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NEUROSPHERES AND MULTIPOTENT ASTROCYTIC STEM CELLS: NEURAL
PROGENITOR CELLS RATHER THAN NEURAL STEM CELLS
GREGORY PAUL MARSHALL, II
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
Gregory Paul Marshall, II
To my beautiful wife Kathleen, whose steadfast love and support made all of this
I would like to thank Ed Scott, Dennis Steindler, and Eric Laywell for all of their
guidance throughout my graduate career; my wonderful family for their love and support;
my fellow lab rats for always being there for me; and the Gainesville Rugby Club for
providing a welcoming diversion from my otherwise hectic life.
TABLE OF CONTENTS
A C K N O W L E D G M E N T S ................................................................................................. iv
LIST OF FIGURE S ............................. ........ ........ ........... ................ viii
A B ST R A C T .......... ..... ...................................................................................... x
1 INTRODUCTION TO ADULT MURINE STEM CELLS.............................. .... 1
H em atop oietic Stem C ells ................................................................. ..................... 1
H em atopoiesis ........................ ............. .......... ........... ............... .
Isolation of the Adult Hematopoietic Stem Cell ................................................3
D term inning Functional Engraftm ent ...................................................................4
Irradiation of the Host Bone Marrow, the Niche of the HSC.............................6
Plasticity of the H em atopoietic Stem Cell.................................... .....................7
Sum m ary ...................................................................................... .. ................... 9
N eural Stem C ells................................... ... ............................. 9
Neurogenesis and the Embryonic Neural Stem Cell ..........................................9
The A dult N eural Stem C ell ......................................... ............ ................... .10
Subependymal-Zone Neural Stem Cells ..... ..............................11
M ultipotent A strocytic Stem Cells ............................ ................................... 14
Potential Identifying M arkers of the N SC ..........................................................15
Transplantation of In Vitro Expanded NS and MASC ........................................16
Adult N S and M ASC: True Stem Cells? ........................................ ...............17
Effects of Radiation on Adult Neurogenesis.....................................................18
S u m m a ry ....................................................................................1 9
2 M ATERIALS AND M ETHOD S ........................................... ........................ 21
R agents ...........................................................................................2 1
Avertin M house Anesthetic Solution ....................................... ............... 21
5-brom o-2'-deoxy-uridine (BrdU) Solution...................................................21
Epidermal Growth Factor (EGF) Stock Solution (1000x) ................................22
Basic Fibroblast Growth Factor (bFGF) Stock Solution (1000x) .....................23
Immunocytochemistry Hybridization Buffer ............................................... 23
Neural Stem Cell (N SC) Culture M edia................................... ............... 23
4% Paraformaldehyde (100 mL) ............................ ....................... 24
Puromycin Stock Solution (1 m g/mL) ..................................... .................24
A n tib o d y L ist ................................................... ................. ................ 2 5
Primary Antibodies............. ............................ 25
Secondary Antibodies............. .............................. 25
M e th o d s ..............................................................................2 5
C ell C u ltu re ...............................................................2 5
Isolation and culture of neurospheres ............... .....................25
Isolation and culture of multipotent astrocytic stem cells............................26
Selection of gfp+ multipotent astrocytic stem cells by puromycin
selection n ..................................................................... 2 7
Transplantation and A analysis ........................................ ....... .................. 27
Transplantation of gfp+ MASC and neurospheres into the lateral
ventricles of adult C57BL/6 mice ............ ........... ..................27
Transplantation of gfp+ MASC and neurospheres into the lateral
ventricles of neonatal C57BL/6 mice ...............................................27
Tissue im m unohistochem istry................................................................... 28
Analysis of engrafted gfp+ MASC and neurospheres into the brains of
C 57B L /6 m ice............. .............. ....... ............ ...................... ........ 29
Analysis of engrafted gfp+ MASC into the olfactory bulb of C57BL/6
m ic e ..................... ..... ... ........ ................. ............... 2 9
Immunohistochemical analysis of neurospheres and MASCs ..................29
R radiation Studies.............. ........ .......................................... ............. 30
Irradiation and bone marrow reconstitution ............ .............................. 30
Identification of proliferative cells by BrdU labeling ...............................31
Blind analysis of the effects of lethal and sublethal irradiation on
neurosphere yield ............ .................................... ............ .... ..... 32
3 LACK OF EVIDENCE FOR THE CLASSIFICATION OF NEUROSPHERES
AND MULTIPOTENT ASTROCYTIC STEM CELLS AS TRUE STEM CELLS .34
Introduction .................. ........ ........................................................ .. 34
Inability of Gfp+ Neural Stem Cells to Survive Re-isolation Following
Transplantation into the Lateral Ventricles of C57BL/6 Mice..............................37
Gfp+ Neurospheres are not Generated from the Brains of Adult C57BL/6
Mice Transplanted with Gfp+ Neurospheres......................................37
Gfp+ Neurospheres are not Generated from the Brains of Neonatal C57BL/6
Mice Transplanted with Gfp+ Neurospheres.............. ......................38
Gfp+ Multipotent Astrocytic Stem Cells are not Generated from the Brains of
Neonatal C57BL/6 Mice Transplanted with Gfp+ Multipotent Astrocytic
Stem C ells .................. ................................ ...... ....... ... .. ................... .4 1
MASC Isolated from the Brains of C57BL/6 Neonatal Mice Transplanted
with Gfp MASC do not Survive Puromycin Treatment..............................43
Long Term Engraftm ent ......................................................... .............. 46
4 IONIZING RADIATION ENHANCES THE ENGRAFTMENT OF
TRANSPLANTED IN VITRO DERIVED NEURAL STEM CELLS..................... 49
In tro d u ctio n ................... .. ... .. ..... ....... ..... ... ............................... .. ............... 4 9
Effects of Lethal Irradiation on Subependymal Zone Neurogenesis..........................52
Lethal Irradiation Severely Depletes Migrating Neuroblasts in the RMS ..........52
Analysis and Quantification ofNeuroblast Depletion in the Subventricular
Zone Following Lethal Irradiation, as Determined by BrdU Incorporation ....54
Diminished Neurosphere Yield from Lethally Irradiated Mice ........................56
Lethal Irradiation Attenuates Engraftment of Transplanted Gfp+ Multipotent
A strocytic Stem C ells ........................ ...................... ............... ... 59
Effects of Sub-Lethal Irradiation on Subependymal Zone Neurogenesis ..................62
Sublethal Irradiation Results in a Transient Decrease in the Number of
Mitotic Subependymal Zone Neuroblasts............................ ............... 62
Diminished Neurosphere Yield from Sublethally Irradiated Mice ...................63
Sublethal Irradiation Enhances Engraftment of Transplanted Gfp+
M ultipotent A strocytic Stem Cells........................................ ............... 63
5 DISCUSSION AND CONCLUSIONS ........................................... ............... 69
L IST O F R E FE R E N C E S ............................................................................. ............. 78
BIO GRAPH ICAL SK ETCH .................................................. ............................... 86
LIST OF FIGURES
1-1. Migratory neuroblasts express PSA-NCAM in the adult mouse rostral migratory
store a m ............................................................................ 1 2
3-1. Gfp+ neurosphere derived cells are evident in the brains of host C57BL/6 mice three
weeks following transplantation................... ....... ............................ 38
3-2. Gfp+ neurospheres are not capable of re-isolation following transplantation into the
lateral ventricle of adult m ice........................................................ ............... 39
3-3. Gfp+ neurospheres display high levels of engraftment following transplantation into
n eo n atal m ice .................................................... ................ 4 0
3-4. Gfp+ neurospheres are not capable of re-isolation following transplantation into the
lateral ventricle of neonatal m ice .................................. ................ ................... 41
3-5. Passage three gfp+ multipotent astrocytic stem cells contain astrocytes but are devoid
o f n eu ro n s ..................................................................... ................ 4 2
3-6. Gfp+ multipotent astrocytic stem cells display high levels of engraftment following
transplantation into neonatal m ice....................................... ......................... 43
3-7. Gfp+ multipotent astrocytic stem cells are not present in the cultures of transplanted
n eo n atal m ice .................................................... ................ 4 4
3-8. Qualitative assessment of the effects of puromycin on multipotent astrocytic stem
cells cultures derived from C57BL/6 and gp+ neonatal mice...............................45
3-9. Transplantation of puromycin selected multipotent astrocytic stem cells isolated
from C57BL/6 mice transplanted with gp+ multipotent astrocytic stem cells
exhibit no gfp donor cell engraftm ent ........................................... ............... 45
3-10. Multipotent astrocytic stem cells isolated from the forebrains of mice transplanted
with gfp+ multipotent astrocytic stem cells do not survive puromycin treatment...47
3-11. Donor-derived cells exist in the brains of gfp+ neurosphere transplanted animals
14 m months after surgery ........................................................................ 48
4-1. Lethal irradiation drastically reduces the volume of PSA-NCAM and P-III Tubulin
positive m igratory neuroblasts ...................................................... .....................53
4-2. Reduction in the volume of 0-III Tubulin positive migratory neuroblasts persists
m months after lethal irradiation ............................................................................ 53
4-3. Region of analysis for quantification of mitotic neuroblast depletion in the
subependymal zone of lethally irradiated mice..................... ............................. 55
4-4. Depletion of mitotic neuroblasts in the subependymal zone of lethally irradiated
a n im a ls ........................................................................... 5 6
4-5. Lethal irradiation significantly decreases the number of BrdU positive SEZ
neuroblasts ............................................................... .... ..... ........ 57
4-6. Depletion of migratory neuroblasts in the rostral migratory stream positive for both
BrdU and P-III Tubulin in lethally irradiated mice ..................... ...............58
4-7. Lethal irradiation significantly reduces the yield of neurospheres cultured from the
adult subependym al zone. ............................................................. .....................60
4-8. N eurosphere differentiation potential ............................................. ............... 60
4-9. Attenuation ofgfp+ multipotent astrocytic stem cell engraftment by lethal
irra d iatio n ......................................................................... 6 1
4-10. Mitotic subependymal zone neuroblasts are transiently depleted following
su b leth al irradiation ................................................................. ........ ... ..... .64
4-11. Sublethal irradiation results in transient, recoverable depletion of mitotic
subependym al zone neuroblasts .................................... ............................. ........ 65
4-12. Sublethal irradiation significantly reduces the generation of neurospheres cultured
from adult subependym al zone ........................................... ......................... 66
4-13. Gfp+ multipotent astrocytic stem cells transplanted into the lateral ventricles of
control and sub-lethally irradiated mice produce donor-derived migratory cells in
the host olfactory bulb ............................................. .. ...... .. ........ .... 67
4-14. Sublethal irradiation enhances engraftment of donor-derived neuroblasts ............68
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
NEUROSPHERES AND MULTIPOTENT ASTROCYTIC STEM CELLS: NEURAL
PROGENITOR CELLS RATHER THAN NEURAL STEM CELLS
Gregory Paul Marshall, II
Chair: Edward W. Scott
Major Department: Molecular Genetics and Microbiology
Adult hematopoiesis is driven by the hematopoietic stem cell (HSC), a cell residing
in the bone marrow that possesses unique, functional characteristics defining it as a true
stem cell. Neural stem cells (NSCs) are reported to exist in the brains of adult mice in
two well characterized regions: the subependymal zone (SEZ) of the lateral ventricles
(LV) and the hippocampal dentate gyms. Mitotic neuroblasts are continuously generated
by the SEZ and migrate along the rostral migratory stream (RMS) toward the olfactory
bulb, where they functionally integrate as intemeurons. SEZ NSCs can be cultured under
specific conditions to generate neurospheres (NS) or multipotent astrocytic stem cells
(MASC), cell types that possess certain stem cell qualities in vitro. However, the in vivo
stem cell characteristics possessed by cultured NSCs (specifically functional engraftment
and serial transplantation) are not fully understood.
Green fluorescent protein (gfp) NS and MASC transplanted into the LV of both
neonatal and adult C57BL/6 mice resulted in engraftment into the recipient brain, with
donor-derived migratory neuroblasts visible in the RMS weeks after transplantation.
Neither displayed the capacity to be re-isolated from adult or neonatal brains even under
rigorous enrichment conditions, nor were they evident in the brains of secondary
recipient mice, indicating that functional, long-term engraftment by the transplanted cells
failed to occur.
Exposure of adult C57BL/6 mice to lethal levels of ionizing radiation resulted in
the ablation of both active hematopoiesis and SEZ neurogenesis, with subsequent
transplantation of gfp MASC into the LV resulting in no observable engraftment.
Exposure to sublethal levels of radiation resulted in a transient depletion of SEZ
neurogenesis and moderately enhanced engraftment levels of transplanted gfp+ MASC,
potentially providing a more ideal model system for NSC transplantation and analysis.
In this study, both NS and MASC failed to meet the criteria of true stem cells as
defined by the properties of the adult HSC. Thus it is possible that neurogenesis in the
adult brain is provided not by an isolatable NSC but rather by neural progenitor cells
derived from a migratory adult stem cell residing in the bone marrow.
INTRODUCTION TO ADULT MURINE STEM CELLS
Stem cells are represented in a variety of organs in the adult mouse. Cells with
"stem-like" properties have been reported to exist in the bone marrow (7), skeletal muscle
(67), liver (13), pancreas (90), intestinal lining (68), skin (74) and the brain (2, 3, 44)
representing all three of the primordial germ layers: endoderm, ectoderm and mesoderm.
Stem cells in the adult mouse are believed to be developmental remnants of germinal
cells that continue to renew the tissues of the organ in which they reside. Adult stem
cells are characterized by the ability to continuously generate all cell types necessary to
perpetuate the existence of their native organ; and the ability to asymmetrically divide, so
that a duplicate stem cell is generated as well as a more lineage-committed daughter cell,
allowing for cell generation to continue for the life of the animal.
Hematopoietic Stem Cells
The hematopoietic stem cell (HSC) is responsible for generating all the cells of the
blood lineage for the life of the animal, a process known as hematopoiesis. During
development, hematopoietic cells arise from the mesoderm after gastrulation and become
organized into blood islands in the extra-embryonic yolk sac near embryonic Day 7.5
(E 7.5) in the mouse (31, 53).
The first isolatable manifestation of the HSC appears in the intra-embryonic aorta-
gonod-mesonephros (AGM) at E 10.5, resulting in the initial onset of definitive
hematopoiesis in the developing animal (48, 54). Via waves of migratory HSCs from the
AGM, hematopoiesis then shifts to the fetal liver (with the presence of HSCs in the AGM
virtually eliminated by E 13). Migration of fetal HSCs is believed to be chemokine
driven, with stromal derived factor 1 (SDF-1) and steel factor (SLF) as the likely agents
of directional migration (11). Numbers of HSCs in the fetal liver begin to decrease
around E 16, presumably as the migratory HSCs leave the liver to seed the developing
bone marrow and spleen however, fetal liver hematopoiesis persists until shortly after
birth; whereas in the adult mouse, the bone marrow becomes the sole region of
hematopoiesis throughout the life of the animal.
Three definitive characteristics define the HSC as a true stem cell: asymmetric
division, pluripotency, and the ability to functionally reconstitute depleted bone marrow
(69). Asymmetric cellular division is a process in which the HSC switches between
generating an exact duplicate of itself and generating a more lineage committed daughter
cell that allows the HSC to perpetuate its existence throughout the life of the animal. The
HSC is capable, by generating lineage-specific hematopoietic progenitor cells (HPCs), of
producing each of the numerous cell types present in the blood. This includes (but is not
limited to) erythrocytes, macrophages, lymphocytes, T-cells, and B-cells. These two
abilities together make up the third criteria: The HSC is capable of repopulating the bone
marrow of a myeloablated mouse (a mouse that has been exposed to sufficient doses of
radiation so that the bone marrow is depleted of viable cells, a lethal condition without
The HSC can home to the injured bone marrow after intravenous transplantation,
repopulating the niche and giving rise to all of the numerous cells within the
hematopoietic lineage. This homing mechanism is believed to be similar to that observed
during development, with an increase in the expression of SDF-1 after myeloablation of
the bone marrow causing an increase in the expression of matrix metalloproteinase-9
(MMP-9). This results in the release of soluble Kit ligand, creating a diffusion gradient
that the migratory c-kit+ HSC follows as it homes to the bone marrow (33). Asymmetric
division of the now engrafted HSCs allows for donor-derived hematopoiesis to continue
for the life of the mouse, deriving from the primary transplanted HSC, later-generated
daughter HSCs and the progeny of the resultant progenitor cells.
A fourth criterion has been proposed, in which isolated candidate HSCs must
display the aforementioned capabilities and the ability to serially rescue a myloablated
animal. In this paradigm, donor-derived HSCs can be re-isolated from the bone marrow
of a mouse rescued by the transplantation and engraftment of a single HSC, and
transplanted into a secondary myloablated host with the same rescue ability conferred to
the animal (40). These four criteria set the "gold standard" for defining the HSC, a
functional standard rather than a phenotypic one. Cell-surface markers unique to only the
HSC have yet to be identified, but techniques exist that allow for the enrichment of cells
extracted from the bone marrow for HSCs.
Isolation of the Adult Hematopoietic Stem Cell
The bone marrow is home to a variety of cell types, from stromal support cells and
mesenchymal stem cells (MSC) to HSCs, HPCs, and their resultant progeny. After
extracting the bone marrow, the MSC is removed by exploiting the ability of the MSC to
adhere to adhesive substrates such as laminin. As the HSC lacks this ability, placement
of the cell suspension onto an adhesive substrate allows the MSCs to adhere to the
substrate, leaving only the cells of the hematopoietic lineage. Removal of terminal
progeny is accomplished by using the surface markers unique to the selected cells: B220
is expressed by B-cells, CD1 lb by macrophages, Terl 19 by T-cells, and Gr-1 by
granulocytes. Fluorescently labeled antibodies specific to these antigens allow for the
selection of cells by a technique known as Fluorescence Activated Cell Sorting (FACS)
in which cells can be selected on the basis of the presence (positive selection) or absence
(negative selection) of definitive cell-surface markers. The FACS sorter is capable of
distinguishing between cell types based on the particular fluorochrome expression pattern
(and the specific antibody conjugated to that fluorochrome) present on treated cell
populations. Exclusion of the terminal daughter cells by negative selections leaves the
remaining cell population consisting of stem and progenitor cells.
To further enrich for the HSC, two specific cell-surface markers (88) can be
utilized: c-Kit and Sca-1. C-kit is the receptor for stem cell factor (SCF) also known as
steel factor (SF), a cytokine shown to inhibit apoptosis in hematopoietic cells potentially
giving HSCs the ability to perpetuate longer than terminally differentiated cells (1).
Sca-1 (stem cell antigen), also known as lymphocyte activation protein-6a (Ly6-A), is a
cell-surface receptor shown to play a role in HSC proliferation; the identity of its binding
ligand has yet to be identified (36). Selecting cells positive for these two markers while
depleted of terminally differentiated daughter cells results in a population of cells 1000-
to 2000-fold enriched for the presence of HSCs. While this process greatly increases the
chances of the resulting cells being HSCs, the aforementioned functional properties of
long-term bone marrow reconstitution must be ascertained before the isolated cell can be
definitively labeled as a true HSC.
Determining Functional Engraftment
After enrichment of whole bone marrow for the presence of HSCs, an assay to
ascertain the rescue capacity of the enriched cell population is critical for the cells
isolated to be deemed HSCs. Mice exposed to levels of radiation sufficient to deplete
the bone marrow of viable hematopoietic cells (lethal irradiation) are generated as
recipients for candidate HSCs. Lethally irradiated mice (also known as myeloablated)
will expire within a few days of exposure without intravenous transplantation of either
whole bone marrow or HSCs, so survival of the irradiated mouse is the first indication of
candidate cell types being HSCs. Duration of survival indicates the identity of the cell
transplanted, as short-term HPCs provide transient protection from myeloablation for a
period of only 3 to 4 mo (69). As previously mentioned, HSCs exist for the life of the
animal and long-term survival of a myloablated mouse (longer than 6 mo) indicates
engraftment of a true HSC.
To ascertain that the engrafted HSC is responsible for the lethally irradiated
animal's hematopoiesis, transplanted HSCs need to differ in some fashion from the host
animal. A common tool to meet this requirement is the gfp mouse; a transgenic animal
with vast experimental capabilities. The gfp mouse contains a transgene that expresses
gfp driven by the chicken beta actin promoter enhanced by a cytomegalovirus enhancer,
resulting in ubiquitous expression of gfp in theoretically every cell of the mouse (32).
Cells isolated from the gf mouse do not initiate an immune response when transplanted
into mice from the C57BL/6 line, as this line was used to generate the gfp transgenic
animal. Furthermore, the expression of gfp continues in vitro after removal of tissue from
the animal, allowing for in vitro manipulation and expansion.
Myeloablated C57BL/6 mice transplanted with candidate HSCs isolated from the
bone marrow of gfp mice result in a chimeric animal whose bone marrow and resultant
progeny are gfp+. By sampling portions of the peripheral blood of transplanted mice, it
is possible to determine which cell types are gfp+ and the percent chimerism contributed
by the transplanted cells. Using fluorescent antibodies against the cell-surface markers
present on terminal daughter cells listed earlier, FACS analysis allows researchers to
determine if circulating progeny are gfp+ and thus the result of an engrafted gfp+ HSC
(donor-derived). The presence of donor-derived progeny 6 mo or longer after
transplantation indicates that the cell type transplanted has characteristics of an HSC.
Serial transplantation provides definitive evidence that the transplanted cell was
indeed an HSC. If a true HSC was transplanted into a myeloablated animal and engrafted
into the bone marrow, resulting in long-term functional hematopoiesis; then it would
stand to reason that the transplanted HSC underwent asymmetric division, producing
additional HSCs. To determine this to be the case, the bone marrow from long-term
reconstituted mice transplanted with gfp+ candidate HSC is extracted and the resulting
cell suspension is again enriched for the presence of HSC. Should the isolated cells
rescue a secondary myloablated mouse, and donor-derived progeny be observed in the
peripheral blood 6 mo or longer after transplantation, it can then be stated that the cell
type transplanted satisfied all the necessary criteria to be classified as an HSC.
Irradiation of the Host Bone Marrow, the Niche of the HSC
Transplantation of enriched bone marrow results in robust, long-term engraftment
only when the host animal has first undergone myeloablation (18, 19, 20). Irradiation
dosage is critical, as insufficient levels will not deplete the bone marrow; and over-
exposure will result in mortal damage to internal organs. While lethal irradiation is
sufficient to deplete the bone marrow of HSC and their progeny, it apparently does little
to damage the support cells that make up the HSC niche. Niche (a term recently applied
to stem cell biology) describes the cellular make-up of the microenvironment in which a
particular stem cell is believed to reside, and the specific mitogens and cytokines needed
to maintain the potency of the native stem cell. As previously mentioned the bone
marrow contains cells of the hematopoietic lineage and also contains stromal support
cells. In vitro studies indicate that osteoblasts, fibroblasts, and endothelial cells are
capable of supporting HSCs and HPCs in culture. While this has yet to be confirmed in
vivo, HSCs in the bone marrow are known to exist in close proximity to the endothelial
vasculature and osteoblasts (87).
The current lack of available information on the exact chemical and cellular
components that comprise the microenvironment of the HSC niche has led to the inability
to expand the HSC in vitro while retaining full potency and rescue ability (14). While it
is possible for HSCs to exist in vitro and to differentiate into different hematopoietic cell
types in certain culture conditions, the HSC has yet to be proven capable of successful
expansion sufficient to daughter HSCs capable of repopulating the bone marrow of a
Plasticity of the Hematopoietic Stem Cell
Because hematopoiesis (and subsequently, the HSC) is the most robust system of
cellular generation in the adult mammal, the HSC is arguably the stem cell with the most
potential for regenerating damaged tissue. The concept of an adult stem cell
transdifferentiating to produce cell types foreign to its native organ is a concept that has
undergone tremendous scrutiny in recent years. At the same time, the concept of HSC
transdifferentiation has elicited excitement in the field of regenerative medicine.
The HSC has been reported to repopulate the bone marrow after transplantation into a
myeloablated mouse, and also sometimes contribute to the brain (8, 17, 49), liver (41, 61,
76, 77), heart (34, 55, 56), muscle (21, 30), and blood vessels (28). HSCs isolated from
mice expressing specific reporter genes were found to generate progeny that appeared
morphologically identical to neighboring cells in these organs and that expressed the
appropriate cell surface markers. The possibility that cellular fusion was the reason
behind these cells appearing donor-derived weakened the argument for
transdifferentiation (75, 86).
Cell fusion is an extremely rare event, with neighboring cells merging to form a
cell that expresses the characteristics of one or both cells involved. This fusion occurs at
the cytoplasmic and nuclear level, with the resultant cell existing in higher than a diploid
state, expressing the cell-surface markers of both contributing cells, and adopting the
morphology of the surrounding tissue. Currently, for an investigator to disprove a fusion
event and thereby support transdifferentiation, specific steps must be implemented in the
experimental design. One such step is to transplant male donor cells into female host
tissue with later analysis for the presence or absence of the y-chromosome in potentially
transdifferentiated cells. Another such step is to use donor animals that constitutively
express a reporter gene downstream (gf for example) of a cre-recombinase gene, and
host animals that constitutively express a second reporter gene (lacZ) immediately
downstream of a stop codon flanked by lox-p sites. In the host animal, lacZ is turned off
until such time as the upstream stop codon is removed. Should a fusion event occur, the
cre-recombinase from the donor cells will excise the stop-codon of the host cell via the
lox-p sites and allow the expression of lacZ along with the expression ofgfp.
Recent investigations involving the long-term (more than 6 mo) contribution of
HSC-derived cells to the brain have yielded positive results, with the transdifferentiated
neurons giving no indication of being the result of fusion (12, 84). The long-term design
of these studies may be a crucial point, in that the HSC could require time to generate
progeny capable of infiltrating the brain's regions of stem cell activity and producing the
observed cell types. Injury to the target region of engraftment as well as the regions of
potential transdifferentiation may also be a critical requirement, as little evidence has
been collected in non-injured recipients (83).
The HSC can very well be classified as the grandfather of adult stem cells.
With almost 50 years of research dedicated to its identification and potential, the HSC
has a nearly insurmountable head start on other adult stem cells. Much of what is known
about stem cells (from their definitive phenotypes and capabilities, to their organs of
residence) has been derived from HSC studies. Candidate stem-cell populations isolated
from other tissues will need to meet the rigorous criteria set forth by the field of
hematopoiesis before they can be deemed to be true stem cells.
Neural Stem Cells
Neurogenesis and the Embryonic Neural Stem Cell
Development of the central nervous system begins after gastrulation, with induction
of the neural plate from the ectoderm to eventually form the neural crest. As
development continues, the structure of the brain emerges via an "inside-out" pattern of
growth (the deepest layers of the brain are formed first and outer layers are then formed
by migratory cells traveling and passing through previously generated layers).
This pattern of migration points toward regions of persistent neurogenesis in the interior
portion of the developing brain.
While opinions still differ as to the existence of a single type of neural stem cell
(NSC) that contributes to the formation of the entire brain, it is generally accepted that a
population of neural stem cells exists during this time, dedicated to producing cells of the
neural and glial lineage. These stem cells presumably generate additional stem cells and
also populations of lineage-specific progenitor cells. Neural progenitors (sometimes
called "neuroblasts") generate the numerous manifestations of neurons such as
interneurons and granule neurons while glial progenitors give rise to astrocytes and
oligodendrocytes. Astrocytes comprise the majority of cell types found in the brain,
offering mechanical and metabolic support to neurons. Oligodendrocytes are responsible
for the production of the myelin (the axonal covering that speeds neuronal transmission).
The Adult Neural Stem Cell
It was believed for many years that while stem cells were required to promote
developmental neurogenesis, the brain of the adult mammal was a static structure without
the ability to repair or modify itself (abilities conferred by stem cells). The presence of
newly generated cells indicated that neurogenesis occurred in the postnatal rat brain, but a
lack of available techniques to positively identify these cells as neurons left the
observations relatively ignored (2, 3).
It is now generally accepted that adult neurogenesis is restricted to two regions: the
subgranule layer of the hippocampal dentate gyms (9, 25, 38) and the ventriclar
subependymal zone (SEZ) (44, 47). During the middle 1990s, numerous reports
emerged showing that (in the adult mammal) cells with stem-like properties can be
isolated from the hippocampus, spinal cord, and SEZ (25, 44, 85). The method of
isolation differs from that seen in the field of hematopoiesis in that no definitive markers
for the NSC are currently available. Instead, candidate stem cells are isolated from
regions of neurogenesis and cultured in defined media conditions to ascertain what (if
any) stem cell properties they may possess.
Subependymal-Zone Neural Stem Cells
In the adult mouse, the SEZ is arguably the most robust region of neurogenesis,
existing as a layer of cells adjacent to the ependyma running along the length of the wall
of the lateral ventricles. Throughout the life of the animal, mitotic neuroblasts are
generated in the SEZ and migrate along a well-defined glial structure known as the rostral
migratory stream (RMS) towards the olfactory bulb (OB), where they integrate as
interneurons (4, 45, 47, 78). Presumably these interneurons allow for the maintenance of
Migration of the mitotic neuroblasts occurs in a rostral orientation until the RMS
enters the OB proper, whereupon migration shifts to a tangential orientation. At this
point, the neuroblasts adopt a neuronal morphology and integrate into the existing
cytoarchitecture of the OB as granule or periglomerular interneurons. The migration of
mitotic neuroblasts is rather rapid (taking 2-6 days to traverse 2-8 mm) and occurs via a
unique form of chain migration (46). The migrating cells adhere to one another
(presumably via the expression of a polysialylated form of the neural cell adhesion
molecule, dubbed PSA-NCAM) and migrate at an estimated rate of 120 pm/h through the
RMS in chains that are devoid of glial cells (Figure 1-1). An estimated 30,000 new
neurons are produced each day in the adult mouse, leading many to conclude that a self-
renewing stem cell must reside in the SEZ in order for this rate to be sustained for the life
of the animal (45).
While the exact identity of the SEZ NSC has yet to be determined, numerous
reports have shed light on its characteristics and function. A heterogeneous population of
cells exists, replicate, and migrate adjacent to the ciliated ependymal layer, a layer of
cells lining the walls of the lateral ventricle that was previously believed to either
Figure 1-1. Migratory neuroblasts express PSA-NCAM in the adult mouse rostral
migratory stream. Sagittal photographic montage (5x images) of migratory
neuroblasts in a 6 week old C57BL/6 female migrating through the RMS to
the OB from the subependymal zone of the LV. (*) denotes chains of
PSA-NCAM positive (red) neuroblasts. OB = olfactory bulb, RMS = rostral
migratory stream, LV = lateral ventricle.
contain or consist of NSCs (37). Little is known of the specific cell types and factors
that comprise the microenvironment of this NSC niche, although recent studies are
helping to better understand the cellular make-up. It has recently been reported that the
SEZ NSC is a type of slowly dividing astrocyte, termed the type B cell (15). The
processes of type B cells form tube-like structures through which migratory neuroblasts
(type A cells) travel toward the OB. Transit amplifying neural progenitor cells (referred
to as type C cells) are interspersed among the type B cells, and it is this cell type that is
believed to be responsible for the production of the type A cells.
Experiments involving the injection of the anti-mitotic agent
cytosine-P-D-arabinofuranoside (Ara-C) into the SEZ of adult rats showed that the cells
initially eliminated were the faster-dividing progenitor cells (the type A and C cells), and
that after removal of Ara-C the first cell to return was the type B cell (16). The type B
cells then generated type C cells, that in turn generated type A cells. Expression of
cell-surface markers of these three cell types are not unique enough to allow selective
isolation or enrichment by FACS, as earlier shown with the HSC. The type B cell
expresses the astrocytic markers glial fibrillary acidic protein (GFAP) and vimentin,
while the type A cell expresses both the migratory marker PSA-NCAM and the pan-
neuronal marker 0-III tubulin (27). Unfortunately, these markers are also expressed on a
variety of terminally differentiated cells in the brain, ruling them out as potential unique
markers for the isolation of the NSC. Interestingly, type C cells appear to exclusively
express Nestin (a marker expressed by primitive neuroepethelium during development)
indicating that this neural precursor is extremely primitive (27). Regardless of its
expression profile, the type B cell is generally accepted to be the NSC of the adult SEZ.
After surgical dissection, adult neural tissue from the SEZ can be dissociated into a
single-cell suspension and cultured in the presence of mitogens; specifically, epidermal
growth factor (EGF) and basic fibroblast growth factor (bFGF). When cultured in the
absence of adhesive substrates, cultured cells form spherical structures with phase
contrast-bright boundaries known as "neurospheres" (NS): presumably clonal structures
consisting of multi-lineage daughter cells in varying stages of maturation (22, 29, 39, 42,
62-65, 83). These NS are multipotent and capable of generating cell types from all three
neural lineages upon induction of differentiation. The NS do not normally form in the
absence of EGF or bFGF, validating the necessity of these mitogens in culture. NSCs
during development express receptors to EGF and bFGF in a temporal- and region-
specific manner, varying as the embryo develops. Expression ofbFGF seems crucial to
normal development, as bFGF knock-out mice have fewer neurons and glia and reduced
tissue volume (57, 81). This observation was supported by a study (73) in which
antibodies designed to neutralize bFGF were injected into neonatal brains resulting in
decreased DNA synthesis in multiple regions of the brain. Conversely, it was observed
that intra-cerebral injections of bFGF results in increased neurogenesis (81). EGF also
plays an important role in neural development: EGF knock-out mice exhibit defects in
cortical neurogenesis, and injecting EGF into the brain has been shown to increase
In addition to their multipotency, another stem cell quality of cultured NS is that
they are capable of in vitro expansion (64). After dissociation and secondary culture,
primary NS yield numerous secondary spheres, with the newly generated NS retaining all
of the pluripotent characteristics of the primary NS. Furthermore, the expansion
capability of the NS is retained even after ten passages, indicating self-renewal of a stem
cell by asymmetric division, although conclusions from conflicting reports would argue
as to the exact number of passages the NS can undergo while retaining potency.
What is not yet known is which cell in the isolated tissue gives rise to the NS (the
sphere-forming cell), if the sphere-forming cell is indeed a true stem cell, or if every NS
in culture is the product of an active NSC. While the results listed above identify the type
B cell as the NSC in vivo, definitive isolation of the adult NSC and its subsequent in vitro
analysis and expansion has yet to be conclusively accomplished.
Multipotent Astrocytic Stem Cells
In addition to the NS, another in vitro manifestation has recently been reported: the
multipotent astrocytic stem cell (MASC) (43). MASC can be isolated from the
cerebellum and forebrain of neonatal mice (up to 2 weeks of age), but are only attainable
from the SEZ in adult mice. Dissociated SEZ tissue forms a monolayer of astrocytes
after culture onto an adhesive substrate in the absence of EGF/bFGF; and a subpopulation
of these astrocytes are believed to be MASC. The theory that the NSC is a form of
astrocyte supports the belief that the MASC is an in vitro manifestation of the NSC (27).
Furthermore, MASC are capable of generating NS upon culture in non-adhesive
conditions in the presence of EGF and bFGF, with resulting NS capable of serial
expansion and multi-lineage differentiation.
Potential Identifying Markers of the NSC
The HSC population can be enriched from the bone marrow cell population using a
series of cell-surface markers, and recent reports indicate that similar protocols can be
utilized to enrich the cell population of the SEZ for NSC. As described earlier, the SEZ
NSC is potentially a form of astrocyte, and because GFAP is expressed by astrocytes
GFAP has been proposed for use as one marker for the isolation of NSC (35). The
inter-filament protein Nestin is expressed by developing neuroepithelium, and is believed
to be expressed on adult NSCs, hinting at the developmental immaturity of these cells
(63, 66). Lewis-X is a carbohydrate moiety expressed on the cell surface of embryonic
stem cells, germinal zones in the developing mouse brain, and on some astrocytes; and
was recently reported to be present on the surface of adult NS-forming SEZ cells (10).
Finally, in the human model, purification of fetal brain using antibodies against CD 133 (a
marker purported to be on the surface of HSCs) greatly enriched for NS (80).
While these protocols theoretically enrich for the presence of NSC (or at the very
least, NS-forming cells), it remains to be seen if one or a combination of these markers
will allow for the isolation of a pure population of NSCs. Furthermore, should these
techniques make it possible for the in vitro enrichment of NSCs, protocols are needed to
determine the in vivo potential possessed by the isolated and enriched NSC populations.
Transplantation of In Vitro Expanded NS and MASC
In addition to pluripotency and expansion, cultured NS and MASC also display the
ability to survive transplantation back into the brain; an important ability, indicating that
extended culture conditions have not adversely affected the cells. Integration
capabilities are limited to the aforementioned regions of neurogenesis gliall cells are
typically the only cell type observed following transplantation into non-neurogenic
regions), and the age of the host animal is also critical.
Neonatal mice (1-3 days post embryonic) are still undergoing active developmental
neurogenesis and presumably offer an environment rich in extra-cellular cues for
enhanced engraftment of transplanted NSC (26), while neurogenesis in the adult animal
is dramatically lower and is limited to the SEZ and hippocampus. In vitro expanded
hippocampal NSCs are capable of integrating into the dentate gyms after transplantation
(24), and also contribute to functional granule cells in the OB after transplantation into
the SEZ (70). Labeled SEZ NS and MASC will survive transplantation to the SEZ,
RMS or lateral ventricles, with donor-derived migratory neuroblasts present in these
regions weeks following transplantation (80, 89).
While the presence of donor-derived neurons and neuroblasts in the OB indicates
engraftment, the degree of functionality is difficult to determine. The transplanted cells
may appear to generate progeny similar to the surrounding cells, yet they do not always
express the appropriate cell-surface markers. Furthermore, the engraftment is typically
only manifested by production of cells of the neuronal lineage as opposed to the multi-
lineage engraftment observed in HSC transplant studies. This would argue that a
progenitor cell was transplanted rather than a NSC, although the limited neurogenesis in
the SEZ could explain the mono-lineage nature of the newly generated daughter cells. It
is also unknown if the transplanted cells truly engraft into the SEZ in a stable fashion and
produce daughter progenitor cells that become evident in the OB as interneurons, or if the
transplanted cells immediately enter into the RMS as neuroblasts and migrate to the OB
(again a phenomenon more consistent with progenitor-cell activity).
Adult NS and MASC: True Stem Cells?
In the beginning of this chapter, four properties were listed as belonging to true
stem cells: multipotency, asymmetric division, functional long term engraftment and
serial transplantation. While it is believed that the endogenous NSC in vivo possesses
these characteristics, little has been done to determine if candidate NSC or their in vitro
manifestations (the NS and MASC), can be defined as true stem cells.
Of the four properties, only multipotency has been conclusively shown to be a
property of NS and MASC, although in vivo studies have indicated that their potential is
limited to interneurons, granule neurons, and neuroblasts. While both NS and MASC
are able to expand following in vitro passages, this ability is often limited to only four to
six passages, though this may be due to deficiencies in the culture conditions rather than a
deficiency on the part of either cell type.
While donor derived cells are observed in host RMS and OB following
transplantation of NS and MASC, their presence is transitory, often ceasing to exist a few
months following transplantation. This is indicative of short-term engraftment of the
transplanted cells, rather than long-term (assuming true engraftment has even occurred).
To date, no data exists to determine if the observed donor-derived cells are the product of
engrafted NSCs in the SEZ, or if the transplanted cells adopted the morphology of
migratory neuroblasts immediately following transplantation.
Finally, serial transplantability is a condition yet to be met by either the NS or the
MASC, shedding doubt on the level of stem-ness possessed by either of these cell types.
There is a distinct possibility that the NS and MASC are in reality neural progenitor cells,
as they do not seem to possess all of the characteristics of a true stem cell.
Effects of Radiation on Adult Neurogenesis
In humans, whole body irradiation prior to bone marrow transplantation is a
requirement for the treatment of many forms of leukemia to enhance the engraftment of
the HSC following bone marrow transplantation, and recent investigations have focused
on the effects this exposure may have on neurogenesis, particularly on the subgranular
zone (SGZ) of the hippocampal dentate gyrus (DG). Throughout the lifespan of the
mouse newly generated neuroblasts generated from subgranular NSC migrate into the
granule cell layer and differentiate into functional granule neurons (9). These neurons
functionally integrate into the DG, potentially playing a role in synaptic plasticity and
learning. This region of neurogenesis appears to be extremely sensitive to x-irradiation in
a dose dependent manner, with the numbers of neuroblasts and granule neurons
significantly depleted by as little as 2Gy of exposure, with little to no recovery observed
months after exposure (50, 59, 71). The death of these cells is reported to be apoptotic
(60) and appears to be linked to an inflammatory response produced by the damaged
tissue, as anti-inflammatory agents injected into the animals before and after irradiation
protected the SGZ from cell loss (52). Exposure to high levels (10Gy) of focused x-rays
results in near total, permanent ablation of mitotic cells in the SGZ and DG, with
pronounced changes in both the vascular microenvironment and cell types as cells with a
glial morphology become predominant following exposure (51).
Relatively few studies have been performed to determine the effects radiation
exposure may have on SEZ neurogenesis. Studies performed only in the rat model have
shown a similar result to that observed in the SGZ and DG in the mouse. Mimicking the
results observed in the hippocampus, radiation exposure adversely effected neurogenesis
in the SEZ of rats in a dose dependent manner, with little to no recovery observed months
following irradiation (72). Interestingly, low levels of focused x-irradiation (1-3Gy)
resulted in an increase of Nestin positive cells in the SEZ in the weeks immediately
following exposure, with levels eventually returning to normal (6).
Researchers have hypothesized that the depletion observed in both the SGZ and
SEZ is due in part to the elimination of NSCs and/or NPCs known to reside in these
tissues. 10Gy of x-irradiation not only permanently depletes the SGZ of mitotic
neuroblasts, but also negatively influences the microenvironment with surviving cells
adopting a glial morphology.
This has also been shown to render the niche unreceptive to transplanted non-
irradiated NPCs, with a significant decrease in the number of transplanted NPCs adopting
a neuronal morphology (51). It is currently believed that the brain is a relatively radio-
sensitive organ compared to the bone marrow, as low doses of radiation only transiently
lower the numbers of HSCs and their resultant progeny while similar doses permanently
diminish neurogenesis in both the SEZ and SGZ.
While it is generally accepted that NSC driven neurogenesis persists in the adult
mammal in the SEZ and SGZ, the exact identity of the NSC remains a mystery.
Candidate NSCs have been extracted from SEZ and SGZ based upon expression of cell
surface markers, and these cells can be cultured in the presence of EGF and bFGF to
generate NS and MASC. Subsequent transplantation of NS and MASC back into regions
of neurogenesis results in minimal engraftment by the donor cells with donor-derived
progeny existing as neuroblasts and/or granule neurons in the host tissue, but it is not
known whether the presence of donor-derived cells indicates functional engraftment or
short-term contribution to the surrounding tissue. Exposure to radiation adversely affects
neurogenesis in the adult rodent in a dose-dependent manner, indicating that the brain is a
more radio-sensitive structure than is the bone marrow. Of the four criteria held by true
stem cells (multipotency, asymmetric division, functional long-term engraftment, and
serial transplantation) the in vitro manifestations of the NSC fulfill only one:
multipotency. Protocols are needed to not only examine the ability of the NS and MASC
to demonstrate robust, long-term engraftment, but also to ascertain their ability to survive
serial transplantation before any claims can be made as to the levels of stem-ness
possessed by these relatively uncharacterized cell types.
MATERIALS AND METHODS
Avertin Mouse Anesthetic Solution
Materials: Avertin (2-2-2 Tribromoethanol, Aldrich, St. Louis, MO; T4, 840-2),
tert-amyl alcohol, 50 mL sterile polystyrene conical tube, 50 mL "Steriflip" disposable
vacuum filtration system (0.22 |tm pore, Millipore, Billerica, MA; SCGP00525),
phosphate buffered saline, aluminum foil, stir bar/plate.
Protocol: Stock solution: Using the bottle in which the avertin was packaged,
added 15.5 mL tert-amyl alcohol and magnetic stirrer and allowed to stir overnight.
NOTE: As avertin is toxic if allowed to photo-oxidize, the stock bottle was kept tightly
capped and wrapped in aluminum foil. Stable at room temperature for well over one
Working solution (20 mg/mL): Combined 0.6 mL of the avertin stock and 39.4
mL ofPBS in 50 mL conical. Agitated briefly to mix the two components, then filtered
through the Steri-flip and wrapped the filtered solution in foil to exclude all light.
Working solution is stable at 40C for several months.
5-bromo-2'-deoxy-uridine (BrdU) Solution
Materials: 5-bromo-2'-deoxy-uridine (Roche, Florence, SC; 280-879),
appropriate size sterile polystyrene conical tube, phosphate buffered saline, 370C water
Protocol: Calculated out the volume of BrdU required for all planned injections
and measured out enough BrdU so that the final concentration was 10mg/mL.
NOTE: BrdU is extremely mutagenic. Be sure that the powder is handled carefully.
Combined the BrdU and PBS in the conical tube and warmed in the water bath for 15
minutes. Vigorous agitation is essential to ensure that all of the BrdU has entered into
suspension. Made fresh for each experiment.
Epidermal Growth Factor (EGF) Stock Solution (1000x)
Materials: Recombinant Human Epidermal Growth Factor (R & D Systems,
Minneapolis, MN; 236-EG, 200 [tg), Glacial Acetic Acid, Bovine Serum Albumin
Fraction V heat shock (Roche Diagnostics, 03 116 999 001), 5 cc syringe w/ regular luer
tip (Becton Dickinson, Franklin Lakes, NJ; 309603), Blunt Needle w/ Aluminum Hub
(Monoject, Becton Dickinson, 202314), Sterile Syringe Filter, 0.45 |tm pore size
(Corning Incorporated, Corning, NY; 31220), 15 mL and 50 mL polystyrene sterile
conical tubes, 1.5 mL sterile micro centrifuge tubes, sterile 200 [tl pipette tips, distilled
water, ice and ice bucket.
Protocol: Generation of EGF suspension solution: 17.24 [tl of Acetic Acid was
mixed with 30 mL of distilled water in a 50 mL conical tube to generate a final
concentration of 10 mM. 0.030 g ofBSA was added to solution (final concentration,
0.1% w/v) and mixed vigorously to re-suspend.
Working on ice in a sterile laminar flow hood, 200 [g of EGF was re-suspended in
10 mL of suspension solution in a sterile fashion then filter sterilized using the 5 mL
syringe with blunt needle and syringe filter. Evenly distributed 500 [L aliquots of the
EGF solution and stored at 800C. Final concentration 20 ng/[tl, used at 1:1000.
Basic Fibroblast Growth Factor (bFGF) Stock Solution (1000x)
Materials: Recombinant-human-FGF (25 ig, R&D systems, 233-FB), Phosphate
Buffered Saline, Bovine Serum Albumin Fraction V heat shock (Roche Diagnostics, 03
116 999 001), 1M Dithioerythritol (DTT) (Sigma, St. Louis, MO; D-8255), 5 cc syringe
w/ regular luer tip (Becton Dickinson, 309603), Blunt Needle w/ Aluminum Hub
(Monoject, 202314), Sterile Syringe Filter, 0.45 |tm pore size (Corning Incorporated,
431220), 15 mL polystyrene sterile conical tube, 1.5 mL sterile micro centrifuge tubes,
sterile 200 [tl pipette tips, distilled water, ice and ice bucket.
Protocol: Generation of bFGF suspension solution: 3 mL of PBS, 0.003 g BSA
(0.1%) and 3 ptL of 1M DTT (final concentration of ImM) were combined and mixed..
Working on ice in a sterilized laminar flow hood, 25[g bFGF was combined in 2.5mL of
suspension solution and filtered sterilized. Aliquots of 100 ptL were stored at -800C.
Final concentration 10 ng/[tL, used at 1:1000.
Immunocytochemistry Hybridization Buffer
Materials: Fetal bovine serum, Phosphate Buffered Saline (PBS), Triton X-100
(Sigma, T-9284), 50 mL sterile polypropylene tube.
Protocol: In sterile laminar flow hood, pipetted 5% of the final volume of FBS
into 50 mL tube. Out of the hood, filled to full volume with PBS and pipetted 0.01%
Triton X-100. Mixed and stored at 40C. Stable for 1 week.
Neural Stem Cell (NSC) Culture Media
Materials: DMEM/F 12 w/ HEPES and L-Glutamine (Gibco BRL, Carlsbad, CA;
11330-032), fetal bovine serum (FBS), N2 supplement 100X (Gibco BRL, 17502-048),
GlutaMAX-1 supplement 100x (Gibco BRL, 35050-061). Endothelial Growth Factor
stock supplement (1000x), Fibroblast Growth Factor-beta stock supplement (1000x), 50
mL sterile polystyrene conical tube, 50 mL "Steriflip" disposable vacuum filtration
system (0.22 |tm pore, Millipore, SCGP00525).
Protocol: Based on the total volume required, the appropriate volumes of the
above serums and supplements were added to DMEM/F 12 to their working concentration
and filter sterilized prior to use.
4% Paraformaldehyde (100mL)
Materials: Paraformaldehyde (Sigma P-6148), 250 mL glass beaker, distilled
water, IN sodium hydroxide (NaOH), IN hydrochloric acid (HC1), 3x PBS, glass Pasteur
pipettes, aluminum foil, 100+mLglass bottle, filter paper, small funnel, stir bar/magnetic
stirrer hot plate, pH meter.
Protocol: 4 g (4% w/v) paraformaldehyde was mixed with 60 mL of distilled
water heated to approximately 55C in a glass beaker and covered with aluminum foil
while mixing for 10 minutes. Two drops of IN NaOH was added to the solution
followed by an additional 5 minutes of mixing, after which the solution became semi-
clear. 30 mL of 3x PBS was added to the solution and the pH adjusted to 7.2 using IN
HC1 and IN NaOH. The solution was then filled to 100mL with distilled water, filtered
through Whatman paper into clean glass bottle and stored at 40C. Solution is stable for
approximately 1 week.
Puromycin Stock Solution (1 mg/mL)
Materials: Puromycin dihydrochloride from Streptomyces alboniger (Sigma
P-8833), 5 cc syringe w/ regular luer tip (Becton Dickinson, 309603), Blunt Needle w/
Aluminum Hub (Monoject, 202314). Sterile Syringe Filter, 0.45 |tm pore size (Corning
Incorporated, 431220), DMEM/F12 w/ HEPES and L-Glutamine (Gibco BRL,
11330-032), sterile polypropylene 1.5 mL tubes.
Protocol: Working in a sterile laminar flow hood, 1 mg of puromycin was added
to 1 mL of sterile DMEM/F12 in 1.5 mL sterile tube and agitated to dissolve the
puromycin powder. Re-suspended solution was filter sterilized and stored at -200C.
* Monoclonal anti-P-III tubulin: Promega, Madison, WI; G712A, dilution 1:1000
* Polyclonal anti-P-III tubulin: Covance; Princeton, NJ; PRB-435P, dilution
* Monoclonal anti-polysialic acid neural cell adhesion molecule [PSA-NCAM]:
Chemicon, Temecula, CA; MAB5324, dilution 1:100
* Monoclonal anti-BrdU: Human Hybridoma Bank, Gainesville, FL; dilution 1:30
* Polyclonal anti-glial fibrillary acidic protein [GFAP]: Shandon Immunon,
Milford MA; 490740, dilution 1:100)
* Rhodamine red-X, goat anti-mouse IgG: Molecular Probes, Eugene, OR; R-
6393, dilution 1:500
* Rhodamine red-X, goat anti-rabbit IgG: Molecular Probes, R-6394, dilution
* Fluoroscein anti-rabbit IgG: Vector Labs, Burlingame, CA; FI-1000, dilution
* Oregon green 514 goat anti-mouse IgG: Molecular Probes, 0-6383, dilution
Isolation and culture of neurospheres
Neurosphere cultures were generated from WT, LI, and SLI animals, as described
(42). Briefly, animals were anesthetized with isofluorane, cervically dislocated and
decapitated. The brain was exposed, surgically removed, and then placed on an ice-cold
sterile dissection board. A rectangular forebrain block containing the SEZ was obtained
by removing the olfactory bulb, cerebellum, hippocampus, lateral portions of the striatum
and lateral and dorsal cerebral cortex. The block was minced with a sterile scalpel, and
placed in ice-cold PBS containing anti-biotic and anti-mycotic agents (Penicillin-
Streptomycin, Gibco, 15140-122, and Fungizone Antimycotic, Gibco, 15295-017) for 10
minutes. Minced tissue was then centrifuged for 5 minutes at 1100 rpm at 40C, re-
suspended in 3 mL 0.25% Trypsin plus EDTA (Gibco, 25200-056), then incubated at
37C for 5 minutes. After neutralizing the trypsin by the addition of 1 mL of fetal bovine
serum, the tissue was triturated into a single cell suspension by pipetting through a series
of descending diameter, fire-polished Pasteur pipettes. The cells were washed in
DMEM/F-12 (Gibco, 11330-032) at 1100 rpm for 5 minutes at 40C, and re-suspended in
neural growth medium. The cells were plated out in non-adhesive 6-well plates (Coming
Costar, 3471) at a density of 1000 cells/cm2. Cultures were supplemented with EGF and
bFGF (20 ng/mL and 10 ng/mL, respectively), every second day.
Isolation and culture of multipotent astrocytic stem cells
Primary SEZ tissue was isolated from neonatal mice transgenic for green
fluorescent protein (gfp+) at post-embryonic day 2 and dissociated to a single cell
suspension in the same manner as listed above. Cells were plated onto tissue culture
flasks at high density in neural growth medium devoid of EGF and bFGF. 3 days
following the initial plating, non-adherent cells were removed and fresh media was
applied. Cultures were passage once the resulting astrocytes had formed a confluent
monolayer, and cultures were deemed suitable for transplantation once they had
undergone three passages (necessary for removal of contaminating neurons in the
Selection ofgfp+ multipotent astrocytic stem cells by puromycin selection
MASC cultures derived from the SEZ of gfp and C57BL/6 neonatal and adult mice
were prepared as described above. Once a confluent monolayer had been established,
cells were passage at 25% confluency with puromycin stock solution added to the neural
growth medium at a final concentration of 1-3 [g/mL. Cells were exposed to puromycin
in the neural growth medium for nine days, with media replaced with fresh neural growth
medium plus puromycin on the fifth day. Cells were analyzed after removal of
puromycin by phase contrast microscopy. In order for surviving cells to generate a
secondary confluent monolayer, cells were collected by trypsinization and re-plated at the
highest possible density in puromycin-free neural growth medium.
Transplantation and Analysis
Transplantation ofgfp+ MASC and neurospheres into the lateral ventricles of adult
Passage three gfp+ MASC or neurospheres were collected via trypsinization and
re-suspended in ImL of growth medium (see above). Once cell number was calculated,
cells were re-suspended in a volume of growth media yielding 50,000 cells per pL.
Recipient mice were anesthetized with Avertin (see above) and the scalp surgically
exposed. 100,000 cells (2 pL) were stereotaxically injected into the lateral ventricle via a
5[L Hamilton syringe attached to a 28 gauge needle at the following coordinates: A-P: -
0.2, M-L: -1.2, H-D: -2.5. Transplanted animals were allowed to recover and placed back
in general housing.
Transplantation of gfp+ MASC and neurospheres into the lateral ventricles of
neonatal C57BL/6 mice
Passage three gf+ MASC or neurospheres were collected via trypsinization and
re-suspended in 1 mL of growth medium (see above). Once the cell number was
calculated, cells were re-suspended in a volume of growth media yielding 100,000 cells
per tl. Recipient C57BL/6 neonatal mice (day post embryonic 1-3) were anesthetized by
placement at -20C for 5 minutes. Cells were transplanted in a volume of 1 [iL via a 5 [iL
Hamilton syringe attached to a 28 gauge needle into the lateral ventricle using the bregma
skull suture as a reference point. Following transplantation, neonatal mice were warmed
to consciousness and returned to the mother's cage prior to return to general housing.
Both wild type (WT), LI and SLI animals were given a lethal dose of the anesthetic
Avertin before being perfused through the left ventricle with 4% paraformaldehyde
(PFA) in PBS. Following perfusion, the brain was removed and post-fixed overnight by
immersion in 4% PFA at 40C. Fixed brains were then serially sectioned through either
the coronal or sagittal plane at 40 [m using a Leica vibratome (model VT-1000-S)
equipped with a sapphire blade. Tissue was prepared for immunohistochemistry by
blocking at room temperature for 1 hour in PBS containing 10% fetal bovine serum and
0.01% Triton X-100. Primary antibodies were applied to the sections overnight with
moderate agitation at 40C. Residual primary antibody was removed by three 5 minute
washes (PBS plus 0.01% Triton X-100), and secondary antibodies were applied at room
temperature for 50 minutes. Finally, sections were washed in PBS three times for 5
minute, mounted on positively charged glass slides (Fisherbrand Superfrost/Plus, 12-550-
15), and allowed to dry for 15 minutes at 37C, before being cover-slipped in Vectashield
(Vector Labs, H-1000) mounting medium. Sections were analyzed and photographed by
fluorescence microscopy using either a Zeiss Axioplan 2 upright microscope (Carl Zeiss
Inc, Thornwood, NY), Leica DMLB (Leica Microsystems AG, Wetzlar, Germany), or a
Leica TCS SP2 AOBS spectral confocal microscope.
Analysis of engrafted gfp+ MASC and neurospheres into the brains of C57BL/6
Three or four weeks following transplantation, brains of transplanted animals were
prepared for tissue immunohistochemistry as described above. The transplanted
hemisphere was sectioned to through the sagittal plane (40 |tm thick) with every section
containing the olfactory bulb collected in a serial fashion for subsequent analysis.
Tissue sections were exposed to antibodies against P-III tubulin as described above and
mounted onto slides using the serial sectioning for visual reconstruction of the brain. All
sections were analyzed at 20x magnification for the presence of gfp+ cells in the SEZ,
RMS, and OB.
Analysis of engrafted gfp+ MASC into the olfactory bulb of C57BL/6 mice
Three weeks following transplantation, brains of transplanted animals were
prepared for tissue immunohistochemistry as described above. For each brain, the
transplanted hemisphere was sectioned through the sagittal plane (40[tm thick), with
every section containing the olfactory bulb collected for analysis.
Resulting sections were exposed to antibodies against P-III tubulin as described above
and mounted for analysis. Gfp+ migratory cells present in the olfactory bulb proper (that
is, the region rostral of the descending horn of the rostral migratory stream) were scored
as engraftedd", with every section analyzed at 20x magnification for each animal.
Immunohistochemical analysis of neurospheres and MASC
NS were picked from their cultures using a handheld pipetter set at 2[tl and placed
in DMEM/F-12 plus 5% FBS atop a laminin/poly-D-lysine coated, chambered culture
slide (Becton/Dickinson, 352688). Spheres or aliquots of MASC were allowed to attach
and differentiate for 2 days, at which time the media was removed and the cells were
fixed by incubation in 4% PFA in PBS at room temperature for 30 minutes. After
fixation, the cells were processed for immunolabeling with antibodies against P-III
tubulin and GFAP, as above.
Irradiation and bone marrow reconstitution
Animals were placed in individual chambers of a plexi-glass container for
irradiation. Lethal irradiation (LI) was induced by exposure to a Cs137 source in a
Gamma Cell 40 irradiator until 850 Rads had been obtained. This amount of radiation is
sufficient to deplete the bone marrow of viable cells, while not inducing immediate death.
Immediately following irradiation, LI mice were anesthetized with isoflurane (Aerrane,
Baxter Deerfield, IL; NDC 10019-773-40) and a "rescue dose" of 1 x 106 whole bone
marrow (WBM) cells was administered to each mouse via retro-orbital sinus (ROS)
injection. WBM was isolated from the femurs of a sacrificed litter mate (anesthetized by
exposure to isoflurane then sacrificed by cervical dislocation). Isolated WBM was then
washed in 10 mL of phosphate buffered saline (PBS), and re-suspended in an appropriate
volume of PBS after quantifying with a hemacytometer. A 32 gauge needle attached to a
1 mL insulin syringe was inserted into the ROS and the WBM was injected in a volume
of 150 pL. Animals were allowed to recover before being returned to conventional
For sublethal irradiation (SLI) studies, the animals were exposed to 450 Rads of
ionizing radiation in the same fashion as listed above. No rescue dose of WBM is
required for survival of the animal at this exposure, and animals were immediately
returned to conventional housing.
Identification of proliferative cells by BrdU labeling
Three days, three weeks or 3 months following irradiation, WT, LI and SLI mice
received 5-bromo-2'-deoxyuridine (BrdU, Sigma, B-5002) three times a day for three
days via intra-peritoneal (IP) injection (3 .tg/300 [iL per injection). The brains were
fixed, removed, and sectioned as above. Sections were prepared for BrdU
immunohistochemistry by first incubating in 2XSSC/Formamide solution (1:1) for 2
hours at 650C. After washing in 2xSSC for 5 minutes at room temperature, sections were
then incubated in 2 N HC1 for 30 minutes at 37C. Sections were washed in 0.1 M borate
buffer for 10 minutes at room temperature prior to processing for immunolabeling with
monoclonal anti-BrdU and polyclonal anti-P-III tubulin antibodies, as described above.
Serial coronal sections were analyzed for BrdU positive cells in the SEZ using a blind
study format (sections coded and scored by separate investigators). The region of the
SEZ analyzed encompassed an area extending from the inferior tip of the lateral ventricle,
superiorly along the wall of the lateral ventricle (approximately 5 cell bodies deep).
This area continued to a point extending 700 [m from the dorsolateral corer of the
The defined region was carefully analyzed at a magnification of 40X, and at both
focal planes of the tissue. To maintain consistency between sections, BrdU labeled cells
were scored as "positive", regardless of the intensity of the antibody fluorescence.
Three adjacent sections per tissue were scored, using the location of the anterior
commissure as a consistent landmark between sections. The cell numbers were collected,
averaged, and placed into a graphical format using a Microsoft Excel spreadsheet.
Statistical significance of values was determined by paired student's t-test analysis with
p-values less than 0.05 deemed to be significant.
Blind analysis of the effects of lethal and sublethal irradiation on neurosphere yield
In order to determine the effects of irradiation on NS generation, a blind paradigm
was utilized to enable unbiased preparation and examination of the cultures from three
WT and three LI mice (two months following lethal irradiation, age matched) and from
four WT and 4 SLI mice (3 weeks following sub-lethal irradiation, age matched).
Briefly, the animals were sacrificed and their brains removed by investigator A,
who gave each brain an identifying number (1 through 8). The brains were then given to
investigator B who removed the SEZ (as described above) from each brain in an identical
fashion. The isolated SEZ tissue was then re-coded with a letter (A through H) by
investigator C, who remained the only individual to know both the letter and number
code. The tissue was then returned to investigator A for culture (as described above) and
quantification. At 21 days in vitro, NS were collected, pelleted, and re-suspended in 2
mL of media.
To determine NS yield, four 50 [tl aliquots from each culture were placed in a 12-
well tissue culture plate. The aliquots were analyzed with a Nikon inverted phase
microscope at both 4x and 10x magnifications. NS diameter was determined by use of
the "SPOT" program (Diagnostic Instruments, Sterling Heights, MI). NS below 40 xtm
in diameter were excluded as a means to avoid potential confusion with hypertrophied
cells. Additionally, spherical aggregates that did not display the NS criteria of a tight
phase contrast-bright perimeter were not counted. The total number of spheres per
aliquot was determined, and the total yield and percent yield of each culture was then
calculated from these numbers. Statistical significance of values was determined by
paired student's t-test analysis withp-values less than 0.05 deemed to be significant. At
the conclusion of the analysis, the code was broken and the identity of the cultures was
LACK OF EVIDENCE FOR THE CLASSIFICATION OF NEUROSPHERES AND
MULTIPOTENT ASTROCYTIC STEM CELLS AS TRUE STEM CELLS
Adult stem cells (ASCs) have been the subject of numerous studies in recent years,
many of which have focused on the isolation and characterization of candidate stem cell
populations. ASCs are represented in a variety of organs in the adult mouse. Cells with
stem-like properties have been reported to exist in the bone marrow (7), skeletal muscle
(67), liver (13), pancreas (90), intestinal lining (68), skin (74) and the brain (2, 3, 44)
representing all three of the primordial germ layers: endoderm, ectoderm and mesoderm.
Perhaps the most robust region of stem cell activity in the adult animal is the bone
marrow, in which the hematopoietic stem cell (HSC) vigorously replenishes the myeloid
and lymphoid lineages of the hematopoietic system for the life of the animal. As
isolation of the HSC has not yet been definitively accomplished, the use of a combination
of antibodies against specific cell surface markers has emerged as a technique to highly
enrich the heterogeneous cell population of the bone marrow for the presence of HSCs.
Bone marrow derived cells (BMDCs) negative for the terminally differentiated markers
B220, CD1 lb, and Terl 19 but positive for stem cell antigen (Sca. 1) and the receptor for
steel factor (C-Kit) yield a population of cells enriched for HSCs. Yet functional
reconstitution of the bone marrow must be established for the isolated population of cells
to be defined as a HSC, making the definition of the HSC reliant on a function rather than
A single highly enriched BMDC transplanted into the peripheral blood of a
myeloablated mouse that displays the ability to home to the injured bone marrow, re-
populate the hematopoietic system, and allow for stable, long-term hematopoiesis to
persist for the life of the animal can be labeled an HSC. The duration of the contributed
hematopoiesis is critical, as this ability is indicative of the transplanted cell undergoing
asymmetric division, a phenomenon where the stem cell shifts between generating an
exact copy of itself and a more lineage committed daughter progenitor cell. This results
in the presence of more HSCs than initially transplanted, with only a small population of
these cells active at any given time allowing for long-term hematopoietic contribution.
This also allows for a vital requirement for the definition of a HSC to be satisfied: serial
transplantation. Work by Krause et al has indicated that a single transplanted HSC can
not only reconstitute the bone marrow of a myeloablated mouse long-term, but also be re-
isolated and transplanted into a secondary myeloablated host and confer the same radio-
protective properties as in the initial transplant (40). From the extensive investigations
into the HSC, a gold-standard definition of a stem cell has emerged: an isolatable cell
capable of asymmetric division, multi-lineage long-term engraftment or contribution to
the native tissue, and serial transplantability. Candidate stem cell populations isolated
from other adult organs would need to meet these same rigorous criteria in order to be
classified as true adult stem cell rather than a progenitor cell.
Neurogenesis in the adult mammalian brain is currently believed to be restricted to
the subependymal zone (SEZ) (44, 47) and the sub-granule cell layer of the hippocampus
(HC) (9, 25, 38). Both regions have been shown to produce migratory daughter cells for
the life of the animal, yet the levels of neurogenesis appear to decrease with age. In vivo
the embryonic NSC has been proposed to contribute to all of the cell types in the
developing brain, but in the adult brain NSCs in both the dentate gyms and SEZ produce
only one cell type: migratory neuroblasts, neural progenitor cells (NPCs) that
differentiate into granule neurons and periglomerular intemeuons. SEZ neuoroblasts
migrate to the olfactory bulb (OB) via a well-defined glial pathway called the rostral
migratory stream (RMS). Migratory neuroblasts divide and differentiate during
migration, eventually functionally integrating into the OB neural circuitry as granule and
periglomerular interneurons (4, 45, 47, 78). Hippocampal NSCs from the subgranule
cell layer generate migratory neuroblasts that migrate a short distance through the granule
cell layer to the mitral cell layer of the dentate gyms, where functional integration as
granule intemeruons occurs (38).
A relative newcomer to the field of stem cell biology, the SEZ NSC has displayed
the potential to be cultured in vitro as both a spherical aggregate of clonal cells known as
a neurospheres (NS) (22, 29, 39, 42, 62-65, 83) and as a monolayer of multipotent
astrocytic stem cells (MASC) (43). Both manifestations display the stem cell properties
in vitro of multi-potency and serial expansion. Upon induction of differentiation, NS and
MASC generated NS are capable of producing neurons, astrocytes, and oligodendrocytes.
They are also capable of increasing in number following repeated passages, although this
expansion is not unlimited.
Transplantation of gp+ NS or MASC into regions of active neurogenesis in the
adult brain results in donor-derived neuroblasts and neurons, while transplantation into
terminally differentiated regions typically results in donor-derived glia, with few
instances of neuronal contribution (23, 26, 80, 89). In SEZ transplantation, engraftment
is often defined by the presence of donor-derived migratory neurons and periglomerular
interneurons in the OB of the host animal. Contrary to HSC engraftment, NSC
engraftment is transient, with the presence of migratory neuroblasts lasting only a few
months, and mono-lineage as only neuroblasts and their resulting neurons are evident
post transplantation (24, 69).
Currently the HSC has not been shown to retain all of the aforementioned
characteristics of a true stem cell in vitro, a shortcoming attributed to an incomplete
reconstruction of the bone marrow microenvironment by the selected culture conditions.
The resulting cell type displays pluripotency and the ability to expand following passages
yet lacks the capacity to reconstitute the bone marrow of a myeloablated mouse, this cell
type has been deemed a hematopoietic progenitor cell (HPC) rather than a true HSC.
While both NS and MASC display the stem cell characteristics of pluripotency,
culture expansion and functional engraftment following transplantation into regions of
proven neurogenesis, little has been done to investigate the potential of the NSC to
survive serial transplantation or to exhibit long-term engraftment. The characteristics of
the NSC with respect to the better-understood HSC were examined to determine the level
of "stem-ness" exhibited by the NSC.
Inability of Gfp+ Neural Stem Cells to Survive Re-isolation Following
Transplantation into the Lateral Ventricles of C57BL/6 Mice
Gfp+ Neurospheres are not Generated from the Brains of Adult C57BL/6 Mice
Transplanted with Gfp+ Neurospheres
Gfp+ NS cultured from day post-embryonic (dpe) 3 gfp+ neonates were dissociated
and transplanted (20,000 cells in 2[tL) into the lateral ventricles (LV) of three month old
C57BL/6 females (n = 15, three independent experiments of 5 animals each) and allowed
to engraft for 3 weeks, at which time the host animals were sacrificed. For each series,
two animals were perfused via intra-cardiac puncture with paraformaldehyde and
prepared for tissue immunohistochemistry to determine levels of engraftment by the
transplanted NS. SEZ tissue was isolated from the remaining 3 animals and cultured for
the generation of NS, with resulting NS analyzed for the presence ofgfp. While a
majority of the transplants resulted in some level of engraftment (Figure 3-1), no gfp+ NS
were evident in any of the cultures, with the NS population consisting only of spheres
similar in appearance to age matched control C57BL/6 NS (Figure 3-2).
Figure 3-1. Gfp+ neurosphere derived cells are evident in the brains of host C57BL/6
mice three weeks following transplantation. Photographic montage of 10x
images from four month C57BL/6 female transplanted with gfp+ NS three
weeks prior to analysis. Arrow depicts the presence of gf+ cells in the neural
tissue surrounding the lateral ventricle (V). Red = P-III tubulin, green = gf.
OB = olfactory bulb, RMS = rostral migratory stream.
Gfp+ Neurospheres are not Generated from the Brains of Neonatal C57BL/6 Mice
Transplanted with Gfp+ Neurospheres
As the yield of engraftment following transplantation of gfp+ NS into the LV of
adult mice is variable and minimal, the assay design was modified to use neonatal
C57BL/6 mice as the recipient animal in hopes that the higher levels of engraftment in
the neonatal model would allow for recovery of the now engrafted, transplanted cells.
As previously mentioned, neurogenesis in the neonatal (dpe 1-3) mouse brain is
Figure 3-2. Gfp+ neurospheres are not capable of re-isolation following transplantation
into the lateral ventricle of adult mice. NS were cultured from the forebrain of
adult C57BL/6 mice transplanted with gfp+ NS 3 weeks prior. Resultant
spheres were more similar in fluorescence to C57BL/6 control NS than gfp+
control NS derived from age-matched gfp mice. A) Gfp+ control NS derived
from gfp mice. B) C57BL/6 control NS. C) NS isolated from C57BL/6 mice
transplanted with gfp+ NS 3 weeks earlier. All images at 10x magnification.
more active than observed in the adult, theoretically providing higher levels of extra-
cellular cues for differentiation and engraftment of transplanted NSCs. Gfp+ NS
cultured from dpe 1-3 neonatal mice were dissociated and transplanted (75,000 cells,
1 tL) into the LV of dpe 1-3 C57BL/6 mice (n = 9) and allowed to engraft for one month.
Immunohistochemical analysis of three transplanted animals indicated engraftment levels
higher than that seen in the previous adult transplants, with donor-derived cells present in
the SEZ, RMS, and OB (Figure 3-3). The morphology of the donor-derived cells in the
OB are similar to granule neurons, indicating that the transplanted NS cells differentiated
from an immature stem cell to that of a terminally differentiated neuron, presumably via a
migratory neuroblast intermediate.
The remaining 6 transplanted animals were sacrificed and cultured for the
generation of NS, with generated NS analyzed for the expression of gfp as compared to
control NS isolated from both gfp and C57BL/6 mice. NS cultures from the forebrain of
the remaining transplanted animals yielded no gfp+ NS (Figure 3-4) with resultant
spheres more similar to C57BL/6 control NS than gfp+ NS.
Figure 3-3. Gfp+ neurospheres display high levels of engraftment following
transplantation into neonatal mice. Four weeks post-transplantation, donor-
derived cells from gfp+ NS are present in the SEZ, RMS, and OB of C57BL/6
recipient mice. A) Fluorescent microscopic analysis reveals the presence of
engrafted donor-derived cells at the site of injection in the lateral ventricle
(photographic montage, 10x light microscope images). B) Donor-derived
cells in the OB display extensive integration of processes into the surrounding
neural architecture of the OB (20x confocal image). C) Donor-derived cells
are present in the SEZ of recipient animals and adopt a migratory appearance
(20x confocal image). OB = olfactory bulb, RMS = rostral migratory stream,
V = lateral ventricle. All images: red = P-III tubulin, green = gfp
Figure 3-4. Gfp+ neurospheres are not capable of re-isolation following transplantation
into the lateral ventricle of neonatal mice. NS were cultured from the
forebrain of adult C57BL/6 mice transplanted with gfp+ NS 4 weeks prior.
Resultant spheres were more similar in fluorescence to C57BL/6 control NS
than gfp+ control NS derived from age-matched gfp mice. A) Gfp+ control
NS derived from gfp mice. B) C57BL/6 control NS. C) NS isolated from
C57BL/6 mice transplanted with gfp+ NS 4 weeks earlier. All images at 10x
Gfp+ Multipotent Astrocytic Stem Cells are not Generated from the Brains of
Neonatal C57BL/6 Mice Transplanted with Gfp+ Multipotent Astrocytic Stem
While NS cultures are an accepted in vitro correlate to the NSC, the protocol for the
generation and dissociation of NS results in low yield and extended culture conditions.
Conversely, the MASC is relatively simple to culture and generates large cell numbers in
a comparatively short time without the presence of mitogens required for NS growth. To
this end, gfp+ MASC were utilized as the transplanted NSC to determine if a cell type
that produces a high yield in culture may reveal the presence of gfp+ cells following
transplantation and re-isolation.
MASC were cultured from the SEZ of dpel-3 gfp neonatal mice, and passage
three times prior to transplantation. This step is necessary for the removal of any
contaminating neuronal cells that may persist in the initial days in vitro but will die off in
the absence of EGF and bFGF. Prior to transplantation, aliquots of prospective MASC
cultures were plated onto adherent chamber slides and allowed to attach. Attached cells
were later stained for the presence of 0-III tubulin positive neurons and GFAP positive
astrocytes (Figure 3-5). Cultures devoid of neurons were collected for transplantation.
Figure 3-5. Passage three gfp+ multipotent astrocytic stem cells contain astrocytes but are
devoid of neurons. Following three passages in vitro, gfp+ MASC were
plated onto adherent chamber slides and analyzed for the presence of 0-III
tubulin positive neurons and GFAP positive astrocytes. Slides analyzed at
20x magnification revealed confluent monolayers of GFAP expressing gfp+
cells with the morphology of astrocytes that contained no observable neurons.
A) Gfp+ MASCs (green) stained for P-III tubulin (red). B) Gfp+ MASCs
(green) stained for GFAP (red). All images at 20x magnification.
C57BL/6 neonatal mice (dpe 1-3, n = 50, 7 independent experiments) were
transplanted with passage-3 gfp+ MASC (100,000 cells per transplant in 1ItL) into the
LV and allowed to engraft for three weeks. In each series of transplants, animals
analyzed for engraftment exhibited levels similar to those seen in previous NS transplants
(Figure 3-6). Donor derived cells were present in the SEZ, RMS and OB, with cells in
the OB adopting a granule neuron morphology. MASC cultures derived from the
forebrain of the remaining animals did not appear to express gfp as compared to controls
MASC Isolated from the Brains of C57BL/6 Neonatal Mice Transplanted with Gfp
MASC do not Survive Puromycin Treatment.
MASC cultures derived from transplanted mice may contain gfp MASC, albeit in
extremely low amounts. In an effort to enrich the resulting cultures for the presence of
Figure 3-6. Gjp+ multipotent astrocytic stem cells display high levels of engrattment
following transplantation into neonatal mice. Three weeks post-
transplantation, donor-derived cells from gfp+ MASC are present in the SEZ,
RMS, and OB. A) Fluorescent microscopic analysis reveals the presence of
engrafted donor-derived cells at the site of injection in the lateral ventricle
(photographic montage, 10x light microscope images). B) Donor-derived
cells in the OB display extensive integration of processes into the surrounding
neural architecture of the OB (20x confocal image). C) Donor-derived cells
are present in the SEZ of recipient animals and adopt a migratory appearance
(20x confocal image). OB = olfactory bulb, RMS = rostral migratory stream,
V = lateral ventricle. All images: red = P-III tubulin, green = gfp
gfp positive cells, MASC monolayers were cultured in the presence of puromycin.
Puromycin was the selective agent utilized during the generation of the gfp transgenic
animal (32), and all cells in the gfp animal theoretically express the puromycin resistance
gene along with gfp. Indeed, MASC cultures isolated from the forebrains of gfp neonatal
mice (dpel-3) survived in the presence of 1, 2, and 3 [g/mL concentrations of
puromycin. MASC isolated from the forebrains of control C57BL/6 neonatal mice failed
to survive at any of the three concentrations (Figure 3-8).
MASC cultures isolated from the brains of 18 animals transplanted with gfp+
MASC three weeks earlier were cultured in the presence of 2[g/mL puromycin for nine
days. Surviving cells were returned to normal culture conditions to generate confluent
monolayers after which they were transplanted into the lateral ventricles of neonatal
C57BL/6 mice (dpe 1-3, 100,000 cells, n = 5). Analysis of the brains of transplanted
animals revealed the presence of auto-fluorescent cellular material in the RMS and walls
of the LV, but the noticeable absence ofgfp+ cells in the SEZ/RMS/OB of recipient mice
three weeks following transplantation into the LV (Figure 3-9).
Figure 3-7. Gfp+ multipotent astrocytic stem cells are not present in the cultures of
transplanted neonatal mice. MASC were cultured from the forebrain of
C57BL/6 mice transplanted with gfp+ MASC 3 weeks prior (n = 50, 7
independent experiments). Resultant cells did not express gfp as compared to
age matched gfp+ MASC control cultures. A) Gfp+ MASC control culture.
B and C) MASC cultures derived from C57BL/6 mice transplanted with gfp+
MASC 3 weeks earlier. All images at 20x magnification.
To ensure that any cells surviving exposure to puromycin were indeed resistant and
not merely remnants of puromycin-negative cells, MASC isolated from primary
transplanted animals (n = 8) were treated with 2 [g/mL puromycin for nine days.
Surviving cells were collected and re-cultured to generate a secondary monolayer of
2 ug/ml Puro
Figure 3-8. Qualitative assessment of the effects of puromycin on multipotent astrocytic
stem cells cultures derived from C57BL/6 and gfp+ neonatal mice. Passage 2
MASC cultures were grown in the presence of puromycin for nine days at 1, 2
and 3 [g/mL puromycin. The above 10x phase-contrast images reveal that
while all three concentrations of puromycin are lethal to C57BL/6 MASC, gfp
MASC proliferate regardless of the concentration of the selective agent.
Figure 3-9. Transplantation of puromycin selected multipotent astrocytic stem cells
isolated from C57BL/6 mice transplanted with gp+ multipotent astrocytic
stem cells exhibit no gfp donor cell engraftment. Three weeks following
transplantation, recipient animals display no evidence of gp+ cells. Auto-
fluorescent cellular material is present in the RMS and LV of transplanted
animals, indicative of lipofusion or apoptotic cellular debris. A) RMS of
transplanted animal, 10x gfp image. B) 10x image of RMS depicted in A, red
= 0-III tubulin. C) Merged image of A and B. D) LV of transplanted animal,
10x gfp+ image. E) 10x image of LV depicted in D, red = P-III tubulin. F)
Merged image of D and E.
1 ug/ml Puro
3 ug/ml Puro
MASC, at which time the cells were again cultured in 2 [g/mL puromycin for nine days.
Of an estimated 1 x 106 cells exposed to a secondary puromycin treatment, only four cells
remained. None of the four cells analyzed expressed gfp, and had a punctate, unhealthy
morphology compared to age matched gfp MASC cultures (Figure 3-10), indicating that
no gfp cells exist in the secondary cultures of animals transplanted with gfp+ MASC.
Long Term Engraftment
As previously mentioned, long-term engraftment is an essential quality possessed
by the HSC and for the NSC to be classified as a true stem cell it must display a similar
capability. Three neonatal C57BL/6 (dpel-3) were sacrificed 14 months following
transplantation of gfp+ NS into the lateral ventricle. Analysis of sagittal sections of these
animals revealed the existence of donor-derived cells in the olfactory bulb, parenchyma,
and SEZ. These donor-derived cells appeared as either granule cells with extensive
processes or as cells with an astrocytic morphology (Figure 3-11). Of particular interest
was the noticeable lack of migratory neuroblasts in the RMS of these animals. The
absence of these cells would argue that long-term contribution to the migratory
neuroblast population was not provided by the transplanted NS, with only transient
contribution to the RMS and OB intemeurons provided by the transplanted cells.
Figure 3-10. Multipotent astrocytic stem cells isolated from the forebrains of mice
transplanted with gfp+ multipotent astrocytic stem cells do not survive
puromycin treatment. Following isolation and culture, MASC from
transplanted forebrains were treated with 2[g/mL puromycin twice for a
duration of nine days per treatment. Surviving cells were not gfp positive as
compared to gfp controls. A) Gfp control MASC, 10x phase contrast image.
B) Gfp control MASC, 10x image, green = gfp. C) Cell which survived
puromycin selection, 10x phase contrast image. D) Same cell depicted in C,
gfp filter. E) Cell which survived puromycin selection, 10x phase contrast
image. F) Same cell depicted in E, gfp filter.
Figure 3-11. Donor-derived cells exist in the brains of gfp neurosphere transplanted
animals 14 months after surgery. Dissociated gfp+ NS were transplanted into
the lateral ventricles of neonatal C57BL/6 mice, and allowed to engraft for 14
months. Donor-derived gfp+ cells were present in the olfactory bulb as
granule neurons with extensive processes and in the walls surrounding the
lateral ventricles and subependymal zone as astrocytic cells. No donor-
derived cells were observed in the rostral migratory stream of these animals.
A) Sagittal photographic montage of C57BL/6 mouse transplanted with gfp+
NS 14 months earlier (10x images). B) 20x image of donor-derived cell in the
SEZ. C) 20x image of donor-derived cell in the parenchyma surrounding the
lateral ventricle. D) and E) 40x images of donor-derived cells in the ob
adopting the morphology of granule neurons. All images: red = P-III
Tubulin, green = gfp. OB = olfactory bulb, RMS = rostral migratory stream,
SEZ = subependymal zone, V = lateral ventricle.
IONIZING RADIATION ENHANCES THE ENGRAFTMENT OF TRANSPLANTED
IN VITRO DERIVED NEURAL STEM CELLS
Neurogenesis in the adult rodent is limited to two well-characterized regions of the
brain: the subgranular layer of the hippocampal dentate gyms, and the subependymal
zone (SEZ) (9, 25, 27, 38). The former produces neurons that functionally integrate into
the granular cell layer of the hippocampus. The SEZ produces neuroblasts in the walls of
the lateral ventricles that migrate along a defined pathway, known as the rostral migratory
stream (RMS), to the olfactory bulb (OB) where they differentiate and functionally
integrate into the existing cytoarchitecture as granule or periglomerular interneurons (45,
46, 47, 78).
These migrating neuroblasts have been well characterized, and are known to be
immunopositive for both the pan-neuronal marker P-III-tubulin, and the polysialylated
neuronal cell adhesion molecule (PSA-NCAM), an antigen restricted mainly to cells in
the brain that are undergoing active migration (15). The number of newly generated
neurons produced daily in the adult mouse has been estimated at 30,000, leading many to
conclude that a self-renewing stem cell must reside in the SEZ in order for this rate to be
sustained for the life of the animal (5). The cell type in the SEZ believed to be the NSC
has been identified by Doetsch and colleagues as a slowly dividing astrocyte known as
the type B cell, that generates the migratory neuroblasts (type A cells) via a transit
amplifying intermediate precursor (type C cell) (4).
Neural stem cells can be isolated from the adult SEZ and cultured in vitro to form
spherical clones known as neurospheres (NS) (22, 29, 39, 42, 62-65, 83), that are capable
of producing the major cell types of the neural lineage (neurons, astrocytes, and
oligodendrocytes) upon differentiation. They are also capable of self-renewal, allowing
them to increase in number following repeated passages in culture while retaining their
multipotency, and have also shown the ability to integrate into host neural tissue upon
transplantation into the host brain (80). It is these traits that have led many to believe that
the NS-forming cell is the in vitro correlate of the in vivo NSC. Multipotent astrocytic
stem cells (MASC) isolated from the SEZ of neonatal mice have also been identified as
an in vitro correlate of the NSC, displaying the potential to not only generate multipotent
NS but also to integrate into host neural tissue upon transplantation in much the same
way as the NS (43, 89). The relatively simple culture conditions required in maintaining
the MASC in vitro offer an attractive alternative to the more complicated conditions
required by the NS.
Transplantation of in vitro NSC (NS or MASC) derived from animals transgenic
for the reporter gene encoding green fluorescent protein (gfp+) into the LV of adult
control animals results in minimal engraftment into the host neural tissue, with few
donor-derived neuroblasts present in the RMS or OB. Transplantation into neonatal
mice, however, results in relatively robust levels of engraftment with comparatively high
numbers of donor-derived neuroblasts and interneurons present in the OB weeks after
transplantation. This presents a problem for research into adult neurogenesis, as the
neonatal and adult brains are dramatically different with respects to active neurogenesis
and results obtained from neonatal transplants may not be applicable to adult studies. A
method for enhancing the engraftment potential of in vitro NSC into adult animals is
crucial in order to better understand the receptiveness of the adult SEZ to transplanted
cells and consequently the functional identity of the transplanted cells.
Perhaps the most well characterized adult stem cell is the hematopoietic stem cell,
which in addition to the aforementioned stem cell characteristics of pluripotency and self-
renewal also has the ability to repopulate the entire hematopoietic system of a
myeloablated animal. Ablation of endogenous HSCs by exposure to high doses of
ionizing radiation is critical for facilitating maximum engraftment of transplanted HSCs,
that cannot normally compete with the native HSC for access to the stem cell niche (18,
19, 20). This reconstitution ability is an essential component in the generally accepted
classic definition of a HSC.
Depletion of the NSC niche has been previously achieved with anti-mitotic agents
(15) and both ionizing and x-irradiation (6, 51, 59, 60, 71, 72) yielding transient and
long-term depletion of neurogenesis in the hippocampus and SEZ. Neurogenesis in the
hippocampus can be attenuated by exposure to varying levels of x-irradiation, as seen by
a decrease in the number of migrating granular neurons out of the subgranular layer (52,
59, 60, 71).
Previous studies investigating the effects of on SEZ neurogenesis following
focused exposure of x-irradiation to the brain of adult rats have reported ablation of of
mitotic cells in the SEZ immediately following irradiation, with a dose dependent
recovery of these cells occurring within 2 months of exposure (6, 72). With the
exception of all but the lowest level of exposure, the observed recovery of neurogenesis
remained well below control levels. Little has been done, however, to characterize the
effects of a single, whole-body lethal or sub-lethal dose of ionizing radiation on SEZ
neurogenesis and subsequent engraftment of transplanted in vitro derived NSC in the
Effects of Lethal Irradiation on Subependymal Zone Neurogenesis
Lethal Irradiation Severely Depletes Migrating Neuroblasts in the RMS
Three weeks following lethal irradiation (LI), bone marrow reconstituted mice
show a marked decrease in the number of P-III tubulin positive neuroblasts in the RMS
(n = 8, Figure 4-1 D). This decrease is variable; some animals were found to have few
or no migrating neuroblasts, while others contained a number of pockets of neuroblasts,
though always drastically fewer than that seen in un-treated littermates (Figure 4-1 B).
To determine if this reduction is permanent, (as opposed to the transient reduction
observed with anti-mitotic treatments) animals were analyzed 3 months after LI. The
results indicate that the depletion of migratory neuroblasts persists even at this time point
(Figure 4-2). To show that the decrease in the number of 0-III tubulin positive
neuroblasts is not due merely to antigen down-regulation, sagittal sections of brain from
LI mice were also stained for the presence ofPSA-NCAM; a marker specific to migrating
neuroblasts (46). Three weeks following LI, the number of migrating PSA-NCAM
positive neuroblasts was severely depleted in the RMS of those mice (Figure 4-1 C) as
compared to un-treated littermates (Figure 4-1 A), again with a small portion of
migratory neuroblasts remaining in the RMS (* in Figure 4-1 C). These findings led to
the conclusion that the exposure to x-irradiation resulted in permanent damage to the
neuroblast-producing cell of the SEZ, but due to the occasional observed cluster of
migratory neuroblasts, accurate quantification of this depletion was performed.
Figure 4-1. Lethal irradiation drastically reduces the volume of PSA-NCAM and P-III
Tubulin positive migratory neuroblasts. Sagittal photographic montages of 5x
light microscopic images. A and B) Untreated C57BL/6 female brain. C and
D) C57BL/6 female, 3 weeks post-lethal irradiation. Note the absence of 0-III
Tubulin positive migratory neuroblasts in the LI brain (D) compared to
control (B) and the diminished volume of PSA-NCAM positive migratory
neuroblasts (C) as compared to control (A). Red = PSA-NCAM, Green = 3-
III Tubulin. OB = olfactory bulb, RMS = rostral migratory stream, LV =
lateral ventricle. (*) denote migratory neuroblasts.
Figure 4-2. Reduction in the volume of 0-III Tubulin positive migratory neuroblasts
persists months after lethal irradiation. Sagittal photographic montages of 10x
light microscopic images. Red = P-III Tubulin. A) C57BL/6 female brain
two months post-LI. B) C57BL/6 female brain three months post-LI. Note
the absence of migratory neuroblasts in both images as compared to non-
irradiated controls (Figure 4-1, B).
Analysis and Quantification of Neuroblast Depletion in the Subventricular Zone
Following Lethal Irradiation, as Determined by BrdU Incorporation
In order to quantify the degree of neuroblast depletion, 5-bromo-2'-deoxyuridine
(BrdU) was used to label mitotic cells within the SEZ. Briefly, 3 weeks and 3 months
following LI (n = 4 for each condition), treated and un-treated mice were given BrdU
intra-peritoneally three times a day for three days allowing for the incorporation of BrdU
into the DNA of dividing cells. SEZ neuroblasts were analyzed by the application of
antibodies against P-III tubulin and BrdU on serial coronal sections at the level of the
anterior commisure (AC). BrdU positive cells were scored blindly, by analyzing three
adjacent sections per brain (scorer was ignorant to the experimental condition of the
animals analyzed). Scoring of adjacent sections was performed only on those sections
where the AC existed as a spherical white-matter structure, directly inferior to the inferior
most extent of the LV. The region of analysis was maintained throughout all animals
scored (Figure 4-3).
In all un-treated animals, the SEZ was observed to contain a robust layer of
dividing neuroblasts (Figure 4-4 A). 3 weeks following LI in age matched mice this
same region exhibited a significant decrease in neuroblasts activity (Figure 4-4 B). This
depletion was observed out to 3 months post-LI (Figure 4-4 C). P-III tubulin
immunolabeling (green, Figure 4-4 insets) was used to confirm that the cells scored were
in fact neuroblasts and not mitotic glial cells of the SEZ. .
Figure 4-5 is a graphical representation of the number of BrdU positive cells for
each condition (4 mice per condition, 3 sections per animal). 3-weeks following LI, the
volume of BrdU positive SEZ neuroblasts decreases to 40% of control. Student's t-test
confirmed that this difference was significant (p value = 0.003).
Figure 4-3. Region of analysis for quantification of mitotic neuroblast depletion in the
subependymal zone of lethally irradiated mice. Coronal photographic
montage of 10x light microscopic images, with the white outline depicting the
region of analysis as encompassing an area from the inferior, descending horn
of the LV to the superior horn of the LV, and continuing 700[tm lateral from
the superior horn while remaining approximately 5 cells in width. Inset:
reprentative image of BrdU positive neuroblasts lining the wall of the lateral
ventricle expressing P-III tubulin. CC = corpus callosum, LV = lateral
ventricle, ST = striatum, SP = septum, and AC = anterior commissure. Red =
BrdU, Green = P-III tubulin.
Figure 4-4. Depletion of mitotic neuroblasts in the subependymal zone of lethally
irradiated animals. The observable volume of BrdU positive (red) neuroblasts
is severely diminished three weeks and three months post-LI as compared to
non-irradiated controls. A) Non-irradiated control. B) 3 weeks post-LI. C) 3
months post-LI. All are coronal photographic montages of 10x light
microscope images. All insets are 40x images of neuroblasts lining the wall of
the ventricles, positive for both BrdU and P-III tubulin (green) identifying
them as neuronal.
There is an approximate 87% decrease in the number of BrdU positive cells at the
3-month time point as compared to control, and this difference is also significant
(p value = 0.002). The 68% decrease in the number of BrdU positive cells between 3
weeks and 3 months post LI was also significant (p value = 0.03), indicating that the
observed depletion is permanent.
The depletion of mitotic cells in the SEZ is also observed in the RMS, with a
marked decrease in the visible BrdU/P-III tubulin positive migratory neuroblasts one
month following LI (Figure 4-6).
Diminished Neurosphere Yield from Lethally Irradiated Mice
As it is well accepted that the NS forming cell is the in vitro manifestation of the
NSC (63), NS from both WT and LI mice were cultured in a blind-study format to
determine if the stem cell pool in the SEZ was affected by exposure to LI.
p = <0.05
3 weeks 3 months
Figure 4-5. Lethal irradiation significantly decreases the number of BrdU positive SEZ
neuroblasts. The total number of BrdU positive neuroblasts in three adjacent
coronal sections of either wild-type or LI mice was tabulated and placed into
the above graphical format. Three weeks following lethal irradiation (n=4),
the number of BrdU positive neuroblasts decreased to 40% of those calculated
to exist in the wild-type controls (n = 4, p value of 0.003). Three months
following lethal irradiation, this decrease is slightly greater, at 13% of control
(n = 4, p value of 0.002). Analysis by student's t-test confirmed these values
to be significant, as compared to non-irradiated controls.
Figure 4-6. Depletion of migratory neuroblasts in the rostral migratory stream positive
for both BrdU and P-III Tubulin in lethally irradiated mice. Sagittal
montages of 20x images reveal that the visible volume of 0-III tubulin/BrdU
positive cells is severely diminished in LI mice as compared to non-irradiated
controls. A) Non-irradiated control. B) LI mouse, one month post-LI.
Animals were injected with BrdU according to the protocol described in
materials and methods. Brains were sectioned and stained for BrdU (red) and
P-III tubulin (green). Ob = olfactory bulb, rms = rostral migratory stream,
V = lateral ventricle.
NS cultured from LI brains displayed a significant decrease in yield of
approximately 77%, when compared to the non-irradiated control cultures (Figure 4-7, p
value = 0.02). This decrease closely corresponds to the decreased levels of BrdU positive
neuroblasts in vivo following lethal irradiation, further supporting the validity of this
finding. It has been reported that the stem cell population of the SEZ is between
approximately 0.02% and 1.0% of the total cells (62, 66, 82), as determined by NS yield
from dissociated SEZ tissue, and the average yield of NS from the wild-type brains in the
present study falls within this range (0.15%). The average yield of NS isolated from the
lethally irradiated brains was significantly lower, at 0.03%, indicating that exposure to
lethal doses of radiation depletes the number of NSC in the brain responsible for the
generation of NS.
Preliminary studies revealed no effect on the potential for differentiation was
observed in NS isolated from LI animals one month following irradiation as compared to
non-irradiated controls. The approximate diameter of the resultant NS was comparable to
controls (Figure 4-8), and when placed on adhesive substrates in the absence of growth
factors to induce differentiation NS from LI mice produced both astrocytes and neurons.
LI appears to only affect the yield of NS isolated from irradiated animals, with the few
spheres formed retaining a normal phenotype, although further studies are needed to
verify that irradiation in no way affects NS differentiation potential.
Lethal Irradiation Attenuates Engraftment of Transplanted Gfp+ Multipotent
Astrocytic Stem Cells
Three weeks after irradiation, LI animals were transplanted with gf+ MASC
(generated from neonatal forebrains) into the lateral ventricle and the cells allowed to
engraft for three weeks.
p = <0.05
Wild Type Lethal Ihadiation
Figure 4-7. Lethal irradiation significantly reduces the yield of neurospheres cultured
from the adult subependymal zone. SEZ tissue isolated in a blind-study
format was cultured to generate NS from both wild-type adult mice and adult
mice LI two months prior, with all tissues treated in identical fashion (n=3).
The resulting NS yield was determined according to the protocol described in
the methods section. The LI cultures averaged 77% fewer NS than did the
identical cultures derived from control mice (significant decrease; p value of
Figure 4-8. Neurosphere differentiation potential. NS isolated from non-irradiated
control animals are capable of differentiation into astrocytes and neurons upon
attachment to adhesive substrates in the absence of EGF and bFGF. A) 10x
phase contrast image of NS derived from non-irradiated control. B) 20x
image of differentiated control NS stained for GFAP (red). C) 40x image of
differentiated control NS stained for P-III tubulin (red) and DAPI (blue).
Figure 4-9. Attenuation of gfp multipotent astrocytic stem cell engraftment by lethal
irradiation. Control animals exhibited normal engraftment, as evidenced by
the presence of donor-derived migratory neuroblasts. A) Photographic
montage of 10x images from a sagittal section of non-irradiated animal two
weeks post transplant (red = P-III tubulin, green = gfp). B and C) 40x images
of donor-derived migratory neuroblasts in non-irradiated control. LI animals
displayed no evidence of engraftment, with no donor-derived cells seen in
either the SEZ, RMS, or OB. D) Photographic montage of 10x images from a
sagittal section of LI animal two weeks post transplant. E) 20x image of 0-III
tubulin positive migratory neuroblasts in the RMS. F) Same image as E, but
with gfp filter, revealing the absence of donor-derived migratory cells.
OB = olfactory bulb, RMS = rostral migratory stream, V = ventricle.
Analysis of 40 |tm sagittal sections revealed the presence of donor-derived cells
only in the corpus callosum, parenchyma and SEZ, but the absence of donor-derived
migratory cells in the RMS or OB (n = 5), while non-irradiated controls were observed to
contain a small but consistent number of donor-derived migratory neuroblasts in the OB
As the lethal dose of ionizing radiation inhibited functional engraftment of the
transplanted cells (possibly by inducing irreparable damage to the radio-sensitive support
cells of the SEZ) a lower exposure dose of 450 rads was assayed as a milder form of
injury for the potential enhancement of gfp MASC engraftment.
Effects of Sub-Lethal Irradiation on Subependymal Zone Neurogenesis
Sublethal Irradiation Results in a Transient Decrease in the Number of Mitotic
Subependymal Zone Neuroblasts.
Unlike LI, SLI (450 rads) does not complete abolish hematopoiesis in the bone
marrow of adult mice, and SLI animals to not require transplantation of bone marrow to
survive exposure. Low levels of focused gamma irradiation (1 to 3Gy) have been shown
to result in a transient increase in mitotic cell activity in the SEZ, with levels eventually
diminishing in the weeks following in a dose dependent fashion when compared to
untreated controls (6, 72).
Contrary to the observations following LI, no decrease was observed in the overall
volume of 0-III tubulin positive migratory neuroblasts in the RMS of irradiated animals 3
weeks following whole body exposure to 450rads (a dose equivalent to 4.5Gy).
BrdU quantification assays revealed that mitotic cell activity in the SEZ was significantly
decreased in the hours immediately following irradiation (Figure 4-10). The levels of
mitotic cell activity returned to near normal at 3 weeks and eventually increased to above
control levels at 6 weeks (Figure 4-11).
Diminished Neurosphere Yield from Sublethally Irradiated Mice
Blind-analysis of NS yield (same protocol as the LI study, n = 4) from SLI brains at
3 weeks revealed a 30% decrease in the number of NS as compared to non-irradiated
controls (Figure 4-12, p = 0.032), with control NS yield again falling into the
aforementioned acceptable range of 0.2 to 1.0% (0.12%). As in the LI studies, no
observable effect was observed in preliminary studies of NS potential in SLI mice,
although further studies are needed to verify this initial observation.
Sublethal Irradiation Enhances Engraftment of Transplanted Gfp+ Multipotent
Astrocytic Stem Cells
Three weeks following irradiation, SLI animals were transplanted with gf+
MASC into the lateral ventricle and the cells were allowed to engraft for three weeks.
Analysis of 40 |tm sagittal sections revealed that in a portion of the irradiated animals a 4
fold higher number of gfp migratory cells were present in the OB as compared to non-
irradiated controls (n = 14, Figure 4-13). Taken as a whole, the average number of g+
migratory cells in the OB of SLI animals was twice the average number seen in non-
irradiated controls (significant value, p = 0.014) indicating that SLI significantly
enhances the engraftment potential of transplanted gfp+ MASC, albeit in a variable
fashion (Figure 4-14).
Figure 4-10. Mitotic subependymal zone neuroblasts are transiently depleted following
sub-lethal irradiation. 6 hours following irradiation, the SEZ is almost
completely devoid of BrdU (red) cells as compared to non-irradiated controls.
3 weeks following irradiation, BrdU positive cells in the SEZ are near to the
levels observed in controls. A) Non-irradiated control. B) 6 hr post-SLI. C)
3 weeks post-SLI. All are coronal photographic montages of 10x light
microscope images. All insets are 40x images of neuroblasts lining the wall of
the ventricles, positive for both BrdU and P-III tubulin (green) identifying
them as neuronal.
0 0 60
neuroblasts than in non-irradiated controls (p = 0.001, n = 3). This depletion<0.05
20 p <0.05
6 hours 24 horn's 48 hours 3 weeks 6 weeks
Time after Irradiation
Figure 4-11. Sublethal irradiation results in transient, recoverable depletion of mitotic
subependymal zone neuroblasts. The total number of BrdU positive
neuroblasts in three adj acent coronal sections of either control or SLI mice
was tabulated and placed into the above graphical format for quantification of
the number of BrdU positive SEZ neuroblasts calculated as percent of control.
Six hours following SLI there are approximately 83% fewer BrdU positive
neuroblasts than in non-irradiated controls (p = 0.001, n = 3). This depletion
persists 24 (78%,p = 0.001, n =3) and 48 hours (90%,p = 0.001, n = 3)
following SLI. Three weeks following irradiation the number of mitotic SEZ
neuroblasts recovers to near control levels, and eventually increases to 13%
above control levels at 6 weeks (p = 0.003, n = 3).
a p = <0.05
Control Sub-Lethal Irradiation
Figure 4-12. Sublethal irradiation significantly reduces the generation of neurospheres
cultured from adult subependymal zone. SEZ tissue was isolated in a blind
format from both non-irradiated control adult mice and adult mice SLI three
weeks prior and cultured to generate NS, with all tissues treated in identical
fashion. The resulting NS yield was determined according to the protocol
described in the methods section. The SLI cultures averaged 30% fewer NS
than did the identical cultures derived from control mice (significant decrease;
p = 0.032, n = 4).
Figure 4-13. Gfp+ multipotent astrocytic stem cells transplanted into the lateral
ventricles of control and sub-lethally irradiated mice produce donor-derived
migratory cells in the host olfactory bulb. Age matched mice (SLI and non-
irradiated controls) were sacrificed three weeks following intra-ventricle
transplantation of passage 3 gf+ MASC. 40 [im sagittal sections of the
transplanted hemisphere were collected in a serial fashion, stained for P-III
tubulin (red) and analyzed by light and confocal microscopy for the presence
ofgfp+ donor-derived cells (green). A and D): 10x photographic montages of
control and SLI mice (respectively). Circles represent the presence of donor-
derived migratory neuroblasts, with representative cells captured at 40x
(insets). B and C) 63x confocal images ofgfp+ migratory cells in the
olfactory bulb of non-irradiated control mice. E and F) 63x confocal images
ofgfp+ migratory cells in the olfactory bulb of SLI mice. Scale bars = 40[m.
p = <0.05
E 5 -----
Sub Lethal Irradiation
Figure 4-14. Sublethal irradiation enhances engraftment of donor-derived neuroblasts.
Quantification of 14 transplanted animals per condition revealed the presence
of approximately twice as many donor-derived migratory neuroblasts in the
olfactory bulb proper of SLI animals as compared to non-irradiated controls (p
= 0.014). Transplanted hemispheres were sectioned three-weeks following
transplantation of passage 3 gfp+ MASC into the lateral ventricle. All
sections containing the olfactory bulb were collected in a serial fashion and
stained for the presence of 3-III tubulin.
DISCUSSION AND CONCLUSIONS
Neurogenesis in the subependymal zone (SEZ) of the adult mouse persists for the
life of the animal, alluding to the existence of a persistent neural stem cell (NSC) pool in
this region. While NSCs cannot yet be prospectively purified like the hematopoietic stem
cell (HSC), they can be cultured under specific conditions to form clonal neurospheres
(NS) and multipotent astrocytic stem cells (MASC); potential NSC manifestations with
multipotent characteristics attributed to stem cells.
The extensive investigation of the HSC has produced a gold-standard definition of
a stem cell: a single cell that is capable of producing all cell types of a particular organ
for the life of the animal via asymmetrical division in which both an exact duplicate of
the stem cell and a lineage-committed progenitor daughter cell are generated.
Additionally, the stem cell can reconstitute its native niche following transplantation, and
this ability is retained following secondary transplantation, a phenomenon known as
serial reconstitution. Functional transplantation of a stem cell and its subsequent
reconstitution of the niche is a vital requirement, as the isolated cell can now be
considered a useful tool for tissue repair. Candidate stem cells from other organs would
need to meet the same criteria if they are to be classified as true stem cells.
Both NS and MASC transgenic for gfp exhibited low levels of engraftment upon
transplantation into the lateral ventricles of adult mice, with few donor derived migratory
neuroblasts in the RMS observed in the weeks immediately following transplantation.
Neither the NS nor the MASC displayed the ability to be re-isolated following
transplantation into proven regions of neurogenesis, a limitation resulting in the inability
of either cell types to survive subsequent serial transplantation. As neurological
development continues shortly after birth and potentially provides an environment rich in
extra cellular cues favored by NSCs, transplantation into the brains of neonatal mice was
performed in an effort to enhance the engraftment of transplanted NS and MASC. While
the levels of engraftment were visibly higher than those seen in adult transplants, neither
cell type survived secondary isolation attempts, even with the use of selective protocols
designed to enrich for gfp+ cells.
Long-term analysis of transplanted animals revealed the existence of donor-derived
granule neurons and astrocytic cells in the olfactory bulb, parenchyma and SEZ, but a
noticeable absence of migratory neuroblasts in the rostral migratory stream (RMS) of the
transplanted animals. This potentially alludes to transient engraftment as opposed to
NSC engraftment could be defined in this case as long-term contribution to
neuroblast pool in the adult animal following transplantation, with functionality exhibited
by the existence of terminally differentiated donor-derived cells in the neural circuitry of
the olfactory bulb. HSC engraftment into the bone marrow of myeloablated mice is
evidenced by the robust generation of daughter cells representing both the myeloid and
lymphoid lineages. The obvious evidence of functional engraftment by the transplanted
HSC is that the myeloablated animal survives the previous lethal dose of radiation; the
depleted bone marrow becomes repopulated by the transplanted HSC and its resultant
progeny. Long-term (i.e. 3 months or longer) engraftment and hematopoietic
contribution is a critical requirement in the definition of the HSC, as short-term, transient
engraftment can be supplied by hematopoietic progenitor cells. The ability to survive
serial-transplantation while retaining the capacity to provide long-term bone marrow
reconstitution fulfills the final requirement for classification as a true stem cell.
Currently, the liver hepatocyte is the only adult stem cell other than the HSC that has
displayed the potential for serial, functional engraftment. In a liver repopulation assay,
transplanted hepatocytes functionally contributed to the regenerating liver in a robust,
serial fashion with contribution observed following the sixth transplantation (58).
The results obtained reported here indicate that the in vitro derived NSCs were not
capable of re-isolation following transplantation into proven regions of neurogenesis.
Whether this is due to the cell population transplanted or the niche into which the cells
were placed is not known. The failure observed in the adult model could be attributed to
the decreased level of neurogenesis in the adult animal, resulting in fewer engrafted cells
and subsequently fewer isolatable cells. However, because transplants into neonatal mice
yielded the same results the cause likely lies in the cells transplanted rather than in the
niche itself. Both NS and MASC cultures are heterogeneous in nature, with cells existing
in varying stages of maturation. It is possible that the cell types observed to be
multipotent and proliferative in culture are progenitor cells derived from a relatively
small number of NSCs, implying that not every NS or MASC in culture is a NSC.
Transplantation into the SEZ results in mono-potent engraftment with only neurons
and neuroblasts observed in the RMS and olfactory bulb, an observation concurrent with
progenitor cell activity. Stable engraftment by the NSC should result in an increase of
donor-derived NSC via asymmetric division, allowing for subsequent re-isolation.
While initial engraftment was observed in the weeks immediately following
transplantation as evidenced by the presence of donor derived cells in the SEZ, RMS and
olfactory bulb, the engraftment ultimately was found to be transitory. As it was not
possible in the experiments performed to re-isolate a donor-derived NS or MASC, the
data presented here would indicate that the in vitro manifestations of the NSC are not true
stem cells, but rather a form of neural progenitor cell lacking the characteristics attributed
to true stem cells and may not be a viable cell source for potential stem cell therapy of
neurological injury and disease.
It is entirely possible that the observed neurogenesis in the adult brain is not the
result of an endogenous, isolatable NSC pool, but rather the product of migratory adult
stem cells that undergo a phenotypic shift upon integration into neurogenic regions of the
brain and subsequently give rise to more lineage committed neural progenitor cells.
Observations supporting the contribution of the HSC to adult neurogenesis are as
numerous as they are conflicting. Donor-derived microglia and astrocytes (17) and
neurons (8, 49) have been reported to exist in the brains of animals exposed to lethal
irradiation and reconstituted with HSC transgenic for reporter proteins (17), while a
recent investigation has argued strongly for the existence of non-fusion product, donor-
derived neurons in the hippocampus of humans following sex-mismatched bone marrow
transplant (12). It has also been suggested that 0.5% of astrocytes in the adult brain are
generated from a bone marrow derived cell (17), and it is generally accepted that the NSC
of the SEZ (the type B cell) is a form of astrocyte (4, 5, 15). With this in mind, the
proposal that adult neurogenesis is driven by an adult stem cell residing in the bone
marrow may not be implausible.
While the neonatal transplant model provides an ideal system for relatively robust
engraftment of transplanted NS and MASC, it is impractical for the investigation of adult
neurogenesis and adult NSC potential as the adult brain is more quiescent than the
developing neonatal brain. A technique is necessary to render the neurogenic niche (in
this case, the SEZ) more receptive to transplanted cells. The current tactic in the field of
hematopoiesis to maximize engraftment of transplanted HSC is to first deplete the host
bone marrow of HSC by exposure to lethal doses of radiation (myeloablation). This
renders the hematopoietic stem cell niche more receptive to transplanted cells, allowing
for stable long-term, multi-lineage engraftment. It was hypothesized that depletion of the
native NSC pool by lethal irradiation would render the SEZ more receptive to
transplanted cells and subsequently enhance the engraftment efficiency of the
Because a single, high dose of ionizing radiation is sufficient to permanently
deplete the bone marrow of viable HSC in adult mice, a similar result should be observed
in the SEZ, with the pool of NSC being similarly depleted. The number of mitotic cells
in the SEZ was decreased by approximately 60% three weeks following irradiation and
this decrease was slightly lower three months later, at 87%, indicating a long-term, if not
permanent depletion. The levels of migrating neuroblasts in the RMS reflected this
decrease, with the chains decreasing in volume at both three week and three month time-
If the NSC pool itself had been diminished by the radiation exposure, the number
of NS cultured from those brains should reflect this decrease in the number of BrdU
positive neuroblasts. In fact, it was observed that there were approximately 77% fewer
NS isolated from the brains of LI adult mice that had been irradiated two months earlier.
Preliminary observations indicated that only the yield of NS appeared to be affected, with
the resulting spheres being similar in size and multipotency to control NS. In addition,
the resulting cultures from control tissue supported earlier findings that the number of
NSC in the SEZ is between 0.02 and 1.0% (62, 66, 82), with the average NS yield in
cultures being approximately 0.15% of the SEZ tissue cultured. The percent yield of NS
cultured from the SEZ of LI mice was diminished, at 0.03%. These observations lead to
the conclusion that the SEZ is significantly depleted of NSC following LI, and that this
depletion is long-term, if not permanent. The reason for this permanent depletion of
neurogenesis is not entirely clear, although recent studies have indicated that the neuro-
inflammatory response to injury in the hippocampus inhibits neurogenesis by disruption
of normal stem cell function (52).
While the most prevalent in vitro manifestation of the NSC is the NS, the MASC
has recently emerged as an alternative to the NS. The NSC in vivo is generally accepted
to be a slowly dividing astrocytic cell described as the type B cell (4) residing in the
ciliated ependymal layer of the SEZ. Work by Laywell et al. demonstrates that by
surgically dissecting the SEZ of neonatal animals and culturing the subsequently
generated dissociated cells in neural media on adhesive substrates, a monolayer of
astrocytes is produced that contains a sub-population of MASC capable of generating
multipotent NS (43). Transplantation of gf+ MASC into the lateral ventricles of both
neonatal and adult C57BL/6 mice results in observable engraftment, with donor-derived
cells present in not only the SEZ but also in the RMS and OB as migratory neuroblasts
(89). Taken as a whole, these observations allude to the MASC being an acceptable in
vitro manifestation of the NSC.
Transplantation of gf+ MASC into the lateral ventricles of LI mice three weeks
following irradiation did not result in the expected increase of donor-derived migratory
cells in OB. Non-irradiated controls exhibited normal engraftment levels while
transplanted LI animals were observed to only contain donor-derived cells in the corpus
callosum and SEZ. While it is unknown why the transplanted cells did not engraft
normally into the neurogenic regions of LI animals, recent work has proposed that
deleterious effects to the microvasculature occur in the dentate gyms following 10Gy of
focused irradiation rendering the region unreceptive to transplanted cells (51) and a
similar phenomenon may occur here.
As LI was not observed to enhance engraftment of transplanted MASC, a milder
form of injury to the SEZ in the form of SLI (450rads) would assessed for enhanced
engraftment of the transplanted cells. Recent studies in stem cell biology have revealed
that injury to the candidate site of transplantation is critical for engraftment and/or
contribution of the transplanted stem cell, as the recipient niche is normally relatively
quiescent in a healthy adult animal and induction of injury often renders the niche more
receptive to transplanted cells (83). Indeed, SLI animals transplanted with gfp+ MASC
were observed to contain significantly more donor-derived migratory cells in the OB as
compared to non-irradiated controls. It is important to note that although this increase
was statistically significant it was highly variable with fully half of the transplanted SLI
animals exhibiting levels of engraftment similar to controls. The reason for this
variability is not known, although it could easily be attributed to the inherent
inconsistency observed to occur in adult transplants.
BrdU incorporation experiments to determine the mitotic cell levels at the time of
transplantation revealed that while the numbers of mitotic SEZ neuroblasts were
significantly depleted immediately following exposure, the levels returned to near normal
by three weeks and were found to be significantly higher at six weeks. This observation
was further supported by blind NS culture assays of SLI animals in which the observed
yield ofNS cultured from brains exposed to SLI 3 weeks earlier was significantly
decreased by 30%. As was observed in the LI study, the NS yield from non-irradiated
controls was again between 0.02 and 1% (0.12%). This mild injury to the brain may
potentially enhance neurogenic activity and allow for increased engraftment by
Candidate NSC have recently been isolated from the brain utilizing antibodies that
bind to unique cell surface antigens, such as CD15 and CD133 (10, 80). Isolated cells are
then subjected to culture conditions that will produce NS, indicating that the cell
population isolated contains NSCs. In the field of hematopoiesis, attempted in vitro
manipulation and subsequent expansion of cultured HSC has been shown to result in
decreased pluripotency and engraftment by the HSC, with only hematopoietic progenitor
cells lacking the capacity for self-renewal being successfully expanded in vitro (14) in
theory due to the incomplete reconstruction of the bone marrow microenvironment in
vitro. Transplantation of non-cultured, primary HSC isolated from donor bone marrow
still yields the most robust, long-term engraftment into the niche, and it is entirely
possible that the in vitro manipulation of NSC results in a similar loss of multipotency
and engraftment ability. Ideally, the NSC would be isolated directly from primary SEZ
tissue prior to manipulation or transplantation, but in order for the NSC to be identified
and isolated, a model system for robust engraftment is necessary for the analysis of the
functional ability of the NSC.
By utilizing the injury model described here, candidate cell populations isolated
according to surface antigen expression could be transplanted to the injury-activated SEZ,
followed by later analysis of the RMS for increased, long-term production of donor
derived neuroblasts. The resulting observations would then allow for a definitive
conclusion to be made as to if the isolated cell population expressed the characteristics of
a true stem cell.
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Gregory Paul Marshall II was born on August 6th, 1976 in Gainesville, FL. After
graduating from Bradford High School in Starke, FL in 1994, he attended St. Andrews
Presbyterian College in Laurinburg, NC where he earned a Bachelor of Science degree
in biology and met Kathleen Kelly, his future wife. After a two year employment with
Dr. Susan Frost as head laboratory technician, Greg entered into the University of
Florida's College of Medicine Interdisciplinary Program in Biomedical Sciences in the
year 2000. After joining the laboratory of Dr. Edward Scott, Greg began his research
into the characterization of neural stem cells. He has presented posters of his research at
the Keystone Symposia in 2003 and at the Annual Society for Neuroscience meeting in
2004, and has recently submitted a first author article for publication in the journal Stem