signaling promotes dif ferentiation, particularly to neural cell lineages (Lowell et al. 2006) In hematopoietic stem cells (HSCs), constitutive activation of the Notch pathway including downstream target Hes1 led to an increased self renewal capacity of long term in vivo repopulating HSCs (Stier et al. 2002; Kunisato et al. 2003) The Notch signaling pathway consists of a transmembrane receptor, Notch, which is cleaved upon the binding of an extracellular ligand. The ligands for Notch are generally also membrane bound to adjacent cells. There are three Jagged proteins and two Delta like ligand (Dll) proteins that serve to activate Notch signaling. Upon ligand binding, Notch undergoes a series of proteolytic cleavages, resulting in the release of the Notch intracellular domain (NICD) which relocates to the nucleus and acts as a transcriptional regulator. To determine what members of the Notch pathway were present in UCB and AdMSC, and to compare that expression with the pathway present in mES, RT PCR was performed for jagged1, jagged2, notch1, notch2, notch2, Dll 1, and Dll 2. Mouse ES exhibited amplicons for all transcripts examined (Fig ure 4 3). Notch 1, Notch3 and Jagged 1 were present in UCB stem cells, indicating the possibility for a functional pathway in these cells. However, only the

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86 ligand Jagged 1 is found in AdMSC. This suggests that adipose derived stem cells may not be full y capable of Notch signaling but could contribute to signaling to surrounding cells. To identify the effects of the Notch pathway on UCB proliferation, cells were cultured for three days in media with or without the Notch inhibitor L685,458. Cell number was determined daily by visual inspection and manual counting. Inclusion of the Notch inhibitor had no significant effect on cell number over the three day period (Figure 44). Furthermore, UCB culture in the presence of the Notch inhibitor had no effe ct on the presence of Oct4, nanog, or Sox2 as determined by RT PCR (data not shown). Notch signaling is mediated by the transcription factors hes and hey. The presence of hes and hey in UCB was determined. 23A2 myoblasts were used as a positive control. No transcript was amplified for hey, however hes transcripts were present in both cell types. Furthermore, there appeared to be no difference in cells treated with L685,458 and control cells of either type (Figure 4 5). To further verify this, real time PCR was performed following three days of treatment with the Notch inhibitor. Delta Ct values are reported in Table 4 4. No difference in expression levels was apparent in either cell type. To verify the efficacy of the Notch inhibitor, 23A2 myoblasts were stimulated to differentiate. The presence of BMP6 abrogates differentiation however, this can be inhibited by the inclusion of L685,458. Control cells formed myotubes within 48 hours in differentiation medium (Figure 4 6). At this time, 76% of nuc lei were included in myosin heavy chain (MyHC) expressing cells. The presence of BMP6 in differentiation media inhibited myotube formation, with fewer than 10% of cells expressing MyHC. However, inhibition of the Notch pathway in the BMP6 stimulated cells abrogated the effects of BMP6 and allowed a partial recovery of

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87 differentiation. Greater than 30% of cells immunostained positive for MyHC. Supplementation of the differentiation media with L685,458 alone had no effect on differentiation. We conclud e from these experiments that while the Notch pathway may play an important role in differentiation of specific stem cell types, it is not crucial for the proliferation of UCB stem cells. Furthermore, we have been unable to demonstrate that UCB stem cells have an active Notch signaling network. Discussion Initial culture strategies for equine UCB stem cells involved growth on uncoated tissue plasticware surfaces (Koch et al. 2007; Reed and Johnson, 2008) but no work has assessed the effects of various substrata or media on the prolonged culture of these cells. Use of UCB stem cells as injury repair aids likely will require an initial expansion in culture to pr ovide sufficient numbers of cells. Several media supplements were assessed for their ability to extend the timeframe that UCB stem cells can be maintained in vitro Expansion of human UCB stem cells in media supplemented with thrombopoietin, Flt3 and c kit ligand results in a population that expresses ES like surface markers, morphology and importantly, a delay in replicative senescence (McGuckin et al. 2003) Passage of equine UCB stem cells in this media did not duplicate the events and phenotypes reported for the human counterparts. Equine UCB stem cells proliferated slowly and exhibited large nuclei with pronounced nucleoli, characteristics of senescent fibroblasts. Close examination of the cultures indicated an absence of any cell clusters with ES like morphology. Because Tpo, Flt3 and c kit were supplemented into a basal media that supports equine UCB stem cell growth, we conclude that the growth factors are detrimental to the routine culture and passage of these cells in vitro Equine UCB stem cells grown at higher densities proliferate more readily than those at lower cell concentrations. Thus, we postulated that secreted factors may play a role in

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88 maintaining growth kinetics in this population. Culture in conditioned media proved deleterious to UCB stem cells, evidenced by poor growth kinetics and morphology suggestive of replicative senescence. This suggests that the improvement seen in growth kinetics at higher cell densities may be a reflection of cell:cell and extracellular matrix interactions rather than the paracrine action of secreted factors. Similar to conditioned media FGF2 appears to be a hindrance to UCB stem cell proliferation. FGF2 is mitogenic for MSCs isolated from adipose and bone marrow tissues (Baddoo et al. 2003; Benavente et al. 2003; Rider et al. 2008) Treatment of mouse BM MSC with FGF2 causes an increase in proliferation without induction of differentia tion (Baddoo et al., 2003) Indeed, FGF2 reversibly inhibited MSC differentiation toward the adipogenic and chondrogenic lineages. However, t reatment of equine UCB stem cells with FGF2 resulted in longer PDTs but no negative effects on the ES marker profile The co ntrasting results may represent specie and tissue source differences. Nanog, Oct4, and Sox2 are key factors in maintaining ES cell pluripotency in addition to being capable of inducing pluripotency in somatic cells (Takahashi and Yamanaka, 2006) In the absence of nanog, mouse ES cells differentiate into presumptive endoderm lineages (Mitsui et al., 2003) Oct4 is required to maintain pluripotency of the inner cell mass; loss of function results in differentiation to trophectodermal lineages (Zaehres et al. 2005) The r equirement of Sox2 to maintain the expression of Oct4 has been demonstrated (Masui et al. 2007) These transcripts, along with c myc, are capable of inducing a pluripotent, ES like state in s omatic cells (Takahashi and Yamanaka, 2006) Notably, the more differentiated adipose derived ste m cells lack expression of N anog and Sox2 (data not shown). Expression of this panel of transcripts, as well as population doubling time was used to monitor the stemness of UCB in different culture

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89 conditions and over time in culture. Cells maintained Oct 4 expression throughout the duration of culture, regardless of media or substrata used. In contrast, the presence of both Nanog and Sox2 decreased with time in culture. The presence of a protein matrix prolonged expression of both nanog and Sox2 transcri pts. This was not unexpected, as most stem cells reside in a very specific niche, a large part of which is composed of extracellular matrix. Extracellular matrix in the stem cell niche provides not only support for adhesion, but instructive signals to ma intain the nave state (Bi et al. 1999; Chen et al. 2007) Additionally, UCB cultured on various substrata had decreased population doubling times compared to those on uncoated plastic ware. As stem cells divide, one daughter cell may proliferate rapidly to expand the progenitor cell population while the other divides much more slowly or not at all to maintain the stem cell population. Thus, slowly dividing stem cells are considered to be the more nave population. However, when cultured for use as a therapeutic aid, cultivation of UCB on a protein substrate decreased population doubling time. This is a beneficial property when considering the expansion of these cells in culture prior to use as a therapeutic aid. The loss of Nanog and Sox2 during extended culture highlights the need for short expansion periods prior to use as a cytotherapeutic tool. Interestingly, despite the increase in population doubling time of cells cultured in medi a containing FGF2, there was no effect of FGF2 on the transcript profile of UCB While FGF2 appears dispensable for the maintenance of pluripotency markers, its negative effects on population doubling time preclude its use in media for UCB expansion. In s ummary, we define d culture conditions sufficient to ensure ES like gene expression patterns for early passage equine UCB stem cells. These include maintenance on matrix protein coated surfaces and cultivation in fetal bovine serum. Genetic markers of plu ripotency decline d

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90 with serial passage suggesting that expansion of UCB stem cells for therapeutic purposes is limited under the current culture conditions

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91 Figure 4 1. GM and GM+FGF support equine UCB stem cell propagation. UCB stem cells cultured i n GM and GM+FGF have faster doubling times than those grown in GM containing thrombopoietin(tpo) Flt3, and c kit, or UCB conditioned media. Population doubling times are presented below their respective phase photograph in hours SEM. Scale bar = 10 GM = growth media; GM + FGF = growth media containing FGF2; GM + Tpo/Flt/kit = growth media containing thrombopoietin, Flt3, and c kit; CM = conditioned media.

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92 Table 4 1. Effect of s ubstrata on e quine UCB1 derived s tem c ell population doubling t ime P value Substrata 2 PDT 3 SEM 4 Con Gel Fib Col Con 35.91 1.89 --<0.0001 <0.0001 <0.0001 Gel 30.67 1.38 ----0.3633 0.7634 Fib 29.72 1.34 ------0.5427 Col 30.36 1.44 --------1 UCB = umbilical cord blood 2 Con= uncoated, Gel = gelatin, Fib = fibronectin, Col = collagen 3 PDT= population doubling time in hours, N=N02(t/ pdt) where N = final cell number, N0 = initial cell number, t=time, and pdt= population doubling time 4 SEM = Standard error of the mean

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93 Table 42. Effect of s ubst rata and m edia on e quine UCB1 derived s tem c ell doubling t ime P value Substrata2 Media3 PDT4 SEM5 Con GM Con G M+FG F Gel GM Gel G M+FG F Fib GM Fib G M+FG F Col GM Col G M+FG F Con GM 30.07 1.59 --<0.0001 0.0628 0.0079 0.0370 0.1055 0.0091 0.0029 Con G M+ FG F 41.74 3.01 ----<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 Gel GM 27.32 1.50 ------<0.0001 0.8187 0.0006 0.4463 <0.0001 Gel G M+FG F 34.01 2.14 --------<0.0001 0.2908 <0.0001 0.7366 Fib GM 26.99 1.57 ----------0.0003 0. 5941 <0.0001 Fib G M+FG F 32.46 2.05 ------------<0.0001 0.1643 Col GM 26.21 1.18 --------------<0.0001 Col G M+FG F 34.50 2.38 ----------------1 UCB=umbilical cord blood 2 Con= uncoated, Gel = gelatin, Fib = fibr onectin, Col = collagen 3 GM = growth media, G M+FGF = growth media + FGF2 4 PDT= population doubling time in hours, N=N02(t/ pdt) where N = final cell number, N0 = initial cell number, t=time, and pdt= population doubling time 5 SEM = Standard error of the mean

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94 Figure 42. Equine UCB stem cells express markers of embryonic stem cell pluripotency. RT PCR was performed on mouse embryonic (n=1, in triplicate) and UCB stem cell (n=4, in duplicate) total RNA using primers specific for Oct4, nanog, Sox2, KL F4 and c myc transcripts Both populations of stem cells express transcripts for the reprogramming genes. UCB = umbilical cord blood stem cells; mES = mouse embryonic stem cells. Representative photo shown.

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95 Table 43. Effects of h orse, passage, m edia and s ubstrata on mRNA e xpression P value mRNA Horse Passage Media Substrata Oct4 0.3535 0.6441 1.0000 0.4272 Nanog 0.0004 0.0001 0.3018 0.0001 Sox2 0.0823 <0.0001 0.5343 0.0574

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96 Figure 4 3. UCB and AdMSC express a limited number of molecules in the N otch signaling pathway. RT PCR was performed for the transcripts listed. mES = mouse embryonic stem cells, UCB = umbilical cord blood derived stem cells, AdMSC = adipose derived stem cells

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97 Figure 4 4. Inhibition of the Notch signaling pathway does not a ffect proliferation. UCB stem cells were cultured in the presence or absence of L685,458 for three days. Cell number was counted daily. CTL = control cells, NI = cells supplemented with L685,458

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98 Figure 4 5. UCB and 23A2 myoblasts express hes R T PCR for the transcripts listed was performed on RNA isolated from cells treated with the Notch inhibitor, L685,458. UCB = umbilical cord blood derived stem cells, Con = control, no inhibitor, NI = cells supplemented with L685,458.

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99 Tabl e 44. Delta Ct v alues for hes r ealtime PCR. Cell Type Treatment 1 dCt 2 23A2 CTL 6.54 0.8 23A2 NI 7.56 0.24 UCB CTL 12.36 0.08 UCB NI 11.11 0 15 1 CTL = control, NI = cells supplemented with L685,458 2 dCt = Delta cycle threshold value standard error of the mean

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100 Figure 4 6. BMP6 inhibits myoblast differentiation in a Notch dependent manner. 23A2 myoblasts were placed in differentiation media in the presence or absence of 50 ng/ml BMP6 and/or the Notch inhibitor L685,458. Cells were fixed after 48 hours and immunostained for myosin heavy chain. Images were captured at 200x. The percent of cells expressing myosin heavy chain was determined and is reported below its respective panel. MyHC = myosin heavy chain

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101 CH APTER 5 CULTURE OF EQUINE UM BILICAL CORD BLOOD A ND ADIPOSE DERIVED S TEM CELLS TO PROMOTE TEN OCYTIC DIFFERENTIATI ON Introduction Tendons are the elastic structures that connect muscle to bone. These fibrous structures provide tensile strength during normal movement as well as during strenuous exercise. Tendon damage is a significant problem in the horse race industry. In the United Kingdom, nearly one half of all injuries are related to failed tendon and ligament function (Pinchbeck et al. 2004) The superficial digital flexor tendon (SDFT) is the anatomical and functional equivalent of the Achilles tendon in humans (Dowling et al. 2000) Historical approaches to SDFT repair are based upon rest and gradual reintroduction to work (Goodship et al. 1994) Recovery times typically are greater than 6 months and the animals are prone to reinjury due to fibrocartilage scar tissue formation (Clegg et al. 2007) Current efforts to improve repair rates and strengthen regenerated tendons include injection of bone marr ow and adipose derived MSCs (Taylor et al. 2007) Anecdotal evidence suggests that these reagents are beneficial but no conclusive data exists. Mononuclear cells isolated from adipose tissue impr oved tendon architecture when implanted into collagenase induced lesions but resulted in no differences in the rate or quality of repair (Nixon et al. 2008) Scleraxis is a class II basic helix loophelix transcription factor expressed early during mouse embryogenesis in the syndetome, a derivative of the somitic sclerotome compartment (Cserjesi et al. 1995; Brent et al. 2003) Expression is associated with connective tissue and skeleton structures during prenatal development and with periodontal ligaments, force generating tendons, brain, lung and Sertoli cells in adult rodent s (Liu et al. 1996; Perez et al. 2003; Muir et al., 2005; Murc hison et al. 2007; Pryce et al. 2007) Genetic ablation of scleraxis is embryonic lethal prior to gastrulation with an absence of mesoderm (Brown et al. 1999) Using transgenic

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102 reporter scleraxis mice, expression of the transcripti on factor is largely confined to tendons early in postnatal development and absent in the structures as they become acellular (Pryce et al ., 2007) Conditional ablation of scleraxis in the developing limb of mice produces an animal with missing tendons in the limb; a few tendons are present but small in size (Murchison et al. 2007) Of importance, scleraxis null animals contain no flexor tendons. In vitro work in tendon fibroblasts suggests that s cleraxis and NFATc cooperatively regulate the activity of the Col1a1 proximal promoter (Lejard et al. 2007) Overexpression of scleraxis in primary cardiac fibroblasts significantly increased the production of Col1a2 through direct binding with the Col1a2 promoter region (Espira et al. 2009) These data suggest a dir ective role of scleraxis in the formation of mature tenocytes. Members of fibroblast growth factor (FGF) family regulate transcription of scleraxis FGFs produced by the myotome supply a paracrine signal that allows formation of the sclerotome and syndetome (Brent and Tabin, 2004) Exogenous FGF4 induces scleraxis and tenascin C mRNA in the developing chick limb (Edom Vovard et al. 2002) FGF8 can substitute partially for myotome suggesting that this FGF is important for tendon progenitor formation. Ectopic expression of RCAS FGF8 resulted in an upre gulation of scx, tnmd and type 1 collagen in the intermuscular tendons associated with visceral smooth muscle cells (Le Guen et al., 2009) Signals transmitted in response to FGF4 and FGF8 include increased activity of MEK1, a requisite kinase for scleraxis expression (Smith et al. 2005) Ectopic expression of FGF5 in the d eveloping chick inhibited muscle enlargement and promoted proliferation of tenasin expressing fibroblasts in the hind limb (Clase et al. 2000) Ectopic expression of kinase defective FGF receptor or constitutive active mitogen activated protein kinase phosphatase 3 (MKP3) inhibits scleraxis transcription in chick embryos. The downstream target of elevated

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103 ex tracellular regulated kinase 1 and 2 (ERK1/2) may be the Ets transcription factor, PEA3 (Brent and Tabin, 2004) Retroviral mis expression of PEA3 in chick embryos causes transcription of scleraxis ; ectopic dominant inhibitory PEA3 represses scleraxis expression. The objective of this experiment was to examine scleraxis and tenascin C mRNA expression in equine umbilical cord blood (UCB) stem cells and equine adipose derived mesenchymal stem cells (AdMSCs) in respo nse to FGF treatment. Results demonstrate that UCB stem cells and AdMSCs express scleraxis and tenascin C Culture in matrigel upregulated expression of both transcripts. UCB stem cells treated with FGF2 or FGF5 causes an increase in MEK dependent phosphoERK1/2, although with different activation kinetics. Scl eraxis mRNA content was measured following 48 hours of treatment with either FGF2 or FGF5. Neither caused an increase in scx over that seen on matrigel alone. However, TnC expression was increased in AdMSC cultured with FGF2 or FGF5. Materials and Methods Stem Cell Culture Umbilical cord blood stem cells ( UCB ) were cultured on plastic tissue culture plates coated with 0.1% gelatin (Fisher Scientific, Pittsburgh, PA) in Dulbeccos Modified Eagle M edium supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and 5 g/ml Plasmocin (InVivogen, San Diego, CA). Equine adipose derived cells (AdMSC) were purchased from ScienCell (Carlsbad, CA). Cells were cultured on 0.1% gelatin coated tissue culture plates in Mesenchymal Stem Cell Medium (ScienCell) according to manufacturers recommendations. Cells were passaged at 70% confluency using 0.025% trypsinEDTA. Three dimensional cultures were achieved by growing cells on Cytodex3 collagen coated beads (Invitrogen) or allowing cells to embed into 30% Matrigel (BD Biosciences, San Jose, CA).

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104 Plasmids and Transfections The 500 base pair minimal promoter of the mouse scleraxis gene was amplified from C2C12 myoblasts with specific primers (F:CAC ACGGCCTGGCACAAAAGACCC R:TTCGGACTGGAGTGGGGCCGCCAGC). Following sequencing to verify the identity of the product, the promoter was cloned into the TOPO Zero Blunt vector prior to subcloning into the pGL3 basic vector such that promoter activity would drive the expression of luciferase (Scx luc). C2C12 mesenchymal cells and UCB stem cells in 12 well plates were transiently transfected with 250 ng Scx luc and 25 ng pRL tk, a Renilla luciferase plasmid, as a monitor of transfection efficiency using FuGene 6.0. Media was replaced after 6 hours to include 50 ng/ml BMP6, 10 ng/ml FGF4, or 10 ng/ml FGF5. Cells were passively lysed in luciferase lysis buffer after 24 hours of cultur e and l uciferase activities measured (Dual Luciferase Reporter kit, Promega, Madison, WI). Scx l uc activity was corrected for pRLtk activity. The mouse minimal Scleraxis promoter was sequence verified. Analysis of the sequence by the Transcription Element Search System (TESS, http://www.cbil.upenn.edu/cgi bin/tess) revealed the presence o f several putative transcription factor binding sites. Confocal Microscopy UCB and AdMSC cultured on gelatin, matrigel, and collagen beads were fixed in 4% paraformaldehyde for 15 minutes. Cells were permeabilized with 0.1% TritonX100 in phosphate buffere d saline (PBS) supplemented with 5% FBS. Actin filaments were visualized using fluorescein conjugated phalloidin (Invitrogen) and nuclei were stained with Hoechst 33342. All immunofluorescence work was completed on glass bottom plates to aid microscopy. Confocal microscopy was performed on a Leica TCS SP5 Laser Scanning Confocal Microscope running Leica LAS AF software for instrument control and image analysis. Images were adjusted for brightness and contrast in Adobe Photoshop CS.

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105 Protein Isolation an d Evaluation Equine UCB and AdMSC were treated with 10 g/ml protamine sulfate for 10 minutes to remove signaling molecules from the extracellular matrix Cells were placed in serum free media for one hour prior to stimulation with FGF2, FGF4, or FGF5 in the presence or absence of the MEK inhibitor PD98059. Cells were lysed directly into SDS PAGE sample buffer. Protein was loaded based on equal cell number and electrophoresed across a 10% polyacrylamide gel. ERK1/2 activity was assessed using phospho a nd total ERK1/2 antibodies (Cell Signaling Technologies, Danvers, MA). Briefly, proteins were transferred to nitrocellulose and nonspecific binding sites were blocked with 10% nonfat dry milk in TRIS buffered saline supplemented with 0.1% Tween20 (TBS T ). Blots were incubated overnight at 4 C with primary antibody (1:1000) in blocking solution. Following extensive washing, blots were incubated with secondary antibody (1:2000). Equal protein loading was ensured by probing membranes with antitubulin (A bcam, Cambridge, MA) for one hour (1:2000) followed by incubation in secondary antibody for one hour (1:5000) prior to visualization. Immune complexes were visualized by chemiluminescence (ECL; GE Life Sciences, Piscataway, NJ) and autoradiography. Assess ment of Proliferation Cells were seeded at equal density and cultured in low serum medium (2% FBS) supplemented with FGF2, FGF4 or FGF5 in the presence or absence of PD98059 for 48 hours. Prior to fixation, cells were pulsed with 10 M bromodeoxyuridine ( BrdU) for two hours. Proliferation index was determined as the proportion of cells expressing BrdU:total cell number. All experiments were performed on UCB collections from four horses in duplicate wells with two replicate experiments.

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106 RNA Isolation, Reve rse Transcription, and Polymerase Chain Reaction Total RNA was isolated by lysis in STAT60 (Iso Tex Diagnostics, Friendswood, TX) and passed over RNeasy Mini columns (Qiagen, Valencia, CA). The RNA was digested with DNase (Ambion, Austin, TX) to remove ge nomic DNA contaminants. One microgram of total RNA was reverse transcribed (SuperScript III, Invitrogen) in 20 l reaction volume. Two microliters of first strand cDNA was amplified with gene specific primers and AccuPrime DNA polymerase (Invitrogen). P rimer sequences are listed in Table 51. PCR products were visualized following electrophoresis through 2% agarose gels impregnated with ethidium bromide. Representative images were captured with a Kodak ImageDoc system and inverted in Adobe Photoshop CS Quantitative PCR Complementary DNA reverse transcribed from 1 g of total RNA was amplified with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and the appropriate forward and reverse primers (Table 51; 20 pM) in an ABI 7300 Real Time PCR System (Applied Biosystems). Thermal cycling parameters included a denature step of 95C for 10 min and 50 cycles of 15 s at 95.0C and 1 min at 60.0C. A final dissociation step included 95C for 15 s, 55C for 30 s, and 95C for 15 s. Serial dilutions of pooled samples were used to generate standard curves to ensure generation of cycle threshold values that were within the linear range of amplification (Castellani et al. 2004) Cycle threshold value ranges for each transcript are reported in Table 52. Results AdMSC and UCB E xpress Markers of Tenocytic C ells Immature tenocytes express the transcription factor scleraxis prior to the upregulation of m ore mature markers such as tenascin C and collagen 1A2. To evaluate adipose and umbilical

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107 cord blood derived stem cells for tenocytic markers, RT PCR was performed using gene specific primers for scleraxis and tenascin C Messenger RNA for both transcripts were present in both adipose and umbilical cord blood derived stem cells (Figure 51). Transcripts were sequenced to verify identity. Scleraxis Minimal Promoter Activity The mouse minimal s cleraxis promoter was sequence verified. Analysis of the sequence by the Transcription Element Search System (TESS, http://www.cbil.upenn.edu/cgi bin/tess) revealed the presence of several putative transcription factor binding sites. The list of binding sites with p values less than or equal to 0.01 is presented in Ta ble 5 3. Figure 5 2 shows a schematic of the promoter highlighting potentially important regulatory sites. Of particular interest is the presence of POU factor, Hes, SP1, and AP1 binding sites. A Klf4 binding site is present at base pair 35, but is not included on the list as the pvalue was given as 0.011. However, there were multiple sites strongly recognized in a small region lending strength to this prediction and its inclusion on the figure. UCB stem cells show much lower basal levels of scleraxis promoter activity than do C2C12s (Figure 53). Supplementation of C2C12 cells with BMP6 reduced luciferase expression driven by the scleraxis promoter. FGF4 and FGF5 had no effect on the scleraxis promoter in these cells. Scleraxis promoter activity in U CB stem cells appears to be unaffected by supplementation with BMP6, FGF4 or FGF5. This may be due to the low basal level of the minimal promoters activity. Other transcription factor binding sites may be required for full activity of this promoter in t his environment. Downstream effectors of FGF signaling include the transcription factors Pea3 and Erm. Activation of Pea3 and Erm is required for scleraxis expression in the developing somite (Brent & Tabin). Thus, we examined the presence of these facto rs by RT PCR in UCB stem cells. RT -

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108 PCR was performed as previously described. UCB stem cells express Pea3 and Erm in addition to scleraxis (Figure 54). AdMSC and UCB S urvive on V arious M atrices The three dimensional environment of a tissue results in dif ferent mechanical stresses and cell contacts which can signal for specific cell identities. To investigate the effects of various matrices, UCB and AdMSC were cultivated on gelatin coated plasticware, collagen coated beads, or allowed to embed into 30% Ma trigel for 48 hours (Figure 55). Cells were fixed in 4% paraformaldehyde and stained with fluorescein conjugated phalloidin to visualize actin structures and Hoechst 33342 to label nuclei. Stem cells grown on gelatin existed in a monolayer and exhibited morphology typical of fibroblasts and MSC with visible actin filaments. Incorporation into a 30% Matrigel resulted in the formation of colonies with compact structure. Cells show fewer stress fibers but maintain filopodia that extend into the surrounding matrix. Culture on collagen beads results in cells which resemble those on gelatin, albeit with apparently smaller amounts of cytoplasmic volume. No differences in morphology were noted between cells derived from adipose or umbilical cord blood. Culture in M atrigel I ncreases T enocyte Gene E xpression To evaluate the effects of different culture matrices on tenocyte gene expression, AdMSC and UCB were cultured for 48 hours on gelatin, collagen coated beads, or matrigel prior to RNA extraction. Realtime PCR revealed increases in scleraxis mRNA in both AdMSC and UCB when cultured on matrigel ( 39.48 and 6.73 fold, respectively, p<0.0001; Figure 56). Scleraxis transcripts were decreased in AdMSC grown on collagen coated beads (p<0.0001). Culture on matrigel increased tenascin C expression 12.32 fold in AdMSC but not in UCB (p<0.0001)

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109 Fibrobl ast Growth Factors Elicit Differing ERK1/2 R esponses in UCB and AdMSC Not only can FGF stimulation promote mitogenesis in stem cells, but FGF signaling through the MAPK pathway can lead to downstream regulation of scleraxis and tenascin C Thus, the ERK1/2 response was examined in UCB and AdMSC following stimulation by FGF2, FGF4, or FGF5. FGF2 elicited ERK1/2 phosphorylation in both cell types, albeit with differing act ivation kinetics (Figure 57). FGF4 did not activate pERK1/2 in UCB, however was responsible for a slight activation in AdMSC. Further experiments with FGF4 were discontinued due to lack of a MAPK response in UCB. Stimulation with FGF5 elicited a transient response in both AdMSC and UCB with similar kinetics. These results indicated that both UCB and AdMSC are capable of downstream signaling elicited by the fibroblast growth factors. The mitogenic effects of the fibroblast growth factors have been shown i n adipose and bone marrow derived stem cells. To clarify the effect of the fibroblast growth factors on UCB and AdMSC proliferation, cells were cultured for 48 hours in media supplemented with FGF2 or FGF5 in the presence or absence of the MEK inhibitor P D98059 (Figure 58). Cells were pulsed with BrdU prior to fixation and quantification. Similar to bone marrow derived stem cells, AdMSC increased proliferation when cultured in the presence of FGF2. This effect was abrogated by the presence of PD98059. In contrast, supplementation of FGF2 to UCB retarded BrdU incorporation in a MAPK dependent manner. Supplementation of AdMSC with FGF5 inhibited proliferation, contrasting with the increased proliferation of UCB. Inclusion of the MEK inhibitor PD98059 reversed the effects of FGF5 in both cell types. To determine the combined effects of three dimensional culture and stimulation of the fibroblast growth factors on stem cell identity, UCB and AdMSC were cultured on gelatin, collagen beads and matrigel and sti mulated with FGF2 or FGF5 for 48 hours. Real time PCR was used to quantify changes in scx and TnC expression. UCB cultured on matrigel express

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110 higher levels of scx ( p<0.0001; Figure 59A). However, Scx tended to be decreased by supplementation with FGF 2. Levels of TnC mRNA are unaffected by culture on different matrices or the inclusion of FGF2 or FGF5 (Figure 59B). In AdMSC, scx expression was increased by culture in matrigel but decreased by culture on collagen beads ( p<0.0001; Figure 59C). When cultured on gelatin or matrigel, FGF5 supplementation increased TnC transcription in AdMSC (p<0.0001) Inclusion of FGF2 in the culture media of cells grown in matrigel increased TnC transcription but to a lesser exte nt than caused by FGF5 (Figure 59D). Inhibition of the MAPK signaling pathway by PD98059 did not reverse the changes in scleraxis or tenascin C mRNA caused by FGF2 or FGF5 stimulation in adipose or umbilical cord blood derived stem cells (data not shown). Effect of FGF5 Supplementation on Ac tin Structure To determine if FGF5 supplementation resulted in changes in cell morphology and actin structure, UCB were stripped of extracellular signaling molecules using 10 g/ml protamine sulfate for 10 minutes. Cells were placed in serum free media fo r one hour prior to the addition of 10ng/ml rhFGF5 or growth media. Those cells receiving neither growth media nor FGF5 were used as controls. UCB stem cells were fixed in 4% paraformaldehyde at times 0, 10, and 30 minutes of treatment prior to permeabil iz ation with 0.1% TritonX100 in phosphate buffered saline (PBS) supplemented with 5% FBS. Actin filaments were immunostained with fluorescein conjugated phalloidin and Hoechst 33342. Morphology was visualized using a Nikon T200 microscope equipped with e pifluorescence. Images were captured with NIS Elements (Nikon Instruments, Melville, NY) software and compiled with Adobe PhotoShop CS. No appreciable differences were visible in UCB receiving any treatment (Figure 510 ). All cells maintained similar size s. No treatment resulted in the formation of extensive filopodia or

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111 stress fibers. Thus, we conclude that FGF5 supplementation does not have an immediate effect on UCB actin structure. Response of the PI3K Pathway to FGF2 and FGF5 Supplementation To det ermine if FGF2 or FGF5 may be signaling through the phosphoinositol 3 kinase pathway, an alternative to ERK1/2, we examined the expression of phosphorylated and total Akt following stimulation with 10 ng/ml of either FGF2 or FGF5. Akt is a downstream effe ctor of PI3K signaling which requires phosphorylation on threonine 308 and serine 473. Western blots were performed as previously described following treatment with FGF2 or FGF5. No phosphorylated Akt was present at any time point following FGF2 or FGF5 s upplementation (Figure 5 11). Total Akt was maintained at similar levels throughout the experiment. This suggests that FGF2 and FGF5 are not activating the PI3K pathway. In conclusion, we demonstrate that AdMSC and UCB react differently to various matr ices and growth factors. To maintain an immature tenocyte like cell, the appropriate culture conditions for UCB appear to be culture on matrigel in the absence of any FGF supplementation. Culture of adipose derived stem cells on matrigel in culture mediu m supplemented with FGF2 promotes proliferation of an early tenocyte like phenotype. Discussion This work highlights the differences between adipose and umbilical cord blood derived stem cells. Previously, we have shown that equine AdMSC express fewer st em cell markers and possess a more limited ability to differentiate compared to UCB (Reed and Johnson, 2008) AdMSC proliferate more rapidly than UCB regardless of the surface substrate (Reed and Johnson, unpublished data). This suggests a difference in regenerative capabilities as more nave stem cells often have longer population doubling times than more differentia ted cells. Few studies have directly compared the two populations in vitro and to date, none have compared

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112 their in vivo ability to assist regeneration. Adipose derived mononuclear cells were capable of improving tendon architecture but not biomechanical properties in a collagenase induced lesion of the SDFT (Nixon et al. 2008) No studies have been completed at this time identifyi ng the contribution of UCB to tendon injury. Transplantation of bone marrow derived stem cells into tendon lesions results in decreased lesion size and greater tendon density (Crovace et al. 2007; Pacini et al. 2007) Because of the innate expression of both scleraxis and the stem cell markers Oct4, nanog, and Sox2, UCB stem cells may provide a better source of regenerative stimulus than adipose der ived cells. The beneficial effects of stem cells in tendon injury may be due to the population of cells expressing scleraxis. Bone marrow, adipose and umbilical cord blood derived stem cells express this transcription factor prior to any in vitro manipul ation ( (Kuo and Tuan, 2008) Figure 1). In AdMSC and UCB, upregulation of scleraxis occurred in response to culture on matrigel. Culture in a three dimensional gelatin environment also upregulated scx in BMSC (Kuo and Tuan, 2008) Expression of scleraxis precedes that of tenascin c and collagen 1a2 in the developing embryo (Kardon, 1998; Schweitzer et al. 2001) Overexpression of scleraxis increased the transcription of col1a2 in NIH 3T3 fibroblasts (Lejard et al. 2007; Espira et al. 2009) Scleraxis appears to bind to the proximal promoter region of col1a2 as a heterodimer with E47 (Lejard et al. 2007) Expression of Col1a2 increases in AdMSC and UCB grown on matrigel (data not shown). These data suggest that the upregulation of scleraxis is consistent with the induction of an early tendonlike cell which expresses t enascin c and collagen1a2 as it matures. Scleraxis may, in fact, drive the expression of the more mature markers of tendon development. The population of cells that initially express scleraxis may give rise to daughter cells which express scleraxis and f urther differentiate to more mature tenocytic cells under appropriate conditions.

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113 Fibroblast growth factors are required for proper syndetome formation and induction of tenocytic lineage. As such, the response of adipose and UCB derived stem cells to FG Fs was investigated. In this work, we show that AdMSC and UCB respond very differently to stimulation by the FGFs. Interestingly, stimulation of AdMSC and UCB with FGF2 or FGF5 resulted in opposing effects on proliferation but similar ERK1/2 activation kinetics. Changes in proliferation were dependent upon ERK1/2, as inclusion of PD98059 abrogates all differences. These differences are likely due to different cellular contexts and possibly different FGF receptor expression. FGF2 can signal through a number of FGF receptor isoforms, however FGF5 is more limited and can only signal through FGFR1c and FGFR2 (Reviewed in Clements et al. 1993; Eswarakumar et al. 2005) It is possible that the differences in response to the fibroblast growth factors may also occur because of variations in ERK1 and ERK2 ratios, priming the cells toward proliferation or differentiation. AdMSC tended to express lower amounts of ERK1 relative to ERK2 than UCB stem cells. It is tempting to speculate that the ratio of ERK1:ERK2 is related to the stemness of the population and the subsequent ability to respond to external signals. AdMSC may express more ERK2 in order to respond to factors signaling for terminal differentiation. Alternatively, UCB may have higher levels of ERK1 to retain the ability to respond to both proliferation and differentiation signals to allow self renewal of the stem cell population as well as creation of daughter cells primed to differentiate. As might be expect ed, activation of ERK1 was also reduced compared to ERK2 phosphorylation following FGF stimulation in both cell types. This may be a preferential response of the cells to growth factors or simply due to kinase availability. When cultured on matrigel, sc x expression increases drastically in both UCB and AdMSC. Matrigel is composed of a mixture of growth factors (including IGF 1, PDGF, TGF

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114 and extracellular matrix components laminin, collagen IV and entactin, which form a complex three dimensio nal matrix at 37 C. Entactin enables the binding of laminin to collagen IV and the formation of a complex structural matrix. Both AdMSC and UCB embedded into the matrigel and formed colonies with distinct cellular morphology compared to cells on gelatin or collagen coated beads. The differences in cell:cell contact or contact with a complex ECM may be responsible for the upregulation of tenocytic genes. Integrin signaling activated by changes in ECM can result in differentiation of a number of stem cell types. Integrin signaling was activated by culture of hES cells on laminin and was related to an increase in ERK1/2 activation and subsequent decrease in Nanog and SSEA1 expression (Hayashi et al., 2007) Laminin signaling through alpha6/beta1 integrin may direct neural differentiation of hES cells (Ma et al. 2008) Additionally in the kidney alpha3/beta1 integrin signaling in coordination with c Met regulates Wnt expression, which is responsible for epithelial cell survival in the developing kidney (Liu et al., 2009) Activation of integrin mediated RhoA and Rhodependent kinase (ROCK) via cyclic strain resulted in the upregulation of tenascin C in primary skin fibroblasts (Chiquet et al. 2004) While no strain was applied to either AdMSC or UCB, the change in tension from a flat to a three dimensional surface may have been significant enough to activate the integrin signa ling system. The ability of UCB to upregulate scleraxis in a complex environment supports the use of these cells as a therapeutic aid. Tendons are composed primarily of type I collagen as well as other minor collagen and noncollagen components. Initia l tendon injuries are poorly organized but gain structure during the healing process. UCB implanted into tendon injuries should be capable of attachment to the tendon matrix and production of collagen and other matrix proteins. The additional strain that occurs with movement may provide additional direction for UCB to produce the required matrix proteins for proper healing.

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115 In conclusion, we have further delineated the differences between adipose and umbilical cord blood derived stem cells. While both c ell types upregulate scleraxis expression in response to culture on matrigel, they respond very differently to stimulation with fibroblast growth factors. We have established culture conditions appropriate to induce an early tenocytic lineage. Further work should clarify the mechanisms behind such changes.

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116 Table 51. Real time PCR p rimers Primer Sequence Expected Product Size, bp Standard Curve Slope Primer Efficiency, % Col1A2 1 F GCACATGCCGTGACTTGAGA R CATCCATAGTGCATCCTTGATTAGG 127 3. 39 97.24 TnC 1 F GGGCGGCCTGGAAATG R CAGGCTCTAACTCCTGGATGATG 70 3.34 99.25 ScxB 1 F TCTGCCTCAGCAACCAGAGA R TCCGAATCGCCGTCTTTC 59 3.35 98.84 Scx 2 F AGGACCGCGACAGAAAGAC R CAGCACGTAGTGACCAGAAGAA 261 n/a n/a 18S 1 F GTAACCCGTTGAACCCCATT R C CATCCAATCGGTAGTAGCG 151 3.35 98.84 Pea3 2 F GTGGCAGTTTCTGGTGGCCCTG R GACTGGCCGGTCAAACTCAGCC n/a n/a Erm 2 F GAGAGACTGGAAGGCAAAGTC R CCCAGCCACCTTCTGCATGATGC n/a n/a 1 (Taylor et al. 2009) 2 Used for endpoint PCR only.

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117 Table 5 2. Cycle t hreshold r anges Cell Type Transcript Mean SEM Min Max Median UCB 18S 13.72 0.06 13.15 15.59 13.59 UCB Scx 30.72 0.21 27.13 33.77 30.71 UCB TnC 21.89 0.29 18.67 28.20 21.27 AdMSC 18S 13.59 0.06 13.13 14.18 13.57 AdM SC Scx 31.19 0.55 27.10 34.31 31.54 AdMSC TnC 22.33 0.64 18.44 25.65 23.10

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118 Figure 5 1. AdMSC and UCB express markers of tenocytic cells. Total RNA was isolated from AdMSC and UCB prior to PCR with gene specific primers. Scleraxis and Tenasin C mRNA are present in AdMSC and UCB stem cells. Scx, scleraxis; TnC, Tenascin C

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119 Figure 5 2. Mouse scleraxis promoter with putative transcription factor binding sites. The minimal promoter is shown with putative transcription factor binding sites highlighted in bold.

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120 Table 5 3. Putative t ranscription fa ctor binding s ites on the m ouse scleraxis m inimal promoter1. Factor Beg 2 Sns 3 Len 4 Sequence L a 5 Lpv 6 Sp1 288 N 11 GtGGGAGGAGC 20 0 EGR2 286 R 10 GCGGGGGcGG 18 0 Sp1 283 R 10 GGgGCGGGGG 18 0 Sp1 330 N 10 GCCCCACcCC 18 0 Sp1 289 N 10 GGGGcGGAGC 18 0 Sp1 289 N 10 tGGGAGGAGC 18 0 Sp1 37 N 10 GGGgCAGGGC 18 0 Sp1 270 R 9 GAGGCGGAG 18 0 Sp1 289 R 10 GGGGcGGAGC 18 0 POU1F1a 92 R 10 TTGATTaATT 18 0 GAGA factor 169 N 16 CGCTCNNNNNNGAgAG 18 0 Sp1 270 R 10 GAGGCGGAGc 18 0 Sp1 17 N 10 AGGGcGTGGC 18 0 Sp1 37 R 10 GGGgCAGGGC 18 0 CACCC binding factor 16 R 10 CAGGGTGgGG 18 0 Sn 222 R 11 RAcAGGTGYAC 18 0 Sp1 288 N 10 GGGGGAGGgG 18 0 HNF 3B 87 N 12 VAWTrTTKRYTY 16.58 0 AP 2alpha 278 R 8 GGCCAGGC 16 0 AP 2alpha 24 R 8 GGCCAGGC 16 0 TEF 2 18 N 8 GGGTGTGG 16 0 HNF 4alpha 53 R 12 RTGRMCYTWGcM 16 0 AREB6 223 R 8 AAAGGTGC 16 0 AP 2 284 R 7 GCGCGGG 14 0 AP 2alpha 284 R 10 SSSNKGGGGA 14 0 AP 1 243 N 7 TGA GTAA 14 0 PuF 121 N 7 GGGTGGG 14 0 AP 1 c Jun 7 N 7 AGAGTCA 14 0 CP2 170 N 11 GCNMNANCMAG 14 0 Nkx6 1 210 N 7 CTATTAA 14 0 MEF 2 210 R 10 YTATTtWWAR 14 0 myogenin 26 R 7 CCAGGCA 14 0 En 1 114 N 7 GTAGAAT 14 0 AP 1 c Fos c Jun 7 R 7 AGAGTC A 14 0 GATA 1 Sp1 121 R 7 GGGTGGG 14 0 GCN4 299 R 6 TGACTG 12 0 ZF5 283 N 6 GGCGCG 12 0 CACCC binding factor 121 N 6 GGGTGG 12 0 FACB 30 R 17 GCANNNNNNNNNNNGGC 12 0 POU3F2 95 R 7 ATTWATK 12 0 GR 54 N 6 TGAACT 12 0 USF1 HES 1 234 N 6 CACGAG 12 0 Sp1 107 R 6 CTGCCC 12 0 RAP1 317 R 10 TGNNNGGNTG 12 0

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121 Table 5 3 Continued. Factor Beg 2 Sns 3 Len 4 Sequence L a 5 Lpv 6 Sp1 329 R 6 GGCCCC 12 0 TTF 1 109 N 9 GCNCTNNAG 12 0 GCN4 8 R 6 GAGTCA 12 0 GCN4 TGA1a 8 N 6 GAGTCA 12 0 NFAT 1 A96 220 N 6 GGAAAA 12 0 c Myb 166 R 6 AAACGC 12 0 Zeste 31 N 6 C ACTCA 12 0 FACB 157 N 15 GCANNNNNNNNNCGC 12 0 AP 1 243 N 7 TGASTMA 12 0 c Myb 55 N 6 GAACTT 12 0 Sp1 43 N 6 GGGCAG 12 0 GCN4 31 R 6 CACTCA 12 0 abaA 116 R 6 AGAATG 12 0 GCN4 86 N 6 TGATTC 12 0 AP 4 E12 46 R 6 CAGCTG 12 0 TTF 1 195 N 9 GCNCTNNAG 12 0 CAC binding protein 158 R 6 CACCCC 12 0 Sp1 121 R 6 GGGTGG 12 0 Sp1 122 N 6 GGTGGG 12 0 Sp1 270 N 9 KRGGCKRRK 12 0 AP 4 E12 46 N 6 CAGCTG 12 0 TBP 306 N 6 TATAAA 12 0 Sp1 192 N 6 GGGGCC 12 0 GR 103 N 6 TCTTCT 12 0 Sp1 2 71 N 6 AGGCGG 12 0 Zeste 334 N 6 CACTCC 12 0 NFAT 1 220 R 6 GGAAAA 12 0 GCN4 244 R 6 GAGTAA 12 0 abaA 116 N 6 AGAATG 12 0 MBF I 228 N 7 TGCRCRC 12 0 EGR 2 286 N 10 GCGGGGGAGG 17 0.0019 CAC BF 17 R 9 AGGGTGTGG 15.18 0.0025 v Jun 8 R 12 GAG TCAGACAGG 15.69 0.0037 EGR 1 286 R 9 GCGGGGGAG 14.64 0.0051 V$GC_01 287 N 14 CGGGGGAGGAGCTG 14.14 0.0065 HAP3 168 N 16 ACGCTCCAACCAGAAA 14.29 0.0092 HNF 4 53 N 12 GTGAACTTAGGC 12.81 0.0093 TEF2 18 N 8 GGGTGTGG 15.72 0.01 SBF 63 R 11 GCATGCCAGG A 14.1 0.01 1Identified using Transcription Element Search System ( http:// www.cbil.upenn.edu/cgi bin/tess) 2 Beginning nucleotide 3 Sense, N = normal, R = reverse 4 Length of motif, in base pairs 5 Log likelihood score, higher is stronger 6 Approximate pvalue for La score

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122 Figure 5 3. Scleraxis promoter activity is not increased by growth factor supplementation in UCB stem cells. C2C12 mesenchymal cells and UCB stem cells were transiently transfected with Scx luc and supplemented with 50 ng/ml BMP 6, 10 ng/ml FGF4 or 10 ng/ml FGF5 for 24 hours. Luciferase activity was measured from cell lysates. Relative luciferase units were calculated by dividing luciferase activity by renilla expression.

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123 Figure 5 4. UCB stem cells express Erm Pe a3, and Scleraxis RT PCR was performed on UCB stem cells with gene specific primers as shown. 18S was included as an internal control. Scx = scleraxis.

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124 30% Matrigel Collagen Bead Gelatin UCB AdMSC Figure 5 5. UCB and AdMSC attach to various culture surfaces. UCB and AdMSC were cultu red on gelatin coated plasticware, 30% Matrigel, or on collagen coated beads for 48 hours prior to fixation. Actin structures were stained with fluorescein conjugated phalloidin and nuclei were identified with Hoechst 33342 dye. Immunofluorescence was vi sualized using a TCS SP5 Laser Scanning Confocal Microscope running Leica LAS AF. Scale bar = 10 m

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125 Figure 5 6. Culture in matrigel increases tenocyte gene expression. AdMSC and UCB stem cells were cultured on gelatin coated plasticware, collagen co ated beads, or 30% matrigel for 48 hours prior to RNA isolation and real time PCR with gene specific primers. Culture on matrigel increased scleraxis expression in AdMSC and UCB. Tenascin C mRNA was also increased by culture on matrigel. Asterisk indicates p<0.05.

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126 Figure 5 7. UCB and AdMSC respond uniquely to FGF stimulation. UCB and AdMSC were stimulated with 10 ng/ml FGF2, FGF4, or FGF5 for the times shown. Protein extracts were probed with antibodies specific to phosphorylated and total E RK1/2. Tubulin antibodies were used as a loading control.

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127 Figure 5 8. Fibroblast growth factors stimulate proliferation of AdMSC and UCB stem cells in a MAPK dependent manner. AdMSC and UCB stem cells were cultured in low serum media supplemented with 10 ng/ml FGF2 or FGF5 in the presence or absence of PD98059. Prior to fixation, cells were pulsed with BrdU. FGF2 inhibited proliferation of UCB but stimulated AdMSC division. FGF5 increased proliferation of UCB stem cells.

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128 Figure 5 9. Culture conditions affect tenocyte gene expression in AdMSC and UCB. AdMSC and UCB stem cells were cultured for 48 hours in low serum media containing 10 ng/ml FGF2 or FGF5. Total RNA was isolated and subjected to real time PCR with gene specific primers for scleraxis and tenascin C Matrigel increased expression of scleraxis in UCB stem cells (A). There was no effect of matrix or FGF stimulation on TnC in UCB (B). Scleraxis expression in AdMSC was increased by culture on matrigel (C). Matrigel increased Tenascin C mRNA in AdMSC (D). This was further increased by supplementation with FGF2 or FGF5.

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129 Figure 5 10. FGF5 supplementation does not affect UCB actin structure. UCB stem cells were serum starved for one hour prior to trea tment with FGF5 or growth media. Cells were fixed at 0, 10 and 30 minutes prior to visualizing actin structures with fluorescein conjugated phalloidin. Nuclei were stained with Hoechst 33342. SF = serum free media, FGF5 = FGF5 containing media, GM = growth media Scale bar = 10 m

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130 Figure 5 11. FGF2 and FGF5 do no activate Akt in UCB (A) or AdMSC (B). Cells were treated with 10 ng/ml of FGF2 or FGF5 and lysed at the time points shown. Blots were probed for phosphorylated and total Akt.

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131 CHAPTER 6 SU MMARY AND CONCLUSIONS A hierarchy exists such that embryonic stem cells retain the most plasticity while bone marrow and adult stem cells are much more limited in their potential. Umbilical cord blood derived stem cells retain characteristics of nave ES cells in that they express Oct4 nanog, Sox2, Klf4 and c myc transcripts. Surface markers Tra1 60, Tra181, SSEA1 and minor amounts of SSEA4 are also present in UCB. This population of equine stem cells can be isolated using conventional human protocols a nd reagents. P roper culture conditions result in cells that can be maintained in a proliferative state for up to fifteen passages or approximately 22 population doublings. When cultured in appropriate media, UCB differentiate into proteoglcyan expressing chondrocytes or calcium producing osteocytes. UCB can also enter the hepatic pathway and form albumin producing hepatocytes. However, these cells possess limited ability to form adipocytes and myocytes. In comparison, equine adipose derived mesenchymal s tem cells express fewer markers of stemness, lacking SSEA1, SSEA4, nanog, Sox2, and Klf4 AdMSC lack the ability to differentiate into the immature myocytes or adipocytes formed by UCB. Early cultures of UCB possess small colonies reminiscent of ES coloni es that are lost with time in culture. Alternate culture conditions were assessed to determine more optimal conditions to maintain stem cell proliferation as well as maintenance of stemness. Culture in a combination of thrombopoietin, Flt3, and c kit or media conditioned by confluent UCB resulted in morphology reminiscent of replicative senescence. Growth in conventional growth media (GM) or growth media supplemented with bFGF (GM+FGF) supported cell prolife ration and morphology typical of MSC. Population doubling times were shorter in cells that did not receive FGF supplementation. Further culture on a protein matrix (collagen, fibronectin, or laminin) increased proliferation rates in cells cultured in GM or GM+FGF. Expression of stem cell

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132 markers nanog and Sox2 were gradually lost with time in culture, however Oct4 expression remained. Unmanipulated UCB and AdMSC express the bHLH transcription factor scleraxis, which is crucial to the development of flexor tendons. Culture on the complex protein matrix M atrigel resulted in the upregulation of scleraxis in both AdMSC and UCB stem cells. In M atrigel cells form tight colonies while they retain their fibroblastlike morphology when grown on gelatin or collagen coated surfaces. Supplementation of FGF2 or F GF5 resulted in changes in proliferation of both cell types, but had limited effects on scleraxis or tenascin c mRNA expression compared to culture on matrigel alone. Overall, this work highlights the potential of equine umbilical cord blood derived stem cells as not only a therapeutic aid for horses but as a model system for human medicine. The cells appear to be more nave than either bone marrow or adipose derived stem cells yet do not retain the tumorigenicity of embryonic stem cells. The ability to form immature myocytes as well as hepatocytes suggests that UCB have greater plasticity than other adult stem cells This m ay be a result of a less restrictive epigenetic status than adipose derived stem cells. This open genome structure likely allows for greater plasticity and naivet. The apparent inability of UCB and AdMSC to efficiently differentiate into adipose tissue was surprising but reflects other work in the literature. In truth, the inefficiency of adipocyte formation is a positive aspect fro m a clinical aspect as these cells may be less likely to precociously differentiate into that cell type if used in vivo While initial populations express all of the common markers of a nave stem cell (Oct4, nanog, Sox2, and Klf4), the majority of these markers are lost with time in culture. This may reflect one of several possibilities: (1) UCB stem cells may be differentiating to a more mature

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133 cell type, (2) a small population of nave stem cells express these markers and this cell type is essentially diluted out in culture due to slower proliferation, (3) a small population of nave stem cells exist but are not maintained due to inappropriate culture conditions. High density, early passage cultures contain a small number of dense colonies with morphol ogy reminiscent of ES cell colonies. These colonies may contain cells that are more nave than those that grow in monolayer culture. It is tempting to speculate that the cells which form these colonies are those that contain the stem cell markers found in early cultures, but with passage are greatly outnumbered by other cell types. This is further supported by the notion that generally more nave stem cells proliferate slowly and thus would not be expected to create as many daughte r cells as the cells whi ch grow on a monolayer. Thus, the colony forming cells may be the true stem cells while those which grow in a monolayer are more limited precursor cells. Further work should evaluate the subpopulations within UCB stem cells to identify cells that may be m ore or less useful in various therapeutic settings or as a model for human medicine The differential response of AdMSC and UCB to stimulation by fibroblast growth factors further highlights the differences between the two cell types. It is interesting to note that while both respond to FGF2 and FGF5 stimulation by phosphorylating ERK1/2 and activating the MAPK pathway, there are some significant differences in the type of response. In general, AdMSC appear to have less total ERK1 than UCB, resulting in a different ratio of the two kinases. This may be a reflection of cells poised either for terminal differentiation or for proliferation prior to differentiation. In other cell types, strong phosphorylation of ERK2 occurs in response to differentiation sig nals, while ERK1 responses are associated with proliferation. The response of ERK1 to stimulation by FGFs was weak in both cell types; ERK2 phosphorylation in response to FGF was predominant. However, UCB retain higher amounts of

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134 ERK1 than AdMSC which may allow them to better respond to other mitogenic factors. This may suggest that a more nave stem cell may have a higher ratio of ERK1:ERK2 than more differentiated cell types. These cells may be poised to respond to extracellular factors differently than AdMSC. The expression of low levels of scleraxis in immature cells is not surprising, as many stem cells exist in a state poised for differentiation and transcribe low levels of mRNAs required for that transformation. Not only is scleraxis upregulated in cells grown on M atrigel, but the morphology of the cells changes drastically. The response of UCB to M atrigel is exciting and presents many possibilities for future research. The changes in gene transcription may or may not be directly related to the change in morphology. Integrin signaling and interaction with other ECM components likely result in changes that may allow differentiation. The growth factor concentrations and/or combinations present in M atrigel may signal for transcriptional changes independent of the structure of the extracellular matrix. Alternatively, the changes may result from changes in tensional stress, ligand:receptor interactions with the extracellular matrix, or differences in cell:cell contact. Likely, a combination of stimul i allow for the formation of a cluster of cells similar to tendon precursor cells. Complete differentiation is not recorded at this stage, likely for a variety of reasons. The combination of growth factors that initiates early tendon precursor development is likely not sufficient for complete maturation. Another combination or different ratios of growth factors could help complete the transition. In vivo tendon cells are subjected to a high degree of strain on a daily basis as a result of movement. Man y cell types require cyclic or tensional stress to completely differentiate and it would be extremely surprising were it not also true for tendon cells. Adding strain to UCB cultured in

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135 Matrigel may stimulate the production and secretion of matrix protein s required for tendon repair and/or maintenance. Equine UCB provide more than a potential therapeutic aid for injuries in the horse. The horse provides an excellent model of athletic tendon injury in the human, as injuries to the SDFT correlate extremely w ell to injuries of the Achilles tendon, a frequent spot of injury in human athletes. Horses are treated as athletes and undergo similar training programs as human athletes. Overtraining and overuse injuries result in setbacks in training and competition in both species. Equine umbilical cord blood derived stem cells are readily available and possess characteristics of human UCB stem cells, making them an attractive choice for a model of human medicine. The horse provides a useful model of injury and can be used to determine the usefulness of UCB in treating tendon and other musculoskeletal injuries.

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136 APPENDIX A SUPPLEMENTARY DATA Mouse E mbryonic S tem C ell C ulture and D ifferentiation Mouse embryonic stem cells (mES) are commonly used to study the developmental processes that occur in an ordered manner in the developing embryo. Under proper conditions, mES cells can be maintained in a proliferative, pluripotent state. However, mES cells can also undergo spontaneous or directed differentiation to form cell s from all three germ layers. In culture medium containing leukemia inhibitory factor ( LIF ) mES cells maintain a pluripotent phenotype. Cells grow in colonies with li ght refractile ed ges (Figure A 1 A). Individual cells can rarely be distinguished from the colony. As colonies grow in an unrestricted manner (i.e. without fresh media or appropriate passaging), the cells will begin differentiating into a wide variety of cell type. Initially, colonies develop a ring of epithelial cells that surround the col ony (Figure A 1 B). Cells remaining in the inner portion during this stage may retain greater levels of pluripotency. However, with continuing differentiation, cells migrate away from the colony, forming a monolayer with cells of a wide v ariety of phenoty pes (Figure A 1C). Formation of embroid bodies (EBs) allows for a more synchronous protocol for differentiation. This process allows an individual cell to form a colony which can then be treated and compared with other colonies (also from a single cell) Nave mES colonies are washed off the tissue culture plate and placed in suspension culture in media lacking LIF. The EBs cannot easily attach and are forced to grow into larger spherical colonies. Individual EBs can then be placed back onto tissue culture dishes and allowed to fully differentiate in media lacking LIF. Spontaneous differentiation results in the formation of a wide variety of cell types, including neural, myogenic, and hepa togenic cells (Figure A 2).

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137 Alternative UCB Differentiation Pro tocols Following is a brief description of differentiation protocols that were tested on UCB stem cells but were not reported in earlier work due to the lack of success. Myogenic Differentiation UCB stem cells were plated on gelatin coated plasticware an d cultured in DMEM containing 10% FBS and 10 M azacytidine for 24 hours. The media was subsequently replaced with low glucose DMEM containing 5% FBS. Cells were continuously cultured in this media for up to three weeks. No evidence of myogenic differenti ation was found. RT PCR for the myogenic regulatory factors MyoD and Myf5 was negative. No immunostaining of desmin or myosin heavy chain could be detected. Neural Differentiation To initiate neural differentiation, three protocols were attempted. The fi rst two protocols contain the same basic components but included either low or high amounts of FBS (2% and 10%, respectively). This medium was based on MEM and contained 10 ng/ml PDGF, 10 ng/ml bFGF and 10 ng/ml EGF in addition to serum. The third media was DMEM supplemented with 15% FBS, 20 ng/ml bFGF, 50 ng/ml neural growth factor (NGF) and 0.5 mM IBMX. Cells were maintained in these media for up to three weeks. Following culture time, cells were immunostained for nestin, glial fibrillary acidic protein (GFAP), and beta 3 tubulin. No immunoreactivity was apparent at any time. Adipogenic Differentiation Three protocols for adipogenic differentiation w ere attempted during this study. The previously described protocol gave the greatest level of success and is thus the protocol of choice for UCB. However, it should be noted that the efficiency of differentiation remained extremely low. A low glucose DM EM based media containing 10% FBS, 1 M dexamethasone, 0.5 mM

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138 IBMX, 100 M indomethacin, and 10 ng/ml insulin was used to culture cells for up to three weeks. The second protocol used dexamethasone, 0.5mM IBMX, 50 M i ndomethacin, and 5 ng/ml insulin. As rabbit serum has been shown to increase the efficiency of adipogenic differentiation, it was substituted for FBS in all media as an additional set of media concoctions. Neither media presented here (whether supplement ed with FBS or rabbit serum) produced cells that stained positive for Oil Red O or contained lipid vacuoles. Only limited success was noted with the previously described protocol, which was not enhanced by the inclusion of rabbit serum. Quantification of Oct4, nanog, and Sox2 Across Time in Culture To quantify expression of Oct4, nanog, and Sox2 in UCB stem cells across time in culture, real time PCR was performed. Multiple primer sets were tested by end point PCR for Oct4. Those primer sets that produced a single band of the appropriate size were then tested by real time PCR, using a standard curve method. While a number of primer sets provided a product after end point PCR, real time PCR results were inconsistent. The dissociation curves for Oct4 had multiple peaks indicating the presence of multiple products Dissociation curves for the positive control samples (mES) contained a single peak. The single peak found in curves from mES samples overlapped one of the peaks found in the dissociation curve f or UCB samples, indicating the presence of the correct transcript. I t is important to note that the magnitude of the peak was much higher in mES samples than UCB samples. Standard curves were performed to ensure that the values obtained were within a li near range. The curves obtained for the internal control (18S) had a slope of 3.325 and R2 value of 0.994 indicating nearly 100% primer efficiency and an appropriate standard curve on which to base quantifications. However, Oct4, nanog and Sox2 lacked st andard curves that were

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139 consistent across multiple plates. Moreover, these curves had slopes indicating less than 75% primer efficiency. Multiple primer sets were tested to ensure the results obtained were not simply due to inefficient primers. All prime rs resulted in inefficient standard curves and poor dissociation curves with multiple peaks Increasing the annealing temperature also had no effect on the multiple peaks of the dissociation curve. Importantly, reactions that lacked reverse transcriptas e in the cDNA amplification process contained no product in either end point or real time PCR. It should also be noted that internal controls amplified a single band in end point that reflects the single peak in the dissociation curve in UCB as well as mE S. Further work on these transcripts was discontinued, as they were not the primary focus of the project.

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140 Figure A 1. Stages of mES colony differentiation. Nave mES colonies maintain a compact shape with no discernable individual cells (A day one ). More differentiated colonies often have a ring of epithelial cells surrounding a more densely packed core (B day two ). Fully differentiating cells adopt the morphology of their mature cell type and are commonly found in monolayers on the culture surface (C day 5). Scale bar = 10 m

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141 Figure A 2. mES embroid bodies differentiate into a variety of cell types. mES stem cells were allowed to spontaneously differentiate. Following differentiation cells were fixed in 4% paraformal dehyde and immunostained for myosin heavy chain (A), nestin (B), and cytokeratin 18 (C). Hoechst 33342 was used to visualize nuclei.

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142 APPENDIX B TABLE OF PRIMER SEQUENCES Table B 1. Primer sequences and sources. Primer Sequence Size, bp Source Co l1A2 F GCACATGCCGTGACTTGAGA R CATCCATAGTGCATCCTTGATTAGG 127 Taylor et al., 2009 TnC F GGGCGGCCTGGAAATG R CAGGCTCTAACTCCTGGATGATG 70 Taylor et al., 2009 ScxB F TCTGCCTCAGCAACCAGAGA R TCCGAATCGCCGTCTTTC 59 Taylor et al., 2009 Scx F AGGACCGCGACAGAAAGAC R CAGCACGTAGTGACCAGAAGAA 26 1 NM_001105150.1 18S F GTAACCCGTTGAACCCCATT R CCATCCAATCGGTAGTAGCG 151 Taylor et al., 2009 Pea3 F GTGGCAGTTTCTGGTGGCCCTG R GACTGGCCGGTCAAACTCAGCC 315 XM_001917508.1 Erm F GAGAGACTGGAAGGCAAAGTC R CCCAGCCACCTTCTGCATGATGC 293 XM_001499099.1 GAPDH F GATTCCACCCATGGCAAGTTCCATGGCAC R GCATCGAAGGTGGAAGAGTGGGTGTCACT 688 XM_001496020 Col2A1 F CA GCTATGGAGATGACAACCTGGC R CGTGCAGCCATCCTTCAGGACAG 240 NM_001081764.1 Sox9 F GCTCCCAGCCCCACCATGTCCG R CGCCTGCGCCCACACCATGAAG 293 XM_001498424.2 Osteonectin F CCCATCAATGGGGTGCTGGTCC R GTGAAAAAGATGCACGAGAATGAG 149 NM_001143953.1 RunX2 F CGTGCTGC CATTCGAGGTGGTGG R CCTCAGAACTGGGCCCTTTTTCAG 350 XM_001502519.2 Albumin F AACTCTTCGTGCAACCTACGGTGA R AATTTCTGGCTCAGGCGAGCTACT 431 NM_001082503.1 Cytokeratin 18 F GGATGCCCCCAAATCTCAGGACC R GGGCCAGCTCAGACTCCAGGTGC 340 XM_001490377.1 GAPDH, real time F GAGATCCCGCCAACATC R CTGACAATCTTCAGGGAATTGTC 207 XM_001496020.1 Oct4 F GCTGCAGAAGTGGGTGGAGGAAGC R GCCTGGGGTACCAAAATGGGGCCC 363 XM_001490108.2 Nanog F GTCTCTCCTCTGCCTTCCTCCATGG R CCTGTTTGTAGCTAAGGTTCAGGATG 267 XM_001498808.1 Sox2 F AACGGCAGCTACAGCATGA R TGGAGTGGGAGGAAGAGGTA 282 NM_001143799.1 Klf4 F TGGGCAAGTTTGTGTTGAAG R TGACAGTCCCTGTTGCTCAG 336 XM_001492956.2 c myc F GACGGTAGCTCGCCCAAG R ACCCCGATTCTGACCTTTTG 240 XM_001497991.2

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143 Table B 1 Continued. Primer Sequence Size, bp Source Jagged 1 F GCCTGGTGACAGCCTTCTAC R GGGGCTTCTCCTCTCTGTCT 305 XM_001495238.2 Jagged 2 F CATGATCAACCCCGAGGAC R CGTACTGGTCGCAGGTGTAG 169 XM_001494763.2 Notch 1 F GAGGACCTGGAGACCAAGAAGGTTC R AGATGAAGTCGGAGATGACGGC 297 XM_001498582.2 Notch 2 F GCAGGAGCAGGAGGTGATAG R GCGTTTCTTGGACTCTCC AG 188 NM_010928.2 Notch 3 F GTCCAGAGGCCAAGAGACTG R CAGAAGGAGGCCAGCATAAG 219 NM_008716.2 Dll 1 F ACCTTCTTTCGCGTATGCCTCAAG R AGAGTCTGTATGGAGGGCTTC 221 NM_007865.3 Dll 4 F CGAGAGCAGGGAAGCCATGA R CCTGCCTTATACCTCTGTGG 378 NM_019454.2

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171 BIOGRAPHICAL SKETCH Sarah Reed was born in Bellefonte, Pennsylvania, to Lisa and Howard Grove, Jr. She grew up riding and showing Quarter Horses and was a member of 4H and the American Quarter Horse Youth Association. Sarah gr aduated as valedictorian from Bellefonte Area High School in Bellefonte, Pennsylvania in 2000. Following high school, Sarah pursued a bachelors degree in equine science at Delaware Valley College where she graduated summa cum laude in December 2003. Whil e at DVC, Sarah was involved in the Equine Science Organization and the agricultural honor society, Delta Tau Alpha. Following graduation, she worked as a laboratory technician for Dr. Sally E. Johnson at the Pennsylvania State University. In July 2004, S arah married Jared Reed. Upon Dr. Johnsons move to the University of Florida, Sarah enrolled as a masters student and completed her degree in Dr. Johnsons laboratory. The title of her masters thesis was Identification of Differentially Expressed Prot eins as a Result of Raf Kinase Activity Sarah continued her education with Dr. Johnson, pursuing her doctoral degree in the Animal Molecular and Cellular Biology program. While working on her degree, Sarah stayed involved with the horse industry by working at Sundaram Farms in Newberry, Florida and taking show jumping lessons from Beth Stelzleni and Ella Rukin. Sarah currently resides in Alachua, Florida, with her husband, Jared, and their two dogs, Annie and Bella. She and Jared are active members of G race United Methodist Church at Fort Clarke. Sarah enjoys photography, horseback riding, and reading fiction in her spare time.