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Characterization of Canine Bone Marrow-Derived Stromal Cells

Permanent Link: http://ufdc.ufl.edu/UFE0021645/00001

Material Information

Title: Characterization of Canine Bone Marrow-Derived Stromal Cells A Potential Cell Source for Treatment of Neurological Disorders
Physical Description: 1 online resource (127 p.)
Language: english
Creator: Kamishina, Hiroaki
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: bone, cell, cns, dog, marrow, medicine, neuron, regeneration, stem, transdifferentiation, veterinary
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Bone marrow-derived stromal cells (BMSCs) represent a promising cell source for treatment of traumatic and ischemic injury of the central nervous system (CNS). Increasing evidence suggests that BMSCs hold multiple modes of action in promoting repair process of various CNS injuries. Based on these findings, initial clinical studies of autologous BMSC transplantation in human spinal cord injury patients are being conducted. The potential therapeutic value of BMSCs is certainly not limited to human applications. For example, dogs can sustain traumatic spinal cord injuries at relatively high incidence. A need thus exists for developing novel treatments and cell-based therapies for veterinary practice. These canine patients also afford an important animal-to-human translational opportunity. Our study first systematically characterized adult canine BMSCs, in an attempt to understand the frequency of BMSCs in canine bone marrow, growth kinetics in culture, phenotypic profile, and differentiation potentials. Next, neural differentiation properties of canine BMSCs were studied in vitro and in vivo. Finally, the effects of canine BMSCs on neurite extension were studied in vitro. Our data suggest that adult canine bone marrow contains approximately 1 BMSC/2.38 ? 104 bone marrow mononucleated cells. Under standard culture techniques, canine BMSCs grow rigorously to generate morphologically heterogeneous populations of plastic-adherent cells. The flow cytometric profile of canine BMSCs was similar to those of rodent and human counterparts. Standard protocols for osteogenesis and adipogenesis induced differentiation of primary canine BMSCs into respective lineages. Canine BMSCs intrinsically express neuronal and glial markers in vitro, and upon transplantation into a neonatal mouse brain, a small portion of canine BMSCs isolated from young donors, but not from adult donors, migrated into the subventricular zone as well as the olfactory bulb where they exhibited neuronal phenotypes. When co-cultured with dorsal root ganglion neurons, canine BMSCs promoted neurite extension via production of extracellular matrix molecules. We conclude that BMSCs can be isolated from adult canine bone marrow and expanded ex vivo. Canine BMSCs have the potential to differentiate into osteoblasts and adipocytes in vitro although the standard culture method does not support expansion of osteogenic cell populations in passaged cultures. Bone marrow of young dogs contains neurogenic cells; however, it is not known whether cells with similar properties exist in adult canine bone marrow. Nonetheless, adult canine BMSCs have the potential for promoting neuritic outgrowth in tissue culture.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Hiroaki Kamishina.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Clemmons, Roger M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021645:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021645/00001

Material Information

Title: Characterization of Canine Bone Marrow-Derived Stromal Cells A Potential Cell Source for Treatment of Neurological Disorders
Physical Description: 1 online resource (127 p.)
Language: english
Creator: Kamishina, Hiroaki
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: bone, cell, cns, dog, marrow, medicine, neuron, regeneration, stem, transdifferentiation, veterinary
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Bone marrow-derived stromal cells (BMSCs) represent a promising cell source for treatment of traumatic and ischemic injury of the central nervous system (CNS). Increasing evidence suggests that BMSCs hold multiple modes of action in promoting repair process of various CNS injuries. Based on these findings, initial clinical studies of autologous BMSC transplantation in human spinal cord injury patients are being conducted. The potential therapeutic value of BMSCs is certainly not limited to human applications. For example, dogs can sustain traumatic spinal cord injuries at relatively high incidence. A need thus exists for developing novel treatments and cell-based therapies for veterinary practice. These canine patients also afford an important animal-to-human translational opportunity. Our study first systematically characterized adult canine BMSCs, in an attempt to understand the frequency of BMSCs in canine bone marrow, growth kinetics in culture, phenotypic profile, and differentiation potentials. Next, neural differentiation properties of canine BMSCs were studied in vitro and in vivo. Finally, the effects of canine BMSCs on neurite extension were studied in vitro. Our data suggest that adult canine bone marrow contains approximately 1 BMSC/2.38 ? 104 bone marrow mononucleated cells. Under standard culture techniques, canine BMSCs grow rigorously to generate morphologically heterogeneous populations of plastic-adherent cells. The flow cytometric profile of canine BMSCs was similar to those of rodent and human counterparts. Standard protocols for osteogenesis and adipogenesis induced differentiation of primary canine BMSCs into respective lineages. Canine BMSCs intrinsically express neuronal and glial markers in vitro, and upon transplantation into a neonatal mouse brain, a small portion of canine BMSCs isolated from young donors, but not from adult donors, migrated into the subventricular zone as well as the olfactory bulb where they exhibited neuronal phenotypes. When co-cultured with dorsal root ganglion neurons, canine BMSCs promoted neurite extension via production of extracellular matrix molecules. We conclude that BMSCs can be isolated from adult canine bone marrow and expanded ex vivo. Canine BMSCs have the potential to differentiate into osteoblasts and adipocytes in vitro although the standard culture method does not support expansion of osteogenic cell populations in passaged cultures. Bone marrow of young dogs contains neurogenic cells; however, it is not known whether cells with similar properties exist in adult canine bone marrow. Nonetheless, adult canine BMSCs have the potential for promoting neuritic outgrowth in tissue culture.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Hiroaki Kamishina.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Clemmons, Roger M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021645:00001


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1 CHARACTERIZATION OF CANINE BO NE MARROW-DERIVED STROMAL CELLS: A POTENTIAL CELL SOURCE FOR TREATM ENT OF NEUROLOGICAL DISORDERS By HIROAKI KAMISHINA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Hiroaki Kamishina

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3 To my wife Harumi for her cons tant encouragement and support.

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4 ACKNOWLEDGMENTS There are a num ber of people whose assistan ce proved invaluable. I first express my gratitude to my supervisory committee chai r, Dr. Roger Clemmons, for giving me the opportunity to learn and develop my skills under his guidance. I would also like to acknowledge my remaining committee members, Dr. James Farese, Dr. Rowan Milner, Dr. Floyd Thompson, and Dr. Paul Reier for their expertise and guidance in a number of areas. I thank the members of Dr. Reep s research group. It was a great honor to be able to work with them. I wish to thank Ms. Margaret Stoll who helped me in preparing histological specimens. Thanks also go to Ms. Linda Lee-Am brose for her technical help and my colleagues Jennifer Cheeseman and Takashi Uemura for thei r encouragement and valuable discussion. I thank my family Harumi and Yuto for their patience and endless suppo rt. Credit is also due to my parents for their encouragement and support. Financial support through the College of Veterinary Medicine, the American German Shepherd Dog Charitable Foundation, and th e Amerman Family Foundation are also acknowledged.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 LIST OF ABBREVIATIONS........................................................................................................ 11 ABSTRACT...................................................................................................................................14 CHAP TER 1 INTRODUCTION AND BACKGROUND INFORMATION.............................................. 16 Definition and Terminology of Stem Cells............................................................................ 16 Classification of Stem Cells................................................................................................... .17 Adult Stem Cells............................................................................................................... ......17 Bone Marrow-Derived Stromal Cells (BMSCs)..................................................................... 19 Historical Background of BMSC Research..................................................................... 19 Isolation, Expansion, and Cell Surface Markers of BMSCs........................................... 21 Multipotentiality of BMSCs: Mesengenesis.................................................................. 23 Neural Transdifferentiation of BMSCs..................................................................................25 Evidence of in vitro Neural Transdifferentiation of BMSCs .......................................... 25 Evidence of in vivo Neural Transdifferentiation of BMSCs ........................................... 27 Controversies in Neural Transdifferentiation of BMSCs ................................................ 28 Potential Mechanisms of Action............................................................................................. 30 Neuronal Replacement by BMSCs.................................................................................. 30 Production of Soluble Factors by BMSCs....................................................................... 31 Axonal Regrowth Stimulatory Effects of BMSCs..........................................................32 Remyelination by Transplanted BMSCs.........................................................................33 BMSC Transplantation for CNS Injury.................................................................................. 34 Experimental Studies in Animal Models of CNS Disorders ........................................... 34 Spinal Cord Injury........................................................................................................... 35 Brain Injury................................................................................................................... ..36 Neurodegenerative Disorders..........................................................................................38 Human Clinical Trials..................................................................................................... 39 2 CHARACTERIZATION OF CANINE BONE MARROW STROMAL CELLS ................. 41 Background and Introduction................................................................................................. 41 Materials and Methods...........................................................................................................43 Bone Marrow Collection.................................................................................................43 BMSC Culture.................................................................................................................44 Colony-Forming Unit Assay........................................................................................... 44

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6 Growth Kinetics...............................................................................................................45 Flow Cytometric Characterization.................................................................................. 45 Differentiation Assays.....................................................................................................46 Results.....................................................................................................................................47 Morphological Observation of Canine BMSCs..............................................................47 The Frequency of Canine BMSCs...................................................................................49 Growth Kinetics of Canine BMSCs................................................................................49 Flow Cytometric Profile of Canine BMSCs .................................................................... 50 Osteogenic and Adipogenic Differe ntiation of Canine BMSCs ......................................52 Conclusion and Discussion..................................................................................................... 56 3 IN VITRO NEURAL DI FFERENTIATION OF CANINE BONE MARROW STROMAL CELLS................................................................................................................ 61 Background and Introduction................................................................................................. 61 Materials and Methods...........................................................................................................62 Preparation of Canine BMSCs........................................................................................62 Neural Differentiation of Canine BMSCs.......................................................................63 Immunocytochemistry for Neural Specific Markers....................................................... 63 Western Blotting and De nsitom etric Analyses................................................................ 64 Electrophysiological Recording...................................................................................... 65 Results.....................................................................................................................................66 Morphological Observation during N eural Induction..................................................... 66 Immunocytochemical Characterization of Neurally Induced Canine BMSCs ................ 66 Western Blot Analysis of Neural Specific Proteins ........................................................ 69 Electrophysiological Recording...................................................................................... 69 Conclusion and Discussion..................................................................................................... 70 4 IN VIVO NE URAL DIFFERENTIATION OF CANINE BONE MARROW STROMAL CELLS....................................................................................................................................76 Background and Introduction................................................................................................. 76 Materials and Methods...........................................................................................................78 Preparation of Canine BMSCs and Fibroblasts ............................................................... 78 Transplantation of Canine BMSCs and Fi broblasts into Neonatal Mouse Brain ............ 79 Immunohistochemistry to Evaluate Tran sdifferentiation of Canine BMSCs ..................79 Chromosome Painting to Evaluate Cell Fusion............................................................... 80 Results.....................................................................................................................................81 Distribution and Phenotypic Fates of Adult Canine BMSCs and Fibroblasts ................. 81 Distribution and Phenotypic Fa tes of Young Canine BMSCs ........................................ 82 Fluorescence In Situ Hybridization for Chrom osome painting....................................... 82 Conclusion and Discussion..................................................................................................... 83

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7 5 EFFECTS OF CANINE BONE MARR OW STROMAL CELLS ON NEURITE EXTENSION FROM DORSAL ROOT GANGLION NEURONS IN VITRO .....................90 Background and Introduction................................................................................................. 90 Materials and Methods...........................................................................................................93 Preparation of Canine BMSCs and Fibroblasts ............................................................... 93 Immunocytochemical Analysis for Expr ession of Extracellu lar and Adhesion Molecules .....................................................................................................................93 Direct Co-culture of BMSCs a nd Dorsal Root Ganglion Neurons ................................. 94 Culture of DRG Neurons in Conditioned Medium......................................................... 94 Measurements of Neurite Outgrowth.............................................................................. 95 Results.....................................................................................................................................95 Expression of Extracellula r Matrix Molecules ................................................................ 95 Direct co-culture of DRG on BMSC Monolayer ............................................................96 DRG Cultured in Conditioned Medium.......................................................................... 98 Conclusion and Discussion..................................................................................................... 99 6 SUMMARY AND CONCLUSION..................................................................................... 104 LIST OF REFERENCES.............................................................................................................107 BIOGRAPHICAL SKETCH.......................................................................................................127

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8 LIST OF TABLES Table page 2-1 Summary of bone marrow samples.................................................................................... 472-2 Number and frequency of CFUF from five bone marrow samples.................................. 49

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9 LIST OF FIGURES Figure page 2-1 Morphological observation of canine primary BMSCs.....................................................482-2 The CFU-F assays from five adult canine bone marrow samples..................................... 492-3 Cell growth kinetics as a f unction of initial cell densities................................................. 512-4 Representative results of flow cy tometric analysis of canine BMSCs.............................. 512-5 Morphological changes of canine P1 BMSCs during osteogenic induction...................... 532-6 Osteogenic differentiati on of canine P0 BMSCs............................................................... 542-7 Morphological changes during osteogenic differentiation of rat P1 BMSCs.................... 552-8 Osteogenic differentiation of rat P1 BMSCs (day12)........................................................ 562-9 Adipogenic differentiation of canine P1 BMSCs.............................................................. 573-1 Phase-contrast photomicrographs of canine BMSCs and fibroblasts................................ 673-2 Immunofluorescent micrographs of canine BMSCs.......................................................... 683-3 Western blotting of neuronal ( II I-tubulin) and glial proteins (GFAP)........................... 693-4 Result of whole-cell voltage clamp r ecording of neurally induced BMSCs.....................704-1 Montage immunofluorescence photomicrogra ph of mouse brain with engrafted adult canine BMSCs...................................................................................................................814-2 DiI-positive BMSCs isolated from yo ung donors present in the olfactory bulb............... 834-3 Various morphologies of BMSCs in the subventricular zone........................................... 844-4 BMSC from a young donor located in the subventricular zone......................................... 844-5 Immunostaining of BMSCs in the subventricular zone for GFAP and NeuN expression..........................................................................................................................854-6 Fluorescence in situ hybridization for chromosome painting............................................ 855-1 Immunofluorescent photomicr ographs of canine BMSCs................................................. 965-2 Representative photomicrographs of DRG neurons cultured on three different substrates for 48 hours.......................................................................................................97

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10 5-3 Quantitative neurite measurements of neurite outgrowth from DRG neurons cultured on lam inin, fibroblasts, or BMSCs.................................................................................... 985-4 Quantitative neurite measurements of neurite outgrowth from DRG neurons cultured in control, fibroblast-conditioned, or BMSC-conditioned media...................................... 99

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11 LIST OF ABBREVIATIONS ALCAM activated leukocyte cell adhesion molecule BBB Basso-Beattie-Bresnahan BDNF brain-derived ne urotrophic factor bFGF basic fibroblast growth factor BHA butylated hydroxyanisole BME -mercaptoethanol BMP-2 bone morphogenic protein-2 BMSC bone marrow-derived stromal cell BNP brain natriuretic peptide BSA bovine serum albumin CFU-F colony-forming unit fibroblast CNS central nervous system DAPI 4,6-diamino-2-phenylindole dbcAMP dibutyryl cyclic AMP DMEM Dulbeccos modified Eagles medium DMSO dimethylsulfoxide DRG dorsal root ganglion DTT dithiothreitol ECM extracellular matrix EDTA ethylenediaminetetraacetic acid EG cell embryonic germ cell EGF epidermal growth factor ES cell embryonic stem cell FACS fluorescence activated cell sorting

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12 FBS fetal bovine serum FITC fluorescein isothiocyanate GDNF glial cell linederive d neurotrophic factor GFAP glial fibrillary acidic protein HGF hepatocyte growth factor HSC hematopoietic stem cell IBMX isobutylmethylxanthine ICAM intercellular adhesion molecule LIF leukemia inhibitory factor MAPC multipotent adult progenitor cell MBP myelin basic protein MHC major histocompatibility complex MNC mononucleated cell MPC mesodermal progenitor cell NCAM neural cell adhesion molecule NeuN neuronal nuclear antigen NGF nerve growth factor NICD notch intracellular domain NSE neuron-specific enolase NT-3 neurotrophin-3 OEC olfactory ensheathing cell PBS phosphate buffered saline PDGF platelet-derived growth factor PNS peripheral nervous system PPARperoxisome proliferator-activated receptor

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13 RA retinoic acid RMS rostral migratory stream RT room temperature RUNX-2 runt-related tr anscription factor 2 SCI spinal cord injury SVZ subventricular zone TBS tris buffered saline TGFtransforming growth factor-beta VCAM vascular cell adhesion molecule VEGF vascular endothelial growth facto

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14 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF CANINE BONE MARROW-DERIVED STROMAL CELLS A POTENTIAL CELL SOURCE FOR TREATMENT OF NEUROLOGI CAL DISORDERS By Hiroaki Kamishina December 2007 Chair: Roger M. Clemmons Major: Veterinary Medical Sciences Bone marrow-derived stromal cells (BMSCs ) represent a promising cell source for treatment of traumatic and ischemic injury of the central nervous system (CNS). Increasing evidence suggests that BMSCs hold multiple modes of action in promoting repair process of various CNS injuries. Based on these findings, initial clinical studies of autologous BMSC transplantation in human spinal cord injury patients are being conducted. The potential therapeutic value of BMSCs is certainly not lim ited to human applications. For example, dogs can sustain traumatic spinal cord injuries at rela tively high incidence. A need thus exists for developing novel treatments and cell-based therapies for veterinary practice. These canine patients also afford an important an imal-to-human translational opportunity. Our study first systematically characterize d adult canine BMSCs, in an attempt to understand the frequency of BMSCs in canine b one marrow, growth kinetics in culture, phenotypic profile, and differentia tion potentials. Next, neural differentiation properties of canine BMSCs were studied in vitro and in vivo Finally, the effects of canine BMSCs on neurite extension were studied in vitro Our data suggest that adult canine bone marrow contains approximately 1 BMSC/2.38 104 bone marrow mononucleated cells. Under standard culture

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15 techniques, canine BMSCs grow rigorously to generate morphologically heterogeneous populations of plastic-adherent cel ls. The flow cytometric prof ile of canine BMSCs was similar to those of rodent and human c ounterparts. Standard protocols for osteogenesis and adipogenesis induced differentiation of primary canine BMSC s into respective lineages. Canine BMSCs intrinsically express neuronal and glial markers in vitro and upon transplantat ion into a neonatal mouse brain, a small portion of canine BMSCs isolated from young donors, but not from adult donors, migrated into the subventricular zone as well as the olfactory bulb where they exhibited neuronal phenotypes. When co-cultured with dorsal root ganglion ne urons, canine BMSCs promoted neurite extension via production of extr acellular matrix molecules. We conclude that BMSCs can be isolated from adult canine bone marrow and expanded ex vivo Canine BMSCs have the potential to differentiat e into osteoblasts and adipocytes in vitro although the standard culture method does not support expansion of oste ogenic cell populations in passaged cultures. Bone marrow of young dogs contains neurogenic cells; however, it is not known whether cells with similar properties exist in adult canine bone marrow. None theless, adult canine BMSCs have the potential for promoting neur itic outgrowth in tissue culture.

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16 CHAPTER 1 INTRODUCTION AND BACKGROUND INFORMATION There have been a num ber of breakthroughs in th e field of cell biology that have led to our current knowledge of cell plastici ty, including the demonstration of the incredible plasticity of the adult somatic nucleus giving birth to the cloned sheep Dolly (Wilmut et al., 1997). Development of the novel technique by Thomson (1998) for isolation and culture of human embryonic stem (ES) cells has been a huge stride as well. Even more intriguing idea, derived from these two findings, is therapeutic cloning where a nucleus from a patients somatic cell may be used to create patient-spe cific ES cells, thus avoiding imm une rejection of transplants. More recently, adult stem cells have gained cons iderable attention on the grounds that the use of adult stem cells may circumvent logistical and et hical issues posed by ES and fetal-derived stem cells. Bone marrow-derived stromal cells (BMSCs) are a particularly promising cell source, and recent evidence suggests that these cells may be effective in treating diseases of the central nervous system (CNS). In this chapter, I pr esent a brief overview of stem cell research, in general and BMSCs in particular. I also presen t scientific rationales for the use of BMSCs in treatment of CNS injury, as well as some of the controversies surrounding the authenticity of the plasticity of BMSCs. In the following chapte rs, I present our findings on characterization of canine BMSCs and their neural transdifferentiation properties in vitro and in vivo as well as their stimulatory effects on neuritic extension in vitro. Definition and Terminology of Stem Cells A stem cell is defined as an undifferentiated cell that is capable of bot h replicating itself (a process termed self-renewal) and producing multipotent daught er cells (a process termed differentiation). Daughter cells produced from stem cells are precursor cells that usually proliferate before giving rise to fully differentiated cells. Ther efore, these precursor cells are

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17 also called transit amplifying cells. The term s precursor cell and progenitor cell are used interchangeably; however, some progenitor cells are considered to have more developmental potential than other precursor cells There is inconsistency as to how to define the self-renewal ability of stem cells; some definitions require stem cells be able to self-renew indefinitely, whereas others do not. Multipotentiality (or multipot ency) refers to the abili ty to give rise to multiple cell types and has been used as another definition of stem cells; however, this is not a required definition of a stem cell. For example, spermatogonial stem cells in the testis produce only spermatozoa (Meachem et al., 2001). Classification of Stem Cells Stem cells can be operationally classified based on their developmental potential. Pluripotent stem cells refer to those that can ge nerate all the cells in the body including germ cells. Embryonic stem cells (ES cells) and embr yonic germ cells (EG cells) are produced in culture from the epiblast cells of a blastocyst and primordial germ cells of an early embryo, respectively. Because ES cells and EG cells cannot produce extra-embryonic tissues that are necessary for embryonic development, these cells ar e not totipotent. Most of the stem cells in animal organs are multipotent, but there are also unipotent stem cells as in the case of spermatogonial stem cells. Stem cells can also be categorized by their embryonic, fetal, or a dult origin. Fetal and adult stem cells can be further divided according to their tissue of origin and referred to as organspecific or tissue-specific stem cells; those that are found in adult organs are specifically called adult stem cells. Adult Stem Cells It was originally thought that ad ult stem cells were only present in certain organs that have high cell turnover rates such as blood gut, skin, testis, and the respir atory tract. These adult stem

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18 cells are responsible for life-long replenishment of damaged and lost cells in the organs in which they reside. The hematopoietic stem cell (HSC ) in the bone marrow is a classical example of adult stem cell and the most exte nsively studied adult stem cell. A single HSC can reconstitute the entire hematopoietic system in an irradiat ed mouse by producing al l the blood cells and the immune system. HSCs were the fi rst adult stem cells to be used clinically and their therapeutic potentials have been fully recognized for numerous hematologic and immune diseases (Kroger et al., 2002). In recent years, it has become increasingly cl ear that most, if not all, adult organs contain stem cells. The discovery of stem cells in th e adult mammalian central nervous system (neural stem cells) was particularly unexpected because the adult central nervous system has a limited regenerative capacity (Gritti et al., 1996; Lois and Alvarez-Buy lla, 1993; Morshead et al., 1994). It is now known that neural stem cells reside in specific areas in the adult brain including the hippocampus and olfactory bulb where adult neurogenesis takes place (Altman and Das, 1966). However, the identity of neural stem cells in vivo is still not fully unde rstood (Laywell et al., 2000). Much of the interest in adult stem cell resear ch relates to its great therapeutic potentials. There are three major rationales for advancing ad ult stem cell research in the context of cell therapy. First, the use of adult stem cells for th erapy avoids ethical problems related to the use of embryos and fetuses for isolation of ES cells and fetal stem cells. Second, it is possible to isolate adult stem cells from the patient requiring the treatment, allowing autologous transplantation to be performed. This will avoid immunological rejection of transplants and the use of immunosuppressive therapy. Third, because adult stem cells are thought to be more restricted in their lineage specification than ES cells and fetal stem cells, the risk of forming tumors is

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19 believed to be reduced. This idea may conflict in one way to the ability of adult stem cells to transdifferentiate into multiple cell types. Among various types of adult stem cells, BMSCs have generated tremendous attention because of their accessibility and multipotency. In particular, the perspectives of the use of BMSC s for neurological diseases ha ve expanded considerably during the last decade (Dezawa et al., 2004). Although promising, many fundamental questions about basic biology of BMSCs remain, such as their true identity and differentiation properties that are clinically meaningful. Bone Marrow-Derived Stromal Cells (BMSCs) Historical Background of BMSC Research Discovery of the presence of bone-form ing cells in bone marrow came from early transplantation studies performed in 1950s and 1960s. In these studies, whole bone marrow was transplanted in ectopic sites (e.g. anterior ch amber of the eye or unde r the renal capsule) and shown to form an osseous tissue (Petrakova et al., 1963; Tavassoli a nd Crosby, 1968; Urist and Mc, 1952) and marrow stromal microenvironment for hematopoiesis (Tavassoli and Crosby, 1968). Initial attempts to identify and isolate these osteogenic cells in bone marrow were made by Friedenstein and colleagues in 1960s and 1970s. They found characteristic fibroblastic colony-forming cells (CFU-F) from the bone marro w of guinea pig, which could be isolated by using their plastic adhe rent property, cultured in vitro, and shown to have osteogenic potential in vivo (Friedenstein et al., 1970; Fr iedenstein et al., 1968; Friede nstein et al., 1966). Thus, adherent, fibroblastic, colony-fo rming cells in the bone marrow were recognized to form bone. In the early 1980s, Ashton et al. (1980) showed that rabbit bone marrow stromal cells consistently generated bone and cartilage in diffusion chambers implanted into the peritoneal cavity of the host animals. It was not clear, however, whether generation of bone and cartilage represented different stages of a skeletal develo pmental process or separate activities by distinct

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20 cell populations. Friedenstein et al. (1987) later reported that a portion of CFU-F colonies have an extensive proliferative capacity after passagin g and proposed that precursor cells with stem cell characteristics reside within the CFU-F compartment. At ar ound this time, the stromal stem cell hypothesis was proposed in which there exists a hierarchy of cellular organization with different developmental stages supported by a small number of self-renewing stem cells, analogous to the organization of the hematopoietic system. It was only in the late 1990s when the first detailed description of multipotential mesenchymal stem cells was published by Pitten ger et al. (1999). This group performed extensive characterization of human BMSCs by means of flow cytometric analysis, gene expression analysis, and multi-lineage differentiati on assays. It was clearly shown that clonal BMSCs derived from single cells demonstrat ed multipotentiality by differentiating into osteoblasts, chondrocytes, and adipocytes, indica ting unequivocally the presence of multipotent mesenchymal stem cells in the adult human bone marrow. They also noted that some of the isolated colonies had limited di fferentiation potential; thus, adhe rent colonies obtained from adult human bone marrow are composed of mixed cell populations of multipotential BMSCs and more lineage restricted progenitor cells. Recent studies suggested the presence of a rare cell population with more primitive stem cell characteristics within the BMSC compartmen t in adult bone marrow. Jiang et al. (2002a) reported that in murine bone marrow, there are po pulations of cells with extensive proliferative and differentiation capacity. These cells were te rmed multipotent adult progenitor cell (MAPC). Murine MAPCs express transcription factors important in maintaining undifferentiated ES cells such as Oct-4, Rex-1, and SSEA-1, and interestingl y, require leukemia inhib itory factor (LIF) for expansion, a feature found in murine ES cells bu t not in human ES cells (Odorico et al., 2001;

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21 Williams et al., 1988). Murine MAPCs differentiated in vitro not only into mesenchymal cells but also visceral mesodermal, neuroectodermal and endodermal cells, and upon transplantation into an early blastocyst, contribut ed to most, if not all, somatic cell types. Reyes et al. (2001) suggested that human bone marrow also contains a primitive cell population, termed mesodermal progenitor cells (MPCs), that have extensive pr oliferative and differen tiation potential. These studies provided evidence that BMSCs are mi xed populations of stem and progenitor cells, which also include pl uripotent stem cells. Isolation, Expansion, and Cell Surface Markers of BMSCs Currently used technique for isolation of BMSCs r elies on the adhesive property of BMSCs to tissue culture plastic, a technique similar to the origin al one described by Friedenstein (1970) and later optimized by Caplan et al. (199 1a). In rodents, bone marrow is typically harvested by flushing excised long bones (e.g. femurs, tibiae, and humeri). In humans and large animals including dogs, bone marrow can be harvested from long bones or the iliac crest of the pelvis by aspiration. After bone marrow colle ction, the marrow is often subjected to fractionation via density gradient centrifugation using Percoll or Ficoll to isolate mononucleated cells. These cells can be cultured in a standard medium such as Dulbeccos modified Eagles medium (DMEM), containing 10-20% fetal bovine serum (FBS). Primary cells form symmetric colonies after low-density plat ing or single-cell sorting (Broc kbank et al., 1985; Colter et al., 2000; Javazon et al., 2001; Kuznetsov et al., 19 97), an important feature of the BMSC. As demonstrated by Owen and Friedenste in (1988) and DiGirolamo et al (1999), these colonies are, however, heterogeneous in both appearance (morphology and size) and differentiation potential. Initially, culture is consiste d of heterogeneous cell populatio ns, but becomes morphologically homogeneous over time by depletion of non-adhesi ve hematopoietic cells (Bruder et al., 1997). Primary cultures are usually maintained for 1014 days, and are then detached by trypsinization

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22 followed by sub-culturing. Passaged cells are sp indle-shaped and fibr oblast-like in their undifferentiated state, which grow in uniform monolayer. Although commonly used isolation and expansion techniques are easy and do not require special equipments, hematopoietic contamina tions remain problematic in some species, particularly in the mouse (Phinne y et al., 1999). Therefore, many attempts have been made to develop techniques for isolation and expansion of a pure population of BMSCs. Most of these techniques are dependent on utilizing cell surf ace antigen specific anti bodies combined with either cell sorting technique or immunological selection methods. However, due to a lack of unique cell markers on BMSCs (Majumdar et al., 2003), most of these selection techniques are designed to eliminate unwanted cell types, mainly hematopoie tic cells, from the starting materials (Tropel et al., 2004), although attempts have been made to purify BMSCs using an antibody Stro-1 (Gronthos et al., 199 4; Simmons and Torok-Storb, 1991). Investigation of cell surface marker profile s of BMSCs has been carried out for the purpose of characterizing BMSCs and developing better purification methods. This task has been difficult since BMSCs do not express uniqu e cell surface markers (Digirolamo et al., 1999; Haynesworth et al., 1992a), in a similar manne r to CD34 positive hematopoietic stem cells. Therefore, BMSCs are identified by a combin ation of immunophenotypic profiles. These include negative profiles against hematopoi etic lipopolysaccharide receptor CD34, CD14, and the leukocyte common antigen CD45, as well as endothelial markers such as CD31, von Willebrand factor, and P-selectin (Pittenger et al., 1999). Human and murine BMSCs express a number of receptors for cytokines (IL-1, IL3, IL-4, IL-6, IL-7), adhesion molecules (ICAM, VCAM, ALCAM, integrins), and growth factors (bFGF, PDGF) (Majumdar et al., 2003). The expression profiles of some of th ese cell surface molecules are not static and influenced by their

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23 developmental stage and other cytokines (Ba rry, 2003; Majumdar et al., 2003). This may explain significant variations of the cell su rface marker profiles of BMSCs reported from different laboratories (Majumdar et al., 1998; Peister et al., 2004). Haynesworth et al. (1992a) developed th e monoclonal antibody SH-2 raised against human BMSCs, which reacts with an epitope pr esent on the transformi ng growth factor-beta (TGFor CD105) and was later used in immunom agnetic selection methods (Barry et al., 1999). The antibodies SH-3 and SH-4 were also raised against human BMSCs, which recognize distinct epitopes on CD73 (membrane-bound ecto-5-nucleotidase) (Barry et al., 2001a). Multipotentiality of BMSCs: Mesengenesis As f irst shown by Pittenger et al. (1999), in vitro differentiation into three major mesenchymal cell types (osteoblasts, chondrocyt es, and adipocytes) has been the widely accepted and perhaps the most reliable single requirement to identify BMSC populations. To induce differentiation into each cell type, different combinations of reagents are used, which slightly vary among different sp ecies. Differentiation along oste oblastic cells typically requires -glycerolphosphate, ascorbic aci d-2-phosphate, and glucocorticoids such as dexamethasone (Jaiswal et al., 1997). BMP-2 has been reported to further stimulate osteogenic differentiation of BMSCs isolated from rodents and dogs (Volk et al., 2005), but not to th e equivalent degree in humans (Einhorn, 2003; Govender et al., 2002). When cultured in m onolayer in the presence of these stimulators, BMSCs acquire osteoblas tic (cuboidal) morphology with concomitant upregulation of related genes (a lkaline phosphatase, osteocalcin, osteopontin, RUNX-2, etc). Chondrogenic differentiation can be induced in a three dimensiona l culture in th e absence of FBS and in the presence of the TGFsuperfamily (Mackay et al., 1998). Under these conditions, BMSCs lose their fibroblastic morpho logy and initiate expressing cartilage-specific extracellular matrix molecules (Barry et al., 2001b). Differen tiation into adipocytes can be

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24 induced in monolayer in the pr esence of isobutylmethylxanthine (IBMX) via activ ation of the ligand-induced transcription factor peroxisome proliferator-activated receptor(PPAR) (Suzawa et al., 2003). Differentiated cells ar e easily identified by their morphology and the presence of large lipid-filled vacuoles. In vivo mesengenic differentiation of BMSCs has been demonstrated in a number of studies by their contribution to a tissue repair process in osse ous and cartilaginous tissues upon transplantation. Transplantation of BMSCs load ed on porous ceramics in a bone defect led to formation of copious amounts of bone and facilita ted bone healing in the canine model (Arinzeh et al., 2003; Bruder et al., 1998; Ka diyala et al., 1997). In initia l clinical trials of allogenic BMSC transplantation in childre n with osteogenesis imperfecta, a genetic disorder in which osteoblasts produce defective type I collagen, lead ing to osteopenia and multiple fractures, new dense bone formation and increases in the to tal body bone mineral content were reported (Horwitz et al., 2002; Horwitz et al., 1999). P onticiello et al. (2000) transplanted scaffolds loaded with human BMSCs in an osteochondral de fect in the rabbit femo ral condyle and showed filling of defective lesion with cartilaginous tissues. In vivo differentiation of BMSCs into cardiomyocytes has also been reported and attracted many investigators as a potential source for ce llular cardiomyoplasty. Toma et al. (2002) reported that human BMSCs, when delivered by infusion to an immunocompromised mouse, could engraft to the normal myo cardium and differentiate into cardiomyocytes. Engrafted cells could persist in the heart a nd displayed normal cellular organization of cardiomyocytes. Preclinical studies of autologous BMSC (Vullie t et al., 2004) or bone marrow cell (Li et al., 2003; Memon et al., 2005) transplantation for ca rdiomyoplasty have been performed in the

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25 canine mode of ischemic myocardium and successfu lly translated into clinical studies on patients with myocardial infarction (Li et al., 2003; Stangel and Hartung, 2002). Neural Transdifferentiation of BMSCs Evidence of in vitro Neural Transdifferentiation of BMSCs Early in vitr o studies (Sanchez-Ramos et al., 2000; Woodbury et al., 2000) reporting differentiation of BMSCs along neuronal phenotypes ha ve aroused considerable interest. This phenomenon was considered to represent trans differentiation based on the fact that BMSCs (mesoderm origin) differentiated into neurons (ectoderm origin), crossing the developmental germ layer boundaries. In these studies, human or mouse BMSCs were treated with different combinations of reagents (e.g. -mercaptoethanol [BME], di methylsulfoxide [DMSO], and butylated hydroxyanisole [BHA]) and growth factors (e.g. epidermal growth factor [EGF], retinoic acid [RA], and BDNF). Neuronal differentiation was confirmed by their morphological changes and expression of a panel of neuronal mark ers such as nestin, ne uron-specific enolase, TrkA, and III-tubulin. Following these st udies, Deng et al. (2001) re ported that human BMSCs differentiated into neurons in vitro when treated with isobuty lmethylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP), two reagents k nown to increase the intr acellular concentration of cAMP. They reported based on the expression pattern of neuronal markers that this treatment induced human BMSCs to commit to early neuronal cells but not mature neurons. Several independent groups re ported that native neuronal cells can instruct BMSCs to differentiate into neurons via ce ll-to-cell contact in addition to their trophic effects. BMSCs were co-cultured with various types of neurons such as fetal mesencephalic cells (SanchezRamos et al., 2000), neonatal hippocampal ne urons (Abouelfetouh et al., 2004), and neonatal cerebellar granule neurons (Wislet-Gendebien et al., 2005). Wislet-Gende bien et al. (2005) was the first to report that when co-cultured with cerebellar granule neurons, rat BMSCs

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26 differentiated into neuron-like cells that seque ntially expressed voltage-gated potassium and sodium channels and became excitable in respon se to depolarizing voltage steps via whole-cell patch clamp. Modification of the gene expression pattern has been used to generate electrophysiologically excitable neurons from BMSCs. Kohyama et al. (2001) transfected mouse BMSCs with Noggin and subsequently cult ured them in a medium supplemented with NGF, NT-3, and BDNF. This treatment gradually induced BMSCs to differentiate into mature neurons which had developed voltage-gated ion ch annels and the ability to uptake calcium in response to potassium and neurotransmitters (e.g acetylcholine and glutamate). Because Noggin is known to play a role in neural developmen t during early embryogenesis (Smith and Harland, 1992) and adult neurogenesis (Lim et al., 2000) by antagonizing bone morphogenic protein signaling (Zimmerman et al., 1996), it was s uggested that Noggin may inhibit osteogenic activities of BMSCs and induce neural differentiation. Dezawa et al. (2004) transfected human and rat BMSCs with Notch intracellular domain (NICD) and subsequently treated them with bFGF forskolin, and ciliary neurotrophic factor. Upon activation by neighboring cell s through Delta/Serrate/Lag-1 lig ands, NICD is cleaved and enters the nucleus where it influences the expressi on of transcription factor s related to progenitor pool maintenance, cell fate decision, and, in cas e of nervous system, terminal specification of cells as neurons and glial cells (Gaiano et al., 2000; Lundkvist and Lendahl, 2001; Morrison et al., 2000). They reported that transfection of NICD was highl y efficient and specific in generating neuronal cells with el ectrophysiological properties from both human and rat BMSCs, without generation of glial cells. Further treatment with glia l cell linederived neurotrophic factor (GDNF) increased the proportion of tyrosine hydroxylase-positive and dopamine-

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27 producing cells, which upon intrastriatal implan tation, improved behavior outcomes in a rat model of Parkinson disease. Evidence of in vivo Neu ral Transdifferentiation of BMSCs Initial evidence that BMSCs produce neural cells in vivo was provided by Pereira et al. who implanted systemically wild-type mouse BM SCs into osetogenesis imperfect transgenic mice (Pereira et al., 1998). In the study, donor-derived DNA was detected in numerous nonhematopoietic tissues including th e brain, although the engraftment rate was low and phenotypes of engrafted cells were not described. Azizi et al. (1998) injected human BMSCs directly into the adult rat brain striatum and reported that BMSCs migrated through the host brain tissue in a manner similar to that of implanted neural stem cells. They reported th at transplanted BMSCs lost immunoreactivity against fibr onectin and collagen-I, putative BMSC markers in culture, but expression of neuronal markers were not examine d. Kopen et al. (1999) transplanted murine BMSCs in the lateral ventricle of neonatal mouse brains and reported that BMSCs migrated throughout the forebrain, cerebellu m, and brain stem where engraf ted cells expressed GFAP or neurofilament. More recently, De ng et al. (2006) tran splanted murine BMSCs into the lateral ventricle of neonatal mouse brains and found that these cells migrated and differentiated into olfactory bulb granule cells and periventricular astrocytes. Th ese events were independent on cell fusion, evaluated by sex chromosome-specific in situ hybridization. These previous studies indicate that BMSCs may be capable of responding to enviro nmental cues provided by the developing and adult CNS and adopting neuronal phenotypes in vivo It remains, however, to be confirmed whether engrafted BMSC-derived neuronal cells possess electrophysiological properties and become integrated into an ex isting or newly establis hed neural circuitry.

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28 Controversies in Neural Transdifferentiation of BMSCs Findings of previous in vitro and in vivo studies, as described above, appeal that under certain conditions adult stem cells may be reprog rammed and generate cells of other germ layer origins including neuroectodermal derivatives. Consequently, neuronal transdifferentiation of BMSCs has generated great excitement since cellular therapy using autologous BMSCs may become possible for diseases of the CNS. Howeve r, this concept has been repeatedly challenged during the last few years and suggested for a need of more careful interpre tations. Some of the findings that led to this cont roversy are described below. Two independent groups reported that adult st em cells can fuse with ES cells and adopt the phenotypes of other cell types. Terada et al. (2002) showed that murine BMSCs spontaneously fused with ES cells in culture und er the presence of interleukin-3. Ying et al. (2002), in the same year, reported that co-cultured neural stem cel ls from transgenic mice fused with ES cells and produced non-neural derivative s. Generated tetraplo id hybrid cells had full pluripotent character and contributed to multilineage chimera formation. Following these studies, several group s have described in vivo cell fusion after transp lantation of BMSCs. Vassilopoulos et al. (2003) implanted transgenic murine BMSCs into livers of mice that lacked fumarylacetoacetate hydrolase. Transplanted BMSCs fused with recipient hepatocytes, which resulted in restoration of norma l liver function. Therefore, this study pointed to the therapeutic potential of cell fusion. Fusion of bone marrow-derived cells with cerebellar Purkinje cells and cardiomyocytes were also reported (Alvarez-Dolado et al., 2003; Weimann et al., 2003a; Weimann et al., 2003b). These stud ies suggest that cell fusion ma y explain some of the early observations that were interprete d as transdifferentiation or cell plasticity and prompt to include genotyping of potentially transdiffe rentiated cells for careful evaluation of such observations.

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29 Contamination of other cell types is another concern in investigation of transdifferentiation. This concern may be pa rticularly important for BMSCs because, as mentioned earlier, adult bone marrow is suggested to contain pluripotent stem cells (Jiang et al., 2002a; Reyes et al., 2001) (e.g. MAPC and MPCs) which may be within the BMSC fraction. These pluripotent cells are t hought to be quiescent, but unde r certain conditions, may be triggered to differentiate into a wide range of cell types, some of which could be cells of other germ layer origins. It was further suggested th at, in addition to bone marrow, adult tissues such as brain and muscle also contai n quiescent pluripotent stem ce lls (Jiang et al., 2002b). If the fraction of bone marrow contain MAPCs and wa s transplanted into the brain, neuronal transdifferentiation could result from differentiation of MAPCs. If the whole bone marrow is to be used for transplantation, hematopoietic stem cells may also contribute to neural transdifferentiation of donor cells as suggested previously (Cogle et al., 2004; Mezey et al., 2003). Therefore, these studies hi ghlight the importance of proper identification of transplanted cells and the origin of differentiated cells. Early in vitro findings (Sanchez-Ramos et al., 2000; Woodbury et al., 2002; Woodbury et al., 2000) claiming neural transdifferentiation of BMSCs have been questioned as to whether the observed morphological and phenotypic changes re presented true neural differentiation or perhaps cytotoxic change s caused by induction medium. In these studies, when BMSCs were cultured in neural induction me dium, conversion of fibroblas tic morphology into neuron-like morphology occurred rapidly (within a few hours). Lu et al. (2004) report ed that using various chemicals (e.g. BME, MHA, DMSO) similar morphological changes can be induced on fibroblasts, HEK293 cells (human embryonic kidney cell line), and PC-12 ce lls (cell line derived from a pheochromocytoma). They noted that th e morphological changes primarily resulted from

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30 shrinkage of cytoskeleton rather than ge nuine neurofilament extension. Further, immunoreactivity against neuronal markers (NSE and NeuN) was detected on these induced cells, which was, however, not detectable as changes in corresponding mRNA levels with RTPCR. Similar results were also reported by Neuhuber et al. (2004). Ri smanchi et al. (2003) found that the initial neural differentiation pr otocol reported by Woodbury (2000) resulted in significantly increased cell death of differentiated cells and that maintenance of neuronal morphology required continued exposure to the i nduction medium. It is thus unlikely that morphological changes into neuronlike cells resulted from tightly regulated process of changes in the gene expression pattern and cellular orga nization in early studies showing rapid neural transdifferentiation of BMSCs. As such, inte rpretation of neural differentiation of BMSCs in vitro needs great caution and systematic approaches including assays at the molecular level. Potential Mechanisms of Action There has been convincing ev idence that transplantation of BMSCs in experim ental models of CNS injury leads to significant improvement in functional as well as electrophysiological measures. Although these studies suggest th erapeutic potentials of BMSC transplantation, mechanisms underlying their eff ects are still largely unknown. A few studies addressing this question propose potential modes of action of transplanted BMSCs, which could be divided into four categories as follows; 1) Neuronal replacement, 2) Production of soluble factors, 3) Axonal regrowth stimulatory effects, and 4) Generation of myelinating cells. Many think that these effects play ro les in combination, potentially s ynergistically to one another. Neuronal Replacement by BMSCs The idea of neuronal rep lacement by BMSCs is based on early studies showing neuronal transdifferentiation of rat and human BMSCs in vitro and in vivo as described above. However, this topic is still under intensive controversy. Even if BMSCs can gene rate functional neurons in

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31 vivo a practical problem still exists on the ground s that BMSC-derived neurons may need to be integrated in the host neural circuitry in order to produce functional improvement. This mode of action itself is therefore unlikely to be sufficien t to explain significan t functional improvements observed in previous studies with experimental CNS injury. On the other hand, it can be said that neuronal transdifferentiation and repl acement by BMSCs may not be the essential requirement for functional recove ry to occur after CNS injury. Production of Soluble Factors by BMSCs Recent stud ies have revealed that BMSCs naturally produce various cytokines, neurotrophic factors, and angioge nic growth factors, including ne rve growth factor (NGF) (Chen et al., 2005; Chen et al., 2002a; Chen et al., 2002b; Crigler et al., 2006; Garcia et al., 2004; Li et al., 2002), glial cell-derived neurotrophic fact or (GDNF) (Chen et al ., 2005), brain-derived growth factor (BDNF) (Chen et al., 2002a; Chen et al., 2002b; Crigler et al., 2006; Li et al., 2002), vascular endothelial growth factor (VEG F) (Chen et al., 2002a; Chen et al., 2002b), hepatocyte growth factor (HGF) (Chen et al., 2002a; Chen et al., 2002b), and brain natriuretic peptide (BNP) (Song et al., 2004) Neurotrophic factors such as NGF and BDNF support survival of damaged neurons (DeKosky et al., 1994; Goss et al., 1998; Hammond et al., 1999; Lu et al., 2001) and stimulate neurite regrowth (Moc chetti and Wrathall, 1995), whereas vasoactive peptides such as VEGF (Chen et al., 2003b; Ch en et al., 2002a; Chen et al., 2002b) and BNP (Song et al., 2004) induce angiogenesis and reduce secondary edema there by participate in the reparative processes after CNS injury. In addition, soluble factors secreted by BMSCs may stimulate differentiation of neural progenitor cells (Wu et al., 2003). A previous observation that transplanted BMSCs lack voltage-gated ion channels necessary for the generation of action potenti als while promoting functional recovery of paralyzed rats supports the hypothesis based on the growth factor-mediated effects (Hofstetter et

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32 al., 2002). Fibroblasts genetically engineered to express neurot rophin-3 (NT-3) (Grill et al., 1997), or to secrete NGF (Nakahar a et al., 1996) or BDGF (Schwa rtz et al., 2003) have been shown to be of therapeutic benefit in rat SCI models. Therefore, these observations further corroborate the effects of neural growth factors secreted by transp lanted cells on the CNS repair. Another possibility is that BMSCs may have the ability to degrade nerve-inhibitory molecules present in the site of CNS injury th ereby allow regenerating axons to grow. Human BMSCs have been shown to produce membrane t ype I matrix metalloproteinase and matrix metalloproteinase-2 (Son et al ., 2006) which degrade nerve-inhib itory molecules (d'Ortho et al., 1997; Fosang et al., 1992; Passi et al., 1999). Axonal Regrowth Stimulatory Effects of BMSCs Perhaps the most plausib le mechanism of BMSC s in promoting recovery from CNS injury is mediated by the ability of BMSCs to stimulate axonal regrowth. This effect is thought to be mediated by several distinct functions of BMSCs, which are likely to work in combination. First, as descried above, BMSCs produce an arra y of neurotrophic factor s, some of which are known to enhance axonal regrowth in vitro and in vivo (Auffray et al., 19 96; Chen et al., 2002a; Chen et al., 2002b; Crigler et al ., 2006; Li et al., 2002). Seco nd, BMSCs produce extracellular matrix and adhesion molecules that are known to have strong influence on the attachment and extension of neuronal bodies a nd their axons. Among these are fibronectin and laminin, which human BMSCs are shown to produce (Grayson et al., 2004; Hofstetter et al., 2002), are potent neurite extension-promoting substr ates. Adhesion molecules such as E-cadherin (Oblander et al., 2007), N-cadherin (Puch et al., 2001; Schense et al ., 2000), and neural ce ll adhesion molecule (NCAM) (Crigler et al., 2006; Kimu ra et al., 2004) have all been implicated to play a role in axonal regeneration. Through production of these molecules BMSCs ar e thought to provide permissive microenvironment for regenerating a xons (Hofstetter et al., 2002). Third, it was

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33 proposed that BMSCs may guide regenerating axons by acting as a cellular bridge and towing axons (Wright et al., 2007 ). In a rat contused spinal cord, BMSCs were shown to form bundles to which regenerating axons were intimately associated (Hofstetter et al., 2002). It has been further suggested that BMSCs may provide a nerve-permissive environment by degrading nerve-inhibitory molecules (such as neural proteoglycans, myelin associated glycoprotein, and Nogo-A) with metalloproteinase and matrix meta lloproteinase-2 (d'Ortho et al., 1997; Fosang et al., 1992; Passi et al., 1999; S on et al., 2006). Thus, BMSCs ma y modify nerve-inhibitory molecules and provide physical, nerve-permissive cellular bridges for axons to regenerate and guide axons to cross the lesion site that is otherwise severely hostile to regenerating axons. Remyelination by Transplanted BMSCs It has been known that peripheral m yelin ating cells, Schwann cells, can myelinate demyelinated axons and enhance axonal regeneration in the spinal cord when transplanted into a lesion site (Blakemore, 1977; Honmou et al., 1996) Olfactory ensheathing cells (OECs) are another type of peripheral myelin ating cells which are found in th e olfactory nerve and the outer layer of the olfactory bulb, which normally do not myelinat e axons but can remyelinate demyelinated axons when transplanted into th e CNS lesion (Franklin et al., 1996; Imaizumi et al., 1998). Although attracting as transplants, harvest of a sufficient number of autologous Schwann cells or OECs poses practical difficu lty. There are, however, interesting studies demonstrating that bone marrow cells may act like peripheral myelinating cells when transplanted in the CNS and rest ore conduction properties of the dama ged spinal cord. Sasaki et al. (2001) transplanted an acutely isolated m ononuclear cell fraction of rat bone marrow into rat spinal cords that were demyelinated by x-irra diation followed by microi njection of ethidium bromide. They found that donor cells remyelinat ed demyelinated host axons with predominately a peripheral myelination patter n. When CD34+ cells were transplanted no remyelination was

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34 observed, indicating that BMSCs remyelinated a xons. Remyelination of the rat demyelinated spinal cord was also reported following systemic delivery of the mononuclear cell fraction of rat bone marrow, which showed both CNS and PNS patterns of myelination and resulted in restoration of conduction velocity (Akiyama et al., 2002a). The same group later demonstrated that rat BMSCs have the ability to form both central and peripheral myelin around demyelinated CNS axons and restore conduction properties of the spinal cord (Akiyama et al., 2002b). An alternative approach may be to coax BMSCs in myelinating cells in vitro prior to transplantation in order to maxi mize the myelination efficacy. Several studies suggest that this may be possible by inducing BMSCs to differentia te into Schwann cells. Dezawa et al. (2001) reported that when rat BMSCs were treated with beta-mercaptoethanol followed by retinoic acid and cultured in the presence of forskolin, bFGF, PDGF and heregulin, these cells differentiated into cells resembling morphologically and immunophenotypically Schwann cells. Upon transplantation into the cut ends of the sciatic nerve, differentiated cells remyelinated and enhanced axon regeneration. Using the same pr otocol, Chen et al. (2006) reported that rat BMSC-derived Schwann cells contributed to reconstruction of th e sciatic nerve and improvement of functional analyses. Kamada et el. (2005) demonstrated th at transplantation of rat BMSC-derived Schwann cells resulted in enhanced axonal regeneration and functional recovery in rats with completely transected spinal cord. BMSC Transplantation for CNS Injury Experimental Studies in Anim al Models of CNS Disorders Following early in vitro studies of BMSC plasticity, th e clinical efficacy and safety of BMSC transplantation for various CNS disorders have been investig ated. Most of these studies are performed in preclinical mode ls of CNS injury, primarily in rodent models, but some have been translated in human clinical trials. Intensive research has been undertaken in the fields of

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35 spinal cord injury (SCI) and brain stroke models but neurodegenerative disorders have also been investigated. Spinal Cord Injury Functional recovery after SCI is very lim ited because of the limited ability of damaged axons and neurons to regenerate in the site of injury. This is largely due to the hostile microenvironment at the injury site containing inflammatory mediators, nerve inhibitory molecules, and lack of trophic factors. As discussed, it has been suggested that neuronal replacement by BMSCs is unlikel y the prime mechanism underlying functional recovery after SCI. Alternatively, evidence suggests that BM SCs help overcome the inhibitory barriers by providing a permissive envir onment for regenerating axons. The efficacy of BMSC transplantation on func tional recovery has been reported in rat contusion SCI models. Chopp et al. (2000) transplanted BMSCs intramedulla ry in rats with SCI 1 week after injury and observed functiona l recovery, as measured by the Basso-BeattieBresnahan (BBB) scores, over a 5-week period. Wu et al. (2003) showed that rat BMSCs promoted differentiation of neurosphere cells in vitro, and upon transplantation in rats with contusion SCI immediately after injury, reduced the size of ca vity and promoted functional recovery. In both studies, aut hors speculated that functional re covery was primarily mediated by trophic support by BMSCs because only a sm all portion of BMSCs showed neuronal phenotypes. In contrast, Hofste tter et al. (2002) reported that significant larger numbers of surviving cells and functional improvement were achieved in rats with contusion SCI when BMSCs were given 1 week after injury compared to immediate treatment. They found 5 weeks after injury that BMSCs formed bundles asso ciated with longitudinally arranged immature astrocytes, bridging the epicenter of the injury. Robust bundles of neurofilament-positive fibers and some 5-hydroxytryptaminepositive fibers we re found mainly at the interface between the

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36 graft and scar tissues. Functional recovery fr om SCI has also been reported after BMSC administration in a mouse model with (Lee et al ., 2003) and without (Kod a et al., 2005) neuronal differentiation of BMSCs. The effects of BMSCs on chronic SCI are less known; so far there ar e 2 studies addressing this question. Zurita et al. (2004) transplanted BMSCs into ra ts with contusion SCI 3 month after the injury and evaluated functional improvement over a 4 w eeks period. They reported that progressive functional recovery was clearly observed in animals treated with BMSCs. Histological sections of the spinal cord revealed that grafted BMSCs formed cellular bridges within the centromedullary cavity and expressed neuronal as well as astrocytic phenotypes. They also noted marked ependymal cell prolifera tion expressing nestin, a marker of neural stem cells, which might have become activated by initia l injury and/or subseq uent grafting of BMSCs as endogenous restorative and regenerative processes. The same group (Zurita and Vaquero, 2006) reported a one year follow-up of the same pro cedures in rats with contusion SCI receiving intralesional BMSC transplantation. They found that functional recovery, evaluated by BBB scores, started a few weeks after transplantatio n, increased over the following months, and stabilized 1 year post transplantation at wh ich point the functional recovery was almost complete. Histochemistry revealed that donor cells formed tissue bundles bridging the central cord cavity. Brain Injury Two types of experim ental brain injury m odel are commonly studied in the context of BMSC transplantation; a contusion model has been used to mimic human traumatic brain injury whereas an ischemic model has been used to mimic brain stroke. In rats with contusion brain injury, intra-arterial and intravenous administration of rat BMSCs resulted in functional recovery (Chopp and Li, 2002). BMSCs administered

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37 intravenously targeted damaged tissues in the br ain and led to functional recovery; these effects were not further promoted by intra-arterial inj ection. As for the case of SCI, the effects of BMSCs on functional recovery were attributed to enhancement of endogenous reparative processes as only a small numbe r of grafted cells adopted ne ural phenotypes (Chopp and Li, 2002; Mahmood et al., 2002). In a subsequent study by the group em phasized the role of NGF and BDNF in neurogenesis, synaptogenesis, an d reduction in apoptotic cells in the boundary zone of the injury site (Mahmood et al., 2004a). Intracerebral administra tion of BMSCs was also shown to be beneficial in rats with traumatic injury, leading to long la sting functional recovery over a 3 months period (Mahmood et al., 2004b; Mahmood et al., 2006). Therapeutic effects were dose dependent and thought be mediated by proliferati on of endogenous progenitor cells (Mahmood et al., 2004b; Mahmood et al., 2006). Add itional studies showed that transplantation of human BMSCs also provided a similar degree of functional recovery in the rat model of traumatic brain injury (Mahmood et al., 2003; Mahmood et al., 2005). BMSC transplantation has also provided promis ing results in the rat model of ischemic brain injury. It was shown that intracerebral tr ansplantation of rat bone marrow with (Chen et al., 2000) and without BDNF (Li et al., 2000) resulte d in neural differentiation of grafted cells and improved motor function. In a following st udy, Chen et al. (2003a) found that rat BMSCs administered intravenously preferentially migrated in the ipsilateral ischemic hemisphere where they reduced apoptosis and promoted proliferation of endogenous progenitor cells, leading to improved function recovery. Long-term effects of rat BMSC tran splantation in stroke were reported by Li et al. (2005). In their study, significant improvement of neurological deficits was observed 4 months post-transplantation. BMSC tr eatment reduced the thic kness of the scar wall and reduced the numbers of microglia/macrophages within the scar wall. Further, they found

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38 increased expression of growth -associated protein-43 (GAP43) (a marker of axonal growth cones), among reactive astrocytes in the scar boundary zone and in the subventricular zone in the treated rats. The beneficial e ffects of BMSC transplantation (1 month post-inju ry) on a chronic model of stroke has also been reported (Shen et al., 2007). Functional recovery following intravenous transplantation of human BMSCs in the rat ischemic model has been reported by several groups. Most of these studies emphasi ze the importance of soluble factors released by BMSCs, including neurotrophic factors (Chen et al., 2002b), vasoactive factors (Chen et al., 2003b; Chen et al., 2002b; Song et al., 2004), and various cytokines (Ki nnaird et al., 2004). Neurodegenerative Disorders Although the potential use of BMSCs for the tr eatm ent of neurodegene rative diseases has been implicated, there is only limited information about the prospect of BMSCs for treatment of neurodegenerative diseases. Unlike other types of CNS injury (e.g. traumatic and ischemic), neurodegenerative diseases ofte n affect specific populations of neurons. Previous studies demonstrated that BMSCs can be induced to differentiate into neurons expressing glutamic acid decarboxylase (Li et al., 2004), choline acetyltransf erase (ChAT) (Li et al., 2004), or tyrosine hydroxylase (Dezawa et al., 2004). Th ese findings encourage researchers to pursue development of a new therapeutic strategy for neur odegenerative diseases using BMSCs. As discussed earlier Dezawa et al. (2004) reported generation of tyrosine hydroxylase positive neurons from rat and human BMSCs by NI CD transfection and GDNF treatment. These neurons produced dopamine and, upon transplant ation in a 6-hydroxy dopamine rat model of Parkinsons disease, promoted functional recovery. Alternatively, BMSCs may be used as a vector to deliver therapeutic molecules to a ta rget tissue. Schwarz et al. (1999) genetically engineered rat and human BMSCs to synthesize L-DOPA using retroviruses encoding tyrosine hydroxylase and GTP cyclohydrolase I. Although transduced BMSCs synthesized L-DOPA and,

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39 upon transplantation into the st riatum of 6-hydroxy dopamine-lesioned rats, led to functional recovery, expression of transgenes ceased at about 9 days and therapeutic effects were shortlived. An attempt to treat Alzheimers disease with BMSCs has also been re ported recently (Wu et al., 2007). Transplantation of BMSCs in a rat model of Alzheimers disease (induced by injection of A amyloid protein in the hippocampus) increased the number of ChAT neurons in the CA1 zone of the hippocampus and improved learning and memory of the lesioned rats. Some of the transplanted GFP-positive BMSCs seemed to have differentiated into ChAT neurons and might have contributed to the functional recovery. Human Clinical Trials The first report on hum an trial of BMSCs tr ansplantation was reported in 2005 by Park et al. (2005). In their study, five human patients with complete acu te SCI received intra-lesional transplantation of autologous whole bone marrow and subcutane ous injection of granulocyte macrophage-colony stimulating factor (GM-CSF). One patient received GM-CSF only. They reported that side effects following the pro cedure were limited to those caused by GM-CSF injection, including transient a fever, myalgic pain, and leukocytosis, and no serious complications increasing mortality and morb idity were found. Sensory improvements were noticed immediately after the operations followed by significant motor improvements noticed 3 to 7 months postoperatively. Neurological impr ovement, judged by the American Spiral Injury Association Impairment Scale (AIS), was achieve d in four of five patients receiving bone marrow transplantation and GM-CSF, and no wors ening of neurological symptoms was found. They concluded that bone marrow transplantatio n and GM-CSF administra tion represent a safe protocol to efficiently manage SCI patients, espe cially those with acute complete injury. It

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40 should be noticed, however, that since they used whole bone marrow, it is not clear which cell populations in the bone marrow primarily contri buted to the observed functional recovery. The same group reported the results of a phase I/II open-label and nonrandomized study of bone marrow transplantation and GM-CSF injection in 35 complete spinal cord injury patients (Yoon et al., 2007). They reported that the proc edures were not associated with any serious adverse events increasing morbidit ies. The AIS grade increased in 30.4% of the acute (within 14 days postinjury) and subacute (between 14 days and 8 weeks postinjury) tr eated patients (AIS A to B or C), whereas no significant improvement was observed in the chronic treatment group (more than 8 weeks postinjury).

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41 CHAPTER 2 CHARACTERIZATION OF CANINE BONE MARROW STROMAL CELLS Background and Introduction Recently, bo ne marrow stromal cells (BMSCs), also referred to as bone marrow-derived mesenchymal stem cells, have gained considerab le attention as a potential source for cellular transplantation therapies for a variety of diseases due to their prolifera tive capacity, accessibility, and multipotentiality. Clinical application of BMSCs depends on development of preclinical animal models. Canine models have been used to evaluate various aspe cts of human diseases, some of which were developed to evaluate the safety and clinical efficacy of BMSC transplantation. The most widely accepted canine models that are being used in the context of BMSC research are myocardiac infarction models (L i et al., 2003; Memon et al., 2005; Vulliet et al., 2004) and bone defect models (Arinzeh et al., 2003; Bruder et al., 1998). These canine models have provided important in sights into stem cell biology under a setting that is perhaps closer to human patients than ma ny laboratory animal models are. Previous studies on canine BMSCs have focused on osteogenic (Arinzeh et al., 2003; Bruder et al., 1998; Kadiyala et al., 1997; Volk et al., 2005) a nd ,to a less extent, chondrogenic (Kadiyala et al., 1997) differentiation properties b ecause of the high prevalence of disorders in the skeletal system in dogs and the potential of su ch disorders to be an animal model in bone and cartilage research. These studies demonstrated proof of concept for the clinical use of BMSCs for bone and cartilage regeneration. Despite the potentials of canine models in BMSC research and their clinical relevance in veterinary medi cine, information regarding the basic biology of canine BMSCs has been rather scarce compared to that of BMSCs from other species. Thus far, little is known about phenotypic pr operties, purification and cultu re expansion techniques, and differentiation abilities of canine BMSCs, all of which will become imperative to develop

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42 preclinical studies using canine BMSCs. In hum an and rodent BMSC research, emphases have been put on the development of prospective iden tification and optimal e xpansion techniques in order to selectively expand authentic BMSCs. Th is relates to the fact that BMSCs have been classically isolated by the use of their physical property to ad here to plastic substrates (Friedenstein, 1995; Goshima et al., 1991b) which results in a high degr ee of hematopoietic contamination. Although there has been much progress in identifying cell surface proteins expressed on BMSCs (Haynesworth et al., 1992a), there is no specific BMSC marker that can be used to prospectively identify, isolate, and expand BMSCs in a similar way to hematopoietic stem cells. Therefore, identif ication of unique BMSC cell surf ace markers and development of culture expansion techniques have been a focus of may investigators. While phenotypic characterization is needed to develop techniques for selective expansion of BMSCs, functional assays are used to demonstrate the multipotency of expanded cell populations. Classically, multipotency of BMSCs has been demonstrated by their ability to differentiate into three major mesodermal cell types in vitro and/or in vivo namely osteoblasts, chondrocytes, and adipocytes (Haynesworth et al ., 1992b; Jaiswal et al., 1 997; Pittenger et al., 1999). Furthermore, it has been shown that in a ddition to these cell types, BMSCs differentiate into myoblasts (Wakitani et al., 1995), cardiomyocytes (Toma et al., 2002) and tendocytes (Jiang et al., 2002a) in vitro. Although demonstration of multipotency has been the gold standard to characterize BMSCs there is evidence that re sponses to given diffe rentiation stimuli are noticeably diverse among different species. Interspecies varia tion of osteogenic differentiation properties of BMSCs has been most intensivel y studied to date (Diefenderfer et al., 2003a; Diefenderfer et al., 2003b; Osy czka et al., 2004; Volk et al., 2005) The differentiation capacity of BMSCs into cell types other th an those of mesodermal origin has been demonstrated in recent

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43 studies and explained in part by their tremendous plasticity. The possibility of generating various cell types from BMSCs has expanded potential applicatio ns of BMSC-based therapies, and we addressed in vitro and in vivo neural transdifferentiation properties of canine BMSCs in Chapter 3 and Chapter 4, respectively. In the present study, we characterized phe notypic and functional pr operties of canine BMSCs. First, we estimated the frequency of BMSCs in canine bone marrow by use of CFU-F assay and investigated the growth kinetics in vitro Next, flow cytometry was used to characterize the phenotypic prope rties and homogeneity of expanded canine BMSCs. Last, we evaluated the differentiation properties of canine BMSCs by observing osteogenic and adipogenic differentiation in vitro and comparing the results to those of rat BMSCs. Materials and Methods Bone Marrow Collection A total of 5 adult m ongrel canine cadavers were collected from a loca l animal shelter after euthanasia. Exact ages of these dogs were unknown; however, we collected only those which had a complete set of adult dentures with minimum dental calc ulus deposition, allowing inclusion of young adults in the study. The use of these animals was approved by the Institutional Animal Care and Use Committee of the University of Florida. Bone marrow was aseptically aspirated from iliac crests into st erilized 10 mL syringes containing 2,000 units of Heparin, using 16-G Jamshidi needles. All bone marrow collections were performed within 30 minutes after euthanasia. Rat bone marrow was obtained from a total of 3 young adult male Sprague-Dawley rats (90 g) that were euthanized for an unrelated project. Specifically, both femurs and tibiae were removed, both ends cut, and bone marrow obtained by flushing it with complete medium through a 21-G needle inserted into the proximal end. The complete medium consisted of

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44 Dulbecco's Modified Eagle's Medium (1g/L glucos e) supplemented with 20% fetal bovine serum (FBS), 100 U/mL penicillin G, 100 g/mL streptom ycin sulfate, and 0.25 g/mL amphotericin B. BMSC Culture Canine BMSCs were isolated and cultured usin g techniques described by Kadiyala et al. (1997) with som e modifications. Briefly, the marrow was washed with Hanks balanced salt solution (HBSS) and mixed with two volumes of complete culture medium. Aliquots of the bone marrow suspension were layered over Ficoll and centrifuged at 400 g for 30 minutes to enrich mononucleated cells. Enriched mononuclear cells were plated in T-75 culture flasks at a cell density of 1.5 l05 cells/cm2 in 15mL of complete medium for each animal and incubated at 37C in a humidified 5% CO2 environment. After 48 hours, fl asks were washed with phosphate buffered saline (PBS) to remove nonadherent cells. The cultures were fed biweekly and passaged upon reaching approximately 80% conf luence by releasing the cells with 0.05% trypsin/0.53 mM EDTA. Released cells were collected by ce ntrifugation at 800 g for 5 minutes, washed twice with HBSS, counted by use of a hematocytometer, and replated at 8 l03 cells/cm2 for subsequent passages. Portions of Fi coll-separated mononucleated cells from each dog were suspended in 10% dimethylsulfoxide (DMS O) and 90% FBS and frozen in aliquots in liquid nitrogen for later use. Rat bone marrow was plated in 2 T-75 flasks and cultured in comple te medium. Upon reaching semiconfluency, primary cells were detached by 0.05% trypsin/0.53 mM EDTA and frozen in liquid nitrogen for later use. Colony-Forming Unit Assay In order to estim ate the frequency of BMSCs in the canine bone marrow, CFU-F assay was performed according to the method described by Meirelles et al. (2003) with modifications.

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45 After Ficoll separation, mononucleated cells were plated in 60mm culture dishes with increasing cell numbers (4.2 105, 8.4 105, and 1.68 106). Cells were cultured for 12 days in complete medium after which colonies were fixed, staine d with Diff-Quick (Dade Diagnostics, Inc.), and observed under an inverted microscope. Colonies containing more than 50 cells were scored. All assays were performed in duplicate. Growth Kinetics The rate of cell growth was m easured in samples obtained from three bone marrow samples. Frozen Ficoll-separated mononucle ated cells were thawed, counted with a hematocytometer, and seeded at 1.5 106/cm2 in T-75 culture flasks. Adherent cells were expanded and first passaged (P1) BMSCs were used to measure cell prolif eration. On day 0, P1 BMSCs were plated in 6-well cu lture plates at low (10 cell/cm2), medium (100 cells/cm2), and high (1,000 cells/cm2) cell densities and cultured for 12 days in complete medium. Medium changes were performed every three days. Each day, 2 wells from each cell density group were trypsinized and the cell numbers were de termined by use of a hematocytometer. Flow Cytometric Characterization To evaluate the cell su rface markers and th e homogeneity of canine BMSCs, P3 BMSCs were analyzed by flow cytometry. Culture me dium was removed and attached cells were washed three times with PBS. The cells were detached from the flasks by incubation with 0.05% Trypsin/0.53mM EDTA. Detached cells were co llected by centrifugation, washed with FACS buffer (PBS containing 0.5% BSA and 0.1% sodium azide), and counted. Aliquots containing 1 106 cells were incubated with primary anti bodies for 30 minutes on ice. The primary antibodies used were R-phycoerythrin (R-PE) conjugated mouse anti-canine CD34 (BD Biosciences; 1:1), FITC-conjugated rat anti-c anine CD45 (Serotec; 1:15), mouse anti-canine CD90 (Thy-1) (VMRD Inc; 1:80), mouse anti-canine MHC-I (VMRD Inc; 1:133), and mouse

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46 anti-canine MHC-II (VMRD Inc; 1:133). AP C-conjugated goat anti-mouse IgM (Caltag Laboratories; 1:5) was used to label anti-CD 90 antibodies and FITC-conjugated rat anti-mouse IgG2a (BD Biosciences; 1:25) was used to label anti-MHC I and MHC II antibodies for 30 minutes on ice. Aliquots containing the equal nu mber of cells were incubated with respective isotype controls under the same conditions. Cells without antibodies were used as a negative control. Labeled cells were then washed w ith FACS buffer, pelleted, and fixed in 0.5% paraformaldehyde and 0.1% sodium azide in PB S. Data were analyzed by collecting 10,000 events on FACS Calibur cytometer (Becton Dick inson Immunocytometry Systems) using Cell Quest software (BD Biosciences). Differentiation Assays Osteogenic and adipogenic differentiation pr operties of canine BMSCs were evaluated using the protocols described by Pittenger et al. (1999) with slight m odifications. To maximize the possibility and rate of cell differentiation, P1 canine BMSCs were used for the assays. Frozen Ficoll-separated canine BMSCs were plated in T-75 culture flasks and expanded in complete medium until semiconfluency. Expanded primary cells were released by 0.05% trypsin/0.53 mM EDTA and subcultured at 8 l03 cells/cm2 as first passage BMSCs in 6-well culture plates. P0 BMSCs from 2 dogs were also subjected to osteogenic differentiation. Rat primary (Po) BMSCs were plated in 6-well culture plates and used for comparative observations. Osteogenic induction was initiated by replac ing the complete medium with medium containing 50M ascorbate 2-phosphate (Sigma), 10mM -glycerophosphate (MP Biomedical), 100nM dexamethasone (MP Biomedical), 10% FBS, in low-glucose (1g/L) DMEM. In selected cultures, bone morphogenic protein-2 (recombinant human BMP-2, Pepro Tech) was added at 50ng/mL as per Volk et al. (2005) Osteogenic induction media we re replaced every three days and continued for 12 days at 37C in a humidified 5% CO2 environment. On day 12,

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47 differentiation of BMSCs into th e osteoblastic phenotype was evaluated under a phase contrast microscope and by Alizarin Red S (Gregory et al ., 2004) as well as Von Kossa (Anselme et al., 2002) staining as described previously. Adipogenic differentiation was induced by replacing the complete medium with adipogenic induction medium containing 1mM dexamethasone, 0.5mM methyl-isobutylxanthine, 10g/mL insulin, 100mM indomethacin, and 10% FB S in DMEM (4.5g/L glucose). Cells were cultured for 3 days and the medium was changed to adipogenic maintenance medium containing 10g/mL insulin and 10% FBS in DMEM (4.5g/L gl ucose) for 24 hours. This cycle of induction and maintenance was repeated three times with last maintenance cycle being extended for 7 days. After 7 days, cells were fixed in 4% paraformaldehyde and stained with Oil Red O staining. Results Bone m arrow was aspirated from each dog a nd mononucleated cells were isolated as described above. The summary of the bone marrow samples used in this study is shown in Table 2-1. The average yield of mononucleated cells per 1mL of bone marrow was 2.27 107 cells. Table 2-1. Summary of bone marrow samples. Dog ID# Dog sex Bone marrow collected (mL) Total MNCs yield* (7) 21 Male 5 16.0 22 Female 3 3.75 23 Female 10 23.9 24 Male 9 18.0 25 Male 10 25.2 All bone marrow samples were obtained from th e iliac crest of young adult canine cadavers. indicates the number of mononucleated cells isolated after Ficoll separation. Morphological Observation of Canine BMSCs Growth characteristics of plastic-adherent cells isolated from canine bone marrow were similar to BMSCs from other species as describe d previously. Approxima tely after 5 days of

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48 plating, adherent cells could be observed in most cultures. Initi ally, there were morphologically two major types of cells in the culture, loos ely attached rounded cells and tightly attached spindle-shape cells. Typically, loosely atta ched rounded cells (Fi g. 2-1A, arrowheads), presumably mature leukocytes and hematopoietic stem/progenitor cells, were observed on top of the spindle-shape cells. Most of these rounded ce lls were washed out over time with changes in medium and almost completely removed from the culture by the time of the first passage (typically after 14 days). After 7 to 10 days of initial platin g, spindle-shape cells rapidly grew, forming discrete symmetric coloni es (Fig. 2-1A). Of the adhe rent colony-forming cells, there were various cell morphologies, including flatte ned fibroblastic cell (Fi g. 2-1B), long spindleshaped cells (Fig. 2-1C), and short spindle-shaped cel ls (Fig. 2-1D). Figure 2-1. Morphological observa tion of canine primary BMSCs. (A) Low magnification view of primary culture on day7. Primary BMSCs grow in colonies with various cell morphologies comprising each colony. Small rounded cells are occasionally observed on top of adherent BMSCs (arrowh eads). There are at least three major types of adherent cells as observed u nder higher magnification views on day14; (B) large flattened fibroblastic ce lls, (C) long spindle-shaped ce lls, and (D) short spindleshaped cells with refractile soma. Scale bar = 100M.

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49 From the first passage culture on, the spindleshape cells became predominant which grew in uniform monolayer and further became fl attened to assume fibroblastic morphology upon further passaging. The Frequency of Canine BMSCs In all sam ples, the numbers of colonies cons istently increased as a function of the number of cells seeded (Fig. 2-2). Ov erall, the frequency of canine BMSCs was estimated to be 0.0042 0.0019 % which equals approximately 1 BMSC in every 2.38 104 mononucleated cells. It was noted, however, that there was variability among bone marrow samples, represented by a wide range of the frequencies of CFU-F (0.0014 0.0057%) (Table 2-2). Figure 2-2. The CFU-F assays fr om five adult canine bone marrow samples. Primary cells were seeded at three different cell densities and cultured for 12 days. Colonies containing more than 50 cells were scored. All cultures were performed in duplicate and the average numbers of colonies were plotted for each dog for each cell density. Growth Kinetics of Canine BMSCs Three bone m arrow samples (Dog#23, #24, #25) were used to evaluate cell growth kinetics at low (10 cells/cm2), medium (100 cells/cm2), and high (1,000 cells/cm2) cell density. Cells seeded at 1,000 cells/cm2 proliferated and produced approximately 4.6105 cells over a 12-day

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50 Table 2-2. Number and fr equency of CFU-F from five bone marrow samples. Seeding density Average Sample # 4.2 x 105 8.4 x 105 1.68 x 106 Frequency Plate 1 2 17 25 Dog 21 Plate 2 5 12 28 Average 3.5 14.5 26.5 Frequency 0.000833333 0.001726 0.001577 0.001379 Plate 1 16 28 49 Dog 22 Plate 2 11 18 52 Average 13.5 23 50.5 Frequency 0.003214286 0.002738 0.003006 0.002986 Plate 1 19 54 78 Dog 23 Plate 2 18 59 69 Average 18.5 56.5 73.5 Frequency 0.004404762 0.006726 0.004375 0.005169 Plate 1 31 54 70 Dog 24 Plate 2 24 40 82 Average 27.5 47 76 Frequency 0.006547619 0.005595 0.004524 0.005556 Plate 1 29 44 105 Dog 25 Plate 2 24 51 72 Average 26.5 47.5 88.5 Frequency 0.006309524 0.005655 0.005268 0.005744 Numbers of colonies for each plate and the aver age are shown. Average frequencies represent the frequency of CFU-F for nucleated cells in bone marrow. culture period, whereas cells seeded at 10 cells/cm2 produced only 7.0 103 cells (Fig 2-3A). However, cells seeded at 10 cells/cm2 expanded approximately 74-fold in 12 days, whereas cells seeded at 1,000 cells/cm2 expanded only 48-fold (Fig 2-3B). Cells seeded at 100 cells/cm2 displayed intermediate growth kine tics patterns (Fig 2-3A and B). Flow Cytometric Profile of Canine BMSCs The surface m arker profile of P3 BMSCs was analyzed by flow cytometry. The results indicated that the cell surface pr ofile of canine BMSCs was comparable to those reported for human and rodent MSCs; they were positive fo r CD90 (mean total % standard deviation, 88.5% 3.9) and MHC-I (85.4% 3.7), and negative for MHC-II (0.9% 0.5) (Fig. 2-4). The

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51 absence of the lipopolysaccharide receptor CD34 (1.1% 0.1) and the leukocyte common antigen CD45 (0.5% 0.1) expression indicated that cells of hematopoietic origin had been excluded during the cell expansion process. Figure 2-3. Cell growth kinetics as a function of initial cell dens ities. BMSCs from three dogs were plated at low (10 cells/cm2), medium (100 cells/cm2), and high (1,000 cells/cm2) cell density. The average tota l cell counts (A) and fold incr eases are plotted for each cell density over a 12-day culture period. Da ta points represent mean values from three samples. Bars represent standard errors. Figure 2-4. Representative resu lts of flow cytometric analysis of canine BMSCs. Canine BMSCs are negative to CD34, CD45, MHC -II and positive to CD90 and MHC-I. Green lines represent counts of the cell population that is positive for the cell surface marker indicated in each panel. Oran ge lines represent corresponding isotype controls. M1 indicates the gated area.

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52 Osteogenic and Adipogenic Diff erentiation of Canine BMSCs To define the osteogenic differentiation pot ential of canine BMSCs, P1 BMSCs were cultured in m edium containing oste ogenic inducers. It was clearly noted that in the presence of osteogenic inducers, BMSCs proliferated at a mu ch higher rate with concomitant morphological changes. Addition of BMP-2 further stimulated cell proliferation. Th e main morphological change appeared at the early stage was transfor mation from fibroblastic cells to spindle-shaped refractive cells (Fig. 2-5E and F). At later stages, proliferated cells became cuboidal, a characteristic morphology of osteoblastic cells (F ig. 2-5H, I, K, and L). However, distinct nodular aggregates were not f ound under any culture conditions. A significant change was not observed in BMSCs in control medium (Fig. 2-5A, D, G, and J). Neither differentiated cells nor control cells were staine d against Von Kossa or Alizarin Red S staining. It was also noted that because of accelerated cell growth in the presen ce of osteogenic inducers, cells often detached from culture plastic around day 6, which precluded further analysis. Then, we tested the osteogenic differentia tion property of primary canine BMSCs under the same induction condition. Primary BMSCs migrated and formed nodular aggregates in osteogenic medium (Fig. 2-6B), whereas cells grew in colonies in control medium (Fig. 2-6A). Larger nodules were consistently seen in cult ure with BMP-2 (Fig. 2-6C) when compared to those without BMP-2 (Fig. 2-6B). The nodular aggregates in osteogenic medium were negative for Von Kossa staining, indicating th at mineralization of the matrix had not occurred (Fig. 2-6E). In contrast, strong Von Kossa staining was found in nodules formed by BMSCs cultured in osteogenic medium with BMP-2 (Fig. 2-6F). Rat P1 BMSCs were used for a comparative observation. Rat BMSCs appeared as flattened and larger polygonal ce lls in the control medium, wh ich proliferated but did not

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53 Figure 2-5. Morphological changes of canine P1 BMSCs during osteogenic induction. Significant morphological cha nges were not observed in BMSCs cultured in control medium (A, D, G, and J) over a 12-da y culture period. Osteogenic medium stimulated cell growth and induced mor phological transformation from fibroblastic cells to spindle-shaped cells between day 6 (E) and day 9 (H), and to cuboidal cells by day 12 (K). These changes were pronounced in cultures in the presence of BMP2 (C, F, I, and L). Nodular aggregate formation was not found in any of the cultures. Scale bar = 250M.

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54 Figure 2-6. Osteogenic differentia tion of canine P0 BMSCs. Primary cells grew in control medium without apparent nodule formation (A and D). In osteogenic medium, small nodules were formed (B), which were Von Kossa-negative (E). BMP-2 stimulated nodule formation (C) which was strongly mineralized as evident by Von Kossa staining (F). Scale bar = 250M. undergo apparent morphological changes during the 12-day culture period (Fig. 2-7A, D, G, and J). In contrast, in the osteogenic medium, cells started to migrate by day 6 and formed nodular aggregates by day 12 (Fig. 2-7B, E, H, and K) Addition of BMP-2 further stimulated cell proliferation and nodule formation (Fig. 2-7C, F, I, L). Mineralization of the cellular matrix was not found in cells grown in control medium (F ig. 2-8A and D). In the osteogenic medium, cellular aggregates showed minima l deposition of mineralized mate rials (Fig. 2-8B and E). In the presence of BMP-2, extensiv e mineralization of the cellular matrix was observed (Fig. 2-8C and F). Next, adipogenic differentiation was induced on canine P1 BMSCs. It was consistently noticed that cells in the induction medium became flattened, losing their refractile appearance by day 3 (Fig. 2-9A). By day 6 of induction, th ere was a small portion of cells containing cytoplasmic vacuoles (Fig. 2-9B). These cytopl asmic vacuoles further enlarged in the induction

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55 Figure 2-7. Morphological change s during osteogenic differentiation of rat P1 BMSCs. Significant morphological cha nges were not observed in BMSCs in control medium (A, D, G, and J) over a 12day culture pe riod. On day3, confluent rat BMSCs were observed in osteogenic media (B and C). On day6, clear nodular aggregates were observed in BMSCs in osteogenic media (E and F). On day9, large nodules were seen in BMSCs (K), which were further st imulated by addition of BMP-2 (L). Scale bar = 250M.

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56 Figure 2-8. Osteogenic differe ntiation of rat P1 BMSCs (day12). Phase contrast photomicrographs show confluent rat P1 BMSCs in control cultures which were negative for both Von Kossa (A) and Alizar in Red (D) staining. In osteogenic induction medium, rat BMSCs migrated a nd formed small nodules. These nodules were partially positive for Von Kossa (B) a nd Alizarin Red (E). Addition of BMP-2 strongly stimulated mineralization of extr acellular matrix evident by strong staining with Von Kossa (C) and Alizarin Red (F). medium by day 9 (Fig. 2-9C) and some of them appeared to fuse by day 12 (Fig. 2-9D). Differentiated adipocytes were found dispersed acro ss the plate, which tended to cluster as seen in Fig. 2-9C, D, and E, indicating that diffe rentiated cells were produced from the common BMSCs. After around day-7 when some of the cells started to differentia te, a significant number of cells detached from culture plastic. Thes e changes were not observed in cells grown in control medium. Oil Red O staini ng showed accumulation of lipid substances in the cytoplasmic vacuoles of differentiated cells on day 12 of induction (Fig. 2-9E). Conclusion and Discussion In the present study, we have characterized canine BMSCs based on their morphological, growth characteristic, phenotypic, and f unctional properties. Morphological characteris tics of canine BMSCs were comparable to BMSCs isolated from rodents and humans as described

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57 Figure 2-9. Adipogenic differen tiation of canine P1 BMSCs. BMSCs cultured in adipogenic medium became flattened after day 3 (A). By day 6, a small number of cells had cytoplasmic vacuoles (B) which further enlarged by day 9 (C). By day 12, these vacuoles fused to form large lipid dropl ets. Oil Red O staining showed lipid substances in the cytoplasm of differentiated cells on day 12 (E). Insets in B, D, and E show higher magnification views of boxed areas. Scale bar = 250M. previously. Similar to BMSCs of rodents and humans, canine primary BMSCs adhered to culture plastic and grew in colonies to form CFU-F. Primary BMSCs were morphologically heterogeneous, containing at leas t three different types of adhere nt cells that could be clearly distinguishable. In passaged cultures, spi ndle-shaped cells progressively became the predominant cell type. These morphological f eatures of primary and passaged canine BMSCs are similar to rodent and human counterparts. FACS analysis showed that th e cell surface marker profile of passaged canine BMSCs was al so comparable to those from rodents and humans. Although canine primary adherent cells were consisted of heterogeneous cell

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58 populations, significant hematopoietic contamination was not evid ent; therefore, the plastic adhesion method seemed sufficient for isolation of can ine BMSCs. This is in contrast to murine BMSCs in which the plastic adhesion method resu lts in significant contamination of granulomonocytic cells in both primary and passaged cult ures (Phinney et al., 1999; Xu et al., 1983), thus requiring preceding immunodepletion usi ng CD11b antibody (Kopen et al., 1999). It is unknown whether morphologically heterogeneou s cell populations in the canine primary adherent cultures represent dist inct classes of stem cells, perh aps containing rare pluripotent stem cell populations present in human and murine bone marrow (J iang et al., 2002a; Jiang et al., 2002b; Reyes et al., 2001), or cells of the same lineage with differe nt developmental stages. At present, the latter is likely the case as human BMSCs are also morphologically heterogeneous even when cloned from single-cell-derived col onies (Colter et al., 2000; Zohar et al., 1997). The frequency of BMSCs in canine bone ma rrow has been reported by Kadiyala (1997) to be approximately 0.004% (1 per 2.5 104 mononucleated cells), al though detailed data was not presented in their study. In the present study, CFU-F assays demonstrated that the frequency of canine BMSCs was approximately 0.0042% (1 per 2.38 104 mononucleated cells). This frequency of canine BMSCs is twofold and four fold higher than the previously reported frequencies of murine (Friedenstein et al ., 1976) and human BMSCs (Bruder et al., 1997), respectively. However, the number of CFU-F can be influenced by culture conditions (e.g. FBS concentration, type of basal medi um, pH of medium, etc) (Xu et al., 1983); therefore, further studies are needed to optimi ze the culture condition for canine BMSCs. We found that the numbers of CFU-F were variable among bone ma rrow samples and several factors could account for this finding. Age is a known factor that has impact on the frequency of BMSCs. In the present study, we did not have information re garding the exact age of the dogs. Although the

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59 dogs in the present study were considered to represent young adult based on their denture, differences in their maturity might have influe nced the frequency of BMSCs. The degree of peripheral blood contamination is another factor since CFU-F is calculated based on the number of mononucleated cells in the sample. This could be significantly in fluenced by postmortem changes such as coagulation of peripheral blood. This problem may arise when bone marrow is collected by aspiration but not by flushing l ong bones which is a common technique for collection of rodent BMSCs. Growth kinetics of canine BMSCs were evaluated under three different cell seeding densities (10 cells/cm2, 100 cells/cm2, and 1,000 cells/cm2). It was clearly shown that initial cell densities have a profound influen ce on the proliferation rate of canine BMSCs, as the case for human BMSCs (Sekiya et al., 2002). Among the three cell densities tested, there was an inverse relationship between the total yiel d of BMSCs and expansion rate (fold increase). This suggests that when BMSCs are seeded at a lower density, they undergo more rapid pr oliferation. It also implies that, because increasing cell density is kn own to trigger cell differentiation (Caplan et al., 1983), there must be a compromise between the total cell yield and the content of undifferentiated cells. Further studies are neces sary to define the optimal culture condition, which generates the maximal cell yield with the greatest content of multipotent canine BMSCs. Our functional assays demonstrated th at passaged canine BMSCs showed limited osteogenic differentiation ability. Although so me morphological changes, which resembled osteoblastic morphology, were observed in passa ged BMSCs cultured in osteogenic medium, formation of nodular aggregates was never f ound in passaged BMSCs, indicating that full differentiation toward osteoblasts did not occur. Osteogenic inducers s timulated cell growth, which often resulted in detachment of ce ll monolayer around after day 6 of induction and

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60 precluded further culture of BMSCs. Despite previous studies (K adiyala et al., 1997; Volk et al., 2005) describing differentiation of passaged canine BMSCs into osteoblasts, these results were not reproducible in our hands. In contrast, primary canine BMSCs differentiated into osteoblasts and formed nodules which were intensively mine ralized. Primary BMSCs cultured in medium without osteogenic inducers did no t show similar changes; thus, contamination of osteoblastic cells in the samples seemed unreasonable. Wh en passaged BMSCs were grown in adipogenic inducers, a small number of cells differentiated into mature adipocytes. Upon exposure to a high concentration of dexamethasone, a significant number of undifferentiated cells detached from the culture plate. These results demonstrated th at a small number of BMSCs possess adipogenic capacity in the passaged BMCS culture. All togeth er, the results of our functional assays suggest that canine bone marrow contains inducible mese nchymal progenitors with the ability to giving rise to osteoblasts and adipoc ytes. Upon passaging, a significant number of these stem cells either lose their multipotency or did not survive in the standard culture condition. Further studies are needed to develop culture techniques whic h enable expansion of canine BMSCs in their undifferentiated state.

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61 CHAPTER 3 IN VITRO NEURAL DI FFERENTIATION OF CANINE BONE MARROW STROMAL CELLS Background and Introduction A variety of cell types such as em bryonic stem cells (McDonald et al., 1999; Wernig et al., 2004) and neural progenitor/stem cells (Oga wa et al., 2002; Pluchino et al., 2003), and developing tissues harboring stem cells such as fetal spinal cord tissues (Anderson et al., 1995; Houle, 1992) have been shown to promote CNS regeneration in experiment al studies. However, clinical application of these s ources is severely limited due to issues associated with the availability of the transplant s, immunological reactions, and mo st importantly with ethical considerations. Cells derived from bone marrow, particularly bone marrow-derived stromal cells (BMSCs, also referred to as mesenchymal stem ce lls), possess potentials to circumvent many of these obstacles; therefore, may repres ent the most realistic cell source. Recent in vitro studies demonstrated that BMSCs can generate not only mesodermal cells but also endodermal and ectodermal cell types, suggesting that thes e cells are less restricted than were previously thought. Neural differentia tion of cells derived from rat and human bone marrow has been reported by several independent groups (Deng et al., 2001; Sanchez-Ramos et al., 2000; Woodbury et al., 2000). These studies ha ve aroused considerab le interest in the potential use of BMSCs for neurol ogical disorders. Unlike ES cells and neural stem cells where embryos or neonatal neural tissues are neces sary to obtain these cells, BMSCs are easily accessible by bone marrow aspiration. Furthermor e, BMSCs are easily expandable in culture without losing their multipotenti ality (Pittenger et al ., 1999). Because of these advantages, BMSCs have received great attention as a promis ing source of cells for a variety of therapies including neuroregeneration.

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62 Early studies on neur al differentiation of bone marrow-de rived stem cells were based on morphological changes and immunoreactivity against some of the neural markers (SanchezRamos et al., 2000; Woodbury et al., 2000). The significance of the morphological changes observed in these studies, however, has been que stioned by others and attributed to actin cytoskeleton shrinkage rather th an genuine neurofilament extensi on (Bertani et al., 2005; Lu et al., 2004; Neuhuber et al., 2004). As such, interpretation of neural di fferentiation of BMSCs in vitro needs great caution and systematic approaches including assays at the molecular level are desirable. While it is still the subject of de bate whether neurogenesis of BMSCs represents genuine trans-differentiation or an epiphenomenon caused by induced chemical stress, functionalities of MSC-derived neurons, such as th eir abilities to secrete neurotransmitters and/or fire action potentials, have been reported by several groups (Hermann et al., 2004; Kohyama et al., 2001; Tao et al., 2005; Wislet-Gendebien et al., 2005). These in vitro studies have raised tremendous interest that BMSCs ha ve the potential to differentiate into functional neural cells. Based on the previous findings in human and rodent BMSC studies, we hypothesized that canine BMSCs can be culture-expanded while maintaining their multipotentiality and that these cells bear neural properties. We induced a rapid neural differentiation on canine BMSCs as previously described by Deng et al. (2001) and evaluated expr ession of neuronal and glialspecific proteins, using immunocytochemical a nd western blotting analyses. We further evaluated the electrophysi ological properties of neurally induced BMSCs using the whole-cell voltage clamp technique. Materials and Methods Preparation of Canine BMSCs Bone m arrow aspirates from 5 canine cadavers were used to isolate and culture canine BMSCs. Detailed descriptions of bone marrow collection, cell isolation, and culture expansion

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63 can be found in Chapter 2. Third passage (P3) BMSCs were used for the assays described in this chapter. Neural Differentiation of Canine BMSCs Neural differentiation of P3 BMSCs was i nduced by replacing the com plete medium by neural induction medium. The neural i nduction medium consisted of 0.5mM methylisobutylxanthine (IBMX) and 1mM dibutyryl cAMP (dbcAMP) in DMEM/F12 as used in neural differentiation of human bone marrow stromal cells (Deng et al., 2001). To support growth of neural cells the medium was supplemented with 1% N2 supplements. For immunocytochemistry, a total of 1 104 P3 BMSCs were suspended in 100 l of complete medium and plated on coverslips placed in each well of 12-well culture plates. After 12 hours, 900 l of complete medium was added and cells were cultured until reaching 70% confluency. Consequently, the medium was changed to 1 ml of neural induction medium and cultured at 37C in a humidified 5% CO2 environment for 5 hours. For Western blot analysis, P3 BMSCs were plated at 8,000 cells/cm2 on 60 mm culture dishes in complete medium. Upon reaching 70% confluency, cells were washed with PBS a nd cultured in neural i nduction medium for 5 hours. Cells were also cultured in complete medi a and served as control. We also used canine fibroblast cultures (also from passage 3) (Coriell Institute for Medical Research) as a control for the assays described below. Immunocytochemistry for Neural Specific Markers We perfor med immunocytochemistry to ev aluate whether canine express neuronal ( tubulin), astrocyte-specific (GFAP), and oligodendrocyte-sp ecific (MBP) proteins. Cells on coverslips cultured in complete medium or neur al differentiation medium were gently washed with PBS and fixed in ice-cold 4% paraformaldehyde for 30 minutes at room temperature (RT). Fixed cells were permeabilized in 1.0% Triton X-100 in PBS for 10 minutes at RT. Non-specific

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64 binding was blocked with a blocking solution (1.0% BSA in PBS) for 30 minutes at RT. Cells were then incubated with pr imary antibodies, including anti-tubulin (Promega Corp, 1:1,000), anti-GFAP (BD Biosciences, 1:1,000), an ti-MBP (HyTest Ltd, 1:50,000), for 1 hour at RT. The primary antibodies were removed, cells wa shed with PBS, and cells incubated with one of secondary antibodies for 1 hour in dark at RT The secondary antibodies (all from Jackson ImmunoResearch Laboratories In c.) used were Cy2 conjugated goat anti-mouse IgG1 (1:400), Rhodamine conjugated goat anti-mouse IgG2b (1 :100), FITC conjugated goat anti-mouse IgG2a (1:400). Cells were also inc ubated without primary antibodies to control for non-specific staining by secondary antibodies. Cells were wa shed with PBS and mounted with a mounting medium containing DAPI (Vector Laboratorie s). Stained cells were observed under a fluorescent microscope (Zeiss Axio plan II, Carl Zeiss MicroImagi ng) with appropriate filters. Western Blotting and Densitometric Analyses To confirm expressi ons of neuronal ( -tubulin) and glial (GFA P and MBP) markers on canine BMSCs, cultures were detached with 0.05% Trypsin/0.53mM EDTA for western blot analyses. Cells were lysed with a lysis so lution (0.18M Tris-HCl, 40% Glycerol, 4% SDS, 0.04% BPB, 0.05M DTT). Canine spinal cord lysa te was obtained using the same lysis solution and used as positive control. Total protein concentrations of each sample were determined by a protein assay kit (Micro BCA pr otein assay, Pierce). Samples c ontaining 20 g of protein were loaded in each lane of 12% polyacrylamide gels and electrophoretically separated. Separated proteins were transferred to nitrocellulose membranes, blocked with TBS containing 5% non-fat dry milk and 0.1% Tween20 for 1 hour at RT, and incubated with primary antibodies ( tubulin; 1:500, GFAP; 1:4,000, MBP; 1:50,000) overnig ht at 4C. The membranes were also probed with -actin (Abcam Inc, 1:1,000) as loading controls. After washing the membranes with TTBS (0.05% Tween20 in TBS solution) for three times, the membranes were incubated

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65 with alkaline phosphate conjugated seconda ry antibodies (Jackson ImmunoResearch Laboratories Inc., 1:5,000) for 1 hour at RT. Th e membranes were washed three times in TTBS and developed in a solution containing NBT and BCIP. Photographs of the membranes were taken using a molecular imager (Flour-S, Multi-imager, Bio-Rad Laboratories) and densitometric analyses performed using a software program (Q uantity One, Bio-Rad Laboratories). Western blotting and densitometric analyses were duplicated in all samples. Electrophysiological Recording Two representative BMSC cultures were used for electrophysiological recordings. Neural differentiation was perform ed as described above. Spontaneous and depolarizing pulse-elicited action potentials were recorded with the wholecell voltage clamp configur ation in current clamp mode (Hamill et al., 1981). Experiments were performed at room temperature (23C) with an Axopatch 200B amplifier and a Digidatal 1200B interface (Axon Instruments, Burlingame, CA). Data acquisition and analyses were perf ormed with the use of Axoscope 7.0 and pClamp 6.2. Differentiated BMSCs were bathed in Tyro des solution containi ng 140 mM NaCl, 5.4 mM KCl, 2.0 mM CaCl2, 2.0 mM MgCl2, 0.3 mM NaH2PO4, 10 mM HEPES, and 10 mM dextrose, pH adjusted to 7.4 with NaOH. BMSCs in the cu lture dish (volume 1.5 ml) were superfused at a rate of 2 ml/min. The patch electrodes (Kimax-5.1, Kimble Glass, Toledo, OH) had resistances of 3 MOhms when filled with an internal pipette solution containing 140 mM KCl, 4 mM MgCl2, 4 mM ATP, 0.1 mM guanosine 59-triphosphate, 10 mM dextrose, and 10 mM HEPES, pH adjusted to 7.2 with KOH. The whole-cell configuration was formed by applying negative pressure to the patch electrode.

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66 Results Morphological Observation during Neural Induction The neural differentiation induction caused rapid m orphological changes of canine BMSCs from fibroblastic morphology to neuron-like mo rphology with multiple processes seen in some cells (Fig. 3-1A and B). Initially, cells becam e rounded leaving some portions of the cytoplasm still attached to the original location. These changes typically appeared after around 3 hours of neural induction and almost completed after 5 hou rs. During the course of the neural induction, cells became less adhesive to the culture flask. As a result, some cells detached during flask manipulation. The changes in cell morphology were, however, not specific to BMSCs as similarly treated fibroblasts assumed neuron-li ke morphology after 5 hour s of induction (Fig. 31C and D). Immunocytochemical Characterization of Neurally Induced Canine BMSCs The results of i mmunocytochemistry demonstrated that canine BMSCs constitutively express neuronal ( III-tubulin) and astrocyte-specific (GFAP) proteins. We observed that almost all BMSCs showed immunoreactivity against GFAP at a relatively low level (Fig. 3-2A) whereas only subsets of these cells were strongly III-tubulin positive (Fig. 3-2B). Double staining of cells showed co-expression of GFAP and III-tubulin (Fig. 3-2C). After the neural induction, expressions of both III-tubulin and GFAP appeared to be pronounced, although these changes might have resulted from condensed localization of these proteins which accompanied with the morphological changes of the cells as described (Fig. 3-2D, E, F). In contrast, MBP positive BMSCs were not found under our culture conditi on either before or after the neural differentiation induction. Canine fibroblasts used as negative control did not show immunoreactivity against all neuronal/glial proteins.

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67 Figure 3-1. Phase-contrast phot omicrographs of canine BMSCs and fibroblasts. Neural differentiation induction caused transfor mation from fibroblastic morphology to neuron-like morphology in both BMSCs (A, be fore; B, after) and fibroblasts (C, before; D, after). Scale bar = 100 M.

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68 Figure 3-2. Immunofluorescent micrographs of can ine BMSCs. Canine BMSCs were positively stained against -tubulin (A) and GFAP (B) be fore neural differentiation induction. C shows merged images of A and B. Co-expression of -tubulin and GFAP is shown in C as double-stained cells (yellow). The expression levels of tubulin (D) and GFAP (E) incr eased after neural differentiation induction, partly due to condensed protein localization. F shows me rged images of D and E. Nuclei in C and F were counterstained with DAPI (blue). Scale bar in A = 100 M, scale bar in C = 50 M.

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69 Western Blot Analysis of Neural Specific Proteins We perfor med western blot anal yses to confirm the presence of neuronal and glial protein expressions on canine BMSCs and to evaluate their expression levels before and after the neural differentiation induction. The presences of distinct bands correspondi ng to III-tubulin and GFAP were observed in untreated canine BMSCs from all sample s (Fig. 3-3). Densitometric analyses confirmed that the expression level of GFAP in BMSCs consistently increased after the neural differentiation induction (39.0 20.6 % increase relative to -actin). On the other hand, the expression level of III-tubulin in BMSCs di d not significantly differ before and after the neural differentiation inducti on (9.6 18.5 % increase relative to -actin). Bands corresponding to MBP were not detected in BMSCs before and after the neural di fferentiation induction. Expressions of these proteins were not detectable in fibroblasts. Figure 3-3. Western blotting of neuronal ( III-tubulin) and glia l proteins (GFAP). Loaded samples were obtained from (A) spinal cord lysate (positive control); (B) fibroblasts before neural induction; (C ) fibroblasts after neural induction; (D) BMSCs before neural induction; (E) BMSC s after neural induction. Electrophysiological Recording In order to assess the electr ophysiological properties of neurally induced canine BMSCs, we applied the whole-cell voltage clam p techni que. We observed neither spontaneous action potentials nor depolarizing pulse -elicited action potentials from induced canine BMSCs. This suggested that although canine BMSCs constitutivel y expressed a neuronal marker and neurally

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70 induced canine BMSCs assumed neuronal morphology these cells did not acquire electrophysiological properties characteristic of mature neurons. Figure 3-4. Result of whole-cell voltage clamp r ecording of neurally induced BMSCs. Canine BMSCs treated with dbcAMP and IBMX fo r 5 hours showed neither spontaneous action potentials nor depolarizing pulse-elicited action potentials. Conclusion and Discussion We have demonstrated, under our culture c onditions, that canine BMSCs constitutively express neuronal and astrocyte-specif ic markers. The results of immunocytochemistry and western blotting revealed that untr eated canine BMSCs strongly express III-tubulin. This observation was consistent with previous repor ts on human MSCs (Deng et al., 2001; Tondreau et al., 2004). Since III-tubulin has been known to be present on early neurons, III-tubulin positive canine BMSCs might have the potential to differentiate into neuronal cells under appropriate conditions. We found that GFAP, a marker fo r mature astrocytes, was also expressed on untreated canine BMSCs. Reports on constitutive expression of GFAP in untreated human BMSCs have been conflicting; for exam ple, Deng et al. (2001) and Woodbury et al. (2000) reported the absence of the expression of GFAP whereas Sanchez-Ramos et al. (2000) and Tondreau et al. (2004) reporte d the expression of GFAP in untreated human BMSCs. These conflicting results from different laboratories ma y reflect the lack of a defined set of surface

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71 markers for BMSCs, resulting in inclusion of unidentified subsets of BMSCs with slightly different phenotypic profiles. In our study, al though expanded canine BMSCs appeared to be morphologically and phenotypica lly homogeneous, it was possibl e that several subsets of BMSCs existed in our culture. This was illustrated by discrete properties of expanded cells in their immunoreactivities against III-tubulin and GFAP; GFAP was expressed in nearly all BMSCs whereas III-tubulin expression was restrict ed in approximately 75% of all BMSCs. It was interesting to note that canine BMSCs expre ssing neuron-specific prot eins also expressed astrocyte-specific proteins (appr oximately 75% of all BMSCs). Therefore, similar to human BMSCs (Reyes et al., 2001; Tondreau et al., 2004) canine MSCs are considered undifferentiated but may also be characterized as multi-differentiated, which may explain their high plasticity. The absence of MBP expression on canine BMSCs wa s somewhat predictable as this protein is only expressed in mature oligodendrocytes and di screte molecular signals may be required to induce BMSCs to differentiate towards this lineage. Markers for earlier stages of oligodendrocytes need to be inve stigated to further characterize the plasticity of canine BMSCs in their neural differentiation capacity. The e ffects of the neural diffe rentiation induction on cell morphology and neuronal/glial marker expressi ons were evaluated on canine BMSCs. The induction protocol using dbcAMP and IBMX ha s been previously described and known to induce a rapid neuron-like mor phological change in human BMSC s as a result of elevated intracellular cyclic AMP levels (Deng et al., 2001). In our study, after the neural induction, canine BMSCs exhibited a neuron-like morphology as early as 3 hours after the induction. However, a similar morphological change was also observed in canine fibr oblasts at about the same rapidity. These observations suggested that the transforma tion of canine BMSCs from the fibroblastic morphology to the neur on-like morphology was not a spec ific event associated with

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72 the multipotentiality of canine BM SCs. Therefore, these drama tic morphological changes should not be overvalued and special care must be taken to interpret results of in vitro neural differentiation of BMSCs. As pointed previously by several investigator s, a rapid morphological change of BMSCs after neural induction may repres ent cytotoxic effects of the reagents in the induction medium, leading to cell shrinkage a nd subsequent adoption of the neuron-like morphology (Bertani et al., 2005; Lu et al., 2004; Neuhuber et al., 2004). Commonly used reagents, solely or in combination, that have effects on BMSCs to induce neuron-like morphology by cytoskeletal shrinkage in clude butylated hyd roxyanisole (BHA), dimethylsulphoxide (DMSO), a nd -mercaptoethanol (BME). Based on the results of immunocytochemistry and western blot anal ysis, we found that neural induction caused up-regulat ion of the expression level of GFAP on canine BMSCs. Therefore, these results may indicate that canine BMSCs can be stimulated to differentiate along mature astrocytes. Similar results have been reported in human BMSCs (Tondreau et al., 2004) and murine BMSCs treated with dbcAMP/IB MX (Deng et al., 2006). Although strong expression of III-tubulin was observed on canine BM SCs before and after neural induction, the expression levels were not affected by this treatment. As mentioned, canine BMSCs became less adhesive to the culture flask and there was a significant reduction of the cell number during the neural differentiation induction. As a result, all analyses we re performed 5 hours after the induction in the present study. Howe ver, it would be interesting to further investigate whether a longer induction period would cause emergence of more mature neuron markers and stable phenotypes. Electrophysiological recordings of neurally induced canine BMSCs revealed that these cells did not acquire the ability to fire s pontaneous or depolariz ing pulse-elicited action

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73 potentials. It may be the case that induced BM SCs committed to the neuronal lineage, as shown by expression of the early neuronal marker, but have not fully differentiated into a mature phenotype. Again, a longer inductio n protocol may be required for full maturation in neural differentiation of canine BMSCs. Alternatively, other techniques may be more useful to evaluate the process of differentiation and maturation of neurally induced BMSCs. For example, expression of voltage-gated ion channels and r ecordings of inward sodium currents are more sensitive methods to monitor the process of ne ural maturation. At current, acquisition of electrophysiological properties by BMSCs was only achieved by gene transfection (Dezawa et al., 2004; Kohyama et al., 2001) and treatment with DMSO and BHA on rat BMSCs was reported to be insufficient in inducing diffe rentiation into mature neuron phenotypes with electrophysiological properties (Hofstetter et al., 2002). With the advancement of our understanding of regulatory mechanisms underlying neural differentiation, it may become possible to genera te a specific neural cell type from canine BMSCs. This approach has been most intensiv ely studied in embryonic stem (ES) cells in an attempt to generate functional neural precursor cells (Zhang et al., 2001). This approach holds an advantage in that pre-induction of ES cells into neural progenito r cells may reduce the possibility of developing tumor (i.e. teratomas) in the transplantation site. Further, induction of ES cells into more specific neur al cell types has been proposed; for example, generation of dopaminergic neurons from human (Brederlau et al., 2006; Perrier et al., 2004), monkey (Kawasaki et al., 2002; Takagi et al., 2005), and murine (Thinyane et al., 2005) ES cells has been reported for potential treatments fo r Parkinsons disease. It has also been shown that purified oligodendrocytes can be generated from human and murine ES cells (Glase r et al., 2005; Nistor et al., 2005). These ES cells-d erived oligodendrocytes have ex tensive myelinating capacity in

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74 vivo therefore, hold promise for treatment of demy elinating diseases in humans. Dezawa et al. (2004) showed that rat and human BMSCs can be specifically induced to generate functional neurons, using gene transfection wi th Notch intracellular domain. In vitro differentiation of rat BMSCs into myelinating cells with phenotypic and functional characteris tics of Schwann cells has been previously described (Dezawa et al., 2001; Keilhoff et al., 2006). Kamada et el. (2005) demonstrated that transplanta tion of rat BMSC-derived Schwann cells resulted in enhanced axonal regeneration and functional r ecovery in rats with complete ly transected spinal cord. These studies on BMSC differentiation into specific neural cells are particularly promising in that if stable functional phenotypes can be ach ieved BMSCs may become the strongest candidate for cellular therapies since autologous transplant ation is clearly advant ageous in a clinical setting. Induction of canine BMSCs into specif ic functional neural cell types may be possible by understanding the transcription factors involve d in neurogenesis. Global gene expression profiling, which has become available through DNA microarray for the canine genome, would aid developing such techniques. In conclusion, a large-scale expa nsion of transplants is one of the critical prerequisites for clinical applications of cellular transplantation th erapies. Canine BMSCs can be readily isolated from bone marrow of the patient, thus allowing autologous transplantati on. These cells can be further expanded in culture while retaining mu lti-differentiation capacity. Canine BMSCs hold neural differentiation properties; therefore, may have the potentia l for usages in treating various neurodegenerative diseases and spinal cord in juries in dogs. Furthe r investigations on mechanisms of neurogenic abilities of canine BMSCs are warranted, which may lead to the developments of novel therapeutic strategies to target specific CNS diseases. Applying these novel therapies in canine patients wi ll provide important insights into the safety and clinical

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75 efficacy of the treatment, which are the essent ial elements in transl ational research of neurological disorders in human patients.

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76 CHAPTER 4 IN VIVO NE URAL DIFFERENTIATION OF C ANINE BONE MARROW STROMAL CELLS Background and Introduction Neurotransplantation has generated trem endous attention as a potential therapeutic approach for disorders of the central nervous sy stem (CNS). Cells and tissues derived from fetuses have been used to treat CNS disorders such as Parkinsons di sease (Kordower et al., 1995; Spencer et al., 1992) and spinal cord inju ry (SCI) (Thompson et al., 2001) and led to significant improvement of clinical signs in some patients. Neural stem cells (Ogawa et al., 2002; Pluchino et al., 2003; Teng et al., 2002) and embryonic stem cells (Keirstead et al., 2005; McDonald et al., 1999; Nistor et al., 2005) have also been ex tensively studied and shown to ameliorate CNS diseases in experimental models. However, these cell/tissue types pose serious obstacles with respect to th e harvesting of donor cells/tissu es and the possibility of tumorigenesis. Transplantation of bone marrow-derived cells, which has been effectively used to treat diseases of the hematopoi etic system, may provide a viab le alternative strategy. Bone marrow contains bone marrow stromal cells (BMS Cs, also referred to as mesenchymal stem cells), nonhematopoietic cells that provide a bone marrow microenvironment and regulate hematopoiesis (Dormady et al., 2001) and also serve as a stem cell reservoir for mesenchymal cells (Pittenger et al., 1999). Due to their multipotency, BMSCs have been shown to regenerate mesenchymal tissues by differentiating into oste oblasts (Bruder et al., 1997; Kadiyala et al., 1997), chondrocytes (Kadiyala et al., 1997; Noth et al., 2007; Williams et al., 2003), or skeletal muscle cells (Dezawa et al., 2005). BMSCs are easily accessible via bone marrow aspiration, extensively expandable ex vivo and amenable for gene transf ection (Lu et al., 2006). BMSCs, therefore, represent a promising candidate cell source for cellular therapy for various diseases including those of the CNS.

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77 Perspectives on BMSCs for neurotransplanta tion have rapidly grown during the last decade, based on several reports on BMSC tran sdifferentiation into neural phenotypes both in vitro (Sanchez-Ramos et al., 2000; Woodbury et al., 2000) and in vivo (Arnhold et al., 2006; Azizi et al., 1998). These studies demonstrated that adult stem cells may be capable of adopting appropriate phenotypic fates in resp onse to environmental cues. Therapeutic benefits of BMSC transplantation has been repeat edly shown in various experimental models of CNS injuries, which include ischemic (Lee et al., 2003; Zhao et al., 2002), traumatic (Wu et al., 2003), and degenerative (Wu et al., 2007) lesions in the br ain or the spinal co rd. Although functional recovery from CNS injury may not result solely from neural tr ansdifferentiation and cellular replacement by transplanted BMSCs, investigations of neural tran sdifferentiation properties of BMSCs provide insights into their potentials in the treatment of CNS diseases and aid in refining the design of new clinical trials. Initial clinical tr ials in humans with SCI using auto logous bone marrow cells have already been reported (Park et al., 2005; Yoon et al., 200 7), and the potential th erapeutic value of bone marrow cells is certainly not limited to human applications. For example, dogs can sustain traumatic SCI at relatively high incidence. A need thus exists for developing novel treatments and cell-based therapies for veterinary practi ce. Based on existing evidence, BMSCs are a reasonable candidate cell platform. Veterinary clinical applicatio ns also afford an important animal-to-human translation oppo rtunity, especially the pathol ogy of clinical SCI in dogs appears very similar to that documented in humans, as well as in cats and numerous examples of experimental SCI (Jeffery et al., 2006; Smith and Jeffery, 2006). The objective of the presen t study was to determine by immunohistochemistry whether canine BMSCs can migrate and adopt neural phe notypes in the developing mouse brain. We

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78 also evaluated by species-specific chromosome painting technique whether cell fusion events contributed to transdifferentiati on properties of canine BMSCs. Materials and Methods Preparation of Canine BMSCs and Fibroblasts Bone m arrow from four adult ca nine cadavers, obtained from a local animal shelter, was used to isolate and culture canine BMSCs. Exact ages of these dogs were unknown; however, we collected from only those which had a complete set of adult dentures with minimum dental calculus deposition, thus meeting inclusion criteria for young adu lts. Bone marrow was also collected from two young dogs (estimated age betw een 3 to 5 months old). The use of these animals was approved by the Institutional Animal Care and Use Committee of University of Florida. In all animals, bone marrow was collect ed from a femur by flushing the medullary canal immediately after euthanasia. Mononucleated ce lls were isolated on a Ficoll density gradient, washed in PBS, and suspended in culture medium consisting of DMEM (1g/L glucose) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/ ml penicillin G, 100 g/ml streptomycin sulfate, and 0.25 g/ml amphot ericin B). Primary adhe rent cells were grown until semiconfluency after which cells were trypsinized and labeled with the fluorescent carbocyanine dye, DiI (Invitrogen). Third passa ge canine fibroblasts (Coriell Institute for Medical Research) were used for comparative purposes. Fluorescent labeling was performed according to a protocol of Laywell et al. (1996) with modifications. Briefly, trypsinized BMSCs or fibroblasts were washed three times in PB S and resuspended in PBS containing DiI (final concentration, 40g/mL). The cells were incuba ted in the DiI-containing PBS for 5 minutes at 37C followed by 15 minutes at 4C before being washed three times in PBS. DiI labeled BMSCs and fibroblasts were frozen at -80C until tran splantation was performed.

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79 Transplantation of Canine BMSCs and Fi broblasts in to Neonatal Mouse Brain Postnatal day-2 immunocompromised mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, The Jackson Laboratory) were used as recipients (n=2 9). On the day of transplantation, DiI-labeled BMSCs or fibroblasts were thawed, washed three times in PBS, and the cell number was determined by use of a hematocytometer. Cell suspensions were made in PBS (2.0 105/L) and transferred on ice to the mous e facility. DiI-labeled BMSCs or fibroblasts were transplanted into the left lateral ventri cle of postnatal day-2 immunocompromised mice as described previously (Deng et al., 2006). Under hypothermic anesthesia, 2.0 105 cells in 1L of PBS were slowly injected into the left lateral ventricle, through a 30G needle attached to a 5L Hamilton syringe. Ten day post-transpla ntation, mice were euthanized with CO2 gas and transcardially perfused with 4% paraformalde hyde in PBS. The brains were excised and postfixed in 0.4% paraformaldehyde containing 30% sucrose for 2-3 days. Brains were sectioned with a freezing microtome into 40M sagittal or coronal slices for immunohistochemistry or 20 M sa gittal slices for fluorescence in situ hybridization. Immunohistochemistry to Evaluate Transdifferentiation of Canine BMSCs Immunohistochem ical staining of free-floating brain slices were performed, using neuronspecific III-tubulin (1:500; Promega, Madison, WI) and AlexaFluor 488-conjugated NeuN (1:50; Chemicon), and astrocytespecific GFAP (1:100; BD Biosciences, Franklin Lakes, NJ) antibodies. Brain slices were washed three times in PBS, and permeabilized in 0.4% Triton X100 in PBS for 30 minutes at 25C. Non-specifi c binding was blocked with a blocking solution (3.0% normal goat serum in PBS) for 60 minutes at 25C. Brain slices were then incubated overnight at 4C with the primar y antibodies. The primary antibodies were removed, slices were washed three times in PBS, and incubated for 60 minutes in dark at 25C with secondary antibodies. The secondary antibodies were Cy2 conjugated goat anti-mouse IgG1 (1:400;

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80 Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or IgG2b (1:200; Jackson ImmunoResearch Laboratories, In c., West Grove, PA). Staine d slices were observed under a fluorescent microscope (Zeiss Axioplan II, Carl Zeiss Microimaging Inc.) to evaluate engraftment and phenotypes of transplanted canine BMSCs. Chromosome Painting to Evaluate Cell Fusion Twenty m icron sagittal sections were used fo r assaying possible fusion events associated with DiI-labeled donor BMSCs in the neonatal mouse brain. Brai n sections were first viewed under a fluorescence microscope to identify secti ons with DiI-positive donor cells. Identified sections were then treated with 0.2N HCl for 30 minutes, and retrieved in 1M sodium thiocyanate (NaSCN) for 30 minutes at 85C. The sections were digested with 4mg/mL pepsin (Sigma; diluted in 0.9% NaCl pH2.0 ) for 60 minutes at 37C. After equilibrating in 2x SSC for 1 minute, the sections were dehydrated through grad ed alcohols. The tissue was then denatured with FITC-conjugated canine X-chromosome probes (Cambio, UK) and biotin-conjugated mouse Xchromosome probes (Cambio, UK) fo r 10 minutes at 60C and hybridized at 37C overnight. After hybridization, slides were wash ed first in 1:1 formamide:2x SSC, then in 2xSSC. Dual color detection was performed usi ng a commercially available kit (Cambio, UK) to visualize mouse X-chromosomes with Cy-5 and enhance FITC signals of canine Xchromosomes. Some slides were reacted only with mouse Xchromosome probes and visualization was performed with streptavidin-conjugated Alexa Fluor 555 (Invitrogen). Slides were then coverslipped in mounting medium containing DAPI (Vector, Burlingame, CA) and evaluated under a fluorescence microscope.

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81 Results Distribution and Phenotypic Fates of Adult Canine BMSCs and Fi broblasts Of 29 recipient mice, 27 survived the 10-da y survival period and were processed for analysis. Transplanted cells could be readily iden tified by the presence of DiI in the cytoplasm. In mice that received adult can ine BMSCs (n=17), most of the engrafted cells remained around the injection site, adhering to th e wall of the lateral ventricle (F ig. 4-1). Some cells were found in the underlying parenchyma of the thalamus and hippocampus; however, it was not possible to determine whether these cells had migrated into th e parenchyma from the injection site or were directly injected into these locations. There were also cells sparsely dispersed in the cerebral cortex distant from the injection site, which s eemed to have migrated there instead of being directly injected. Additionall y, a small number of cells were widely distributed around the periphery of the brain, appearing to have attached to the pia ma tter but not penetrated into the tissue. Figure 4-1. Montage immunofluor escence photomicrograph of mouse brain with engrafted adult canine BMSCs. This sagittal sectio n was stained with neuron-specific III-tubulin and visualized with Cy2 (green). Most of DiI-positive BMSCs (red) were located around the injection site along the wall of the lateral vent ricle while some were found around the periphery of the brain and sp arsely in the cortical parenchyma.

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82 The majority of the engrafted adult BMSCs e xhibited a spindle-shaped appearance similar to their in vitro characteristic morphology. A small populatio n of cells that migrated into the parenchyma underlying the ventricle showed process-bearing morphology. However, immunohistochemistry showed that engrafte d cells expressed neither neuron-specific ( IIItubulin and NeuN) nor astrocyte-sp ecific (GFAP) markers. Engr afted canine fibroblasts (n=3) showed a similar tendency of distribution and phenotypic patterns to adult BMSCs with the exception that fibroblasts rema ined spindle-shaped morphology. Distribution and Phenotypic Fa tes of Young Canine BMSCs In contrast to adult BMS Cs and fibroblas ts, BMSCs isolated from young donors (n=7) demonstrated a different beha vior. Although most BMSCs of young donors remained around the injection site in the lateral ventricle similar to adult BMSCs, a small number of cells were located in the olfactory bulb (Fig 4-2). These cells in the olfactory bulb were III-tubulin positive and thought to have migrated from the lateral ventricle through the rostral migratory stream (RMS), a known pathway of neuronal precur sors migrating from the subventricular zone (SVZ) to the olfactory bulb. There were also DiI-positive donor cells in the RMS. In the SVZ, most of engrafted BMSCs exhibi ted typical fibroblastic morphol ogy (Fig 4-3A), but some cells assumed astrocyte-like (Fig 4-3B) or bipolar neuron-like (Fig 4-3C) morphology. Immunostaining revealed that a signifi cant number of BMSCs in the SVZ were III-tubulin positive (Fig 4-4). Expression of neither NueN nor GFAP was found in any region containing BMSCs (Fig 4-5). Fluorescence In Situ Hy bridization for Chromosome painting We first attempted to visualize canine X-ch romosomes and mouse Xchromosomes using different fluorescence-conjugate d antibodies to evaluate possible fusion events between transplanted canine BMSCs and host neural cells. However, due to different required

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83 hybridization conditions for chromosome probes for each species this was not possible. Consequently, we stained sections only with mouse Xchromosome probes and looked for colocalization of DiI and mouse Xchromosomes. Regardless of th e transplanted cell types (adult BMSCs, young BMSCs, or fibrobl asts) we did not find any cells containing both DiI and mouse Xchromosomes, indicating that cell fusion did not occur in any in stances (Fig 4-6). Figure 4-2. DiI-positive BMSCs isolated from young donors presen t in the olfactory bulb. A small number of DiI positive BMSCs were found in the olfactory bulb (arrows). Inset shows a higher magnification view of a BMSC in the boxed area. Conclusion and Discussion Our data suggest that canine BMSCs isolated from young donors but not from adult donors may have the capacity to under go neural transdifferentiation in vivo in response to environmental cues provided by the developing mouse brain. Young canine BMSCs injected in the lateral ventricle penetrated into the SV Z and were integrated in the pos tnatal neurogenic pathway of the RMS/olfactory bulb system. Our observation that a small number of young canine BMSCs migrated to the olfactory bulb a nd expressed neuron-specific marker III-tubulin suggests that these cells may possess neural transdifferentiation capability. This finding also suggests that a

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84 Figure 4-3. Various morphologies of BMSCs in the subventricular zone. Most of engrafted BMSCs exhibited typical fibroblastic morphology (A). Some cells assumed astrocyte-like (B) or bipolar neuron-like morphology (C) in the SVZ. Figure 4-4. BMSC from a young donor located in the subventricular zone DiI positive BMSC seen in the SVZ (A) is immunopositive against III-tubulin (B). C shows a merged image of A and B.

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85 Figure 4-5. Immunostaining of BMSCs in th e subventricular zone for GFAP and NeuN expression. Engrafted BMSCs isolated from young donors (red) did not express GFAP (green in A) in the SVZ or NeuN (green in B) in the cerebral cortex. Figure 4-6. Fluorescence in situ hybridization for chromosome painting. A cell containing DiI (red) in its cytoplasm is a transplanted BMSC from a young donor. This DiI positive cell does not contain a mouse X-chromosome. Other cells in this view are neural cells of this male recipient mouse as they all contain a single mouse X-chromosome (pink dots in the nucleus). Some X-chro mosomes cannot be seen because of the selected focal plane. Nuclei are stained with DAPI (blue). population of young canine BMSCs may behave like ne ural precursor cells that contribute to postnatal neurogenesis. In contrast to young canine BMSCs, most adult canine BMSCs remained in the injection site and did not migrat e to the olfactory bulb. Some adult BMCSs were found in the SVZ and distant s ites and assumed process-bear ing morphology; however, these cells expressed neither neuronnor astrocyte-specific markers. Canine fibroblasts used as a control showed no migration capacity a nd remained as spindle-shaped cells.

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86 Our observation of the migration and differe ntiation properties of young canine BMSCs is similar to those of previous studies in which murine BMSCs were engrafted into the lateral ventricle of the neonatal mouse brain (Deng et al., 2006; Kopen et al., 1999). However, the extent of migration and adopted phenotype was much more restricted in our study compared to these previous studies. For example, Kopen et al. (1999) reported that murine BMSCs injected into the lateral ventricl e of neonatal mouse brains migrated extensively throughout the forebrain and cerebellum and differentiated into both neurons and astrocytes They found that engrafted BMSCs preferentially populated neuron rich re gions including the Islands of Calleja, the olfactory bulb, and the internal gr anular layer of the cerebellum, events similar to those occur in the ongoing developmental processes in early postn atal life. Based on these observations, they suggested that BMSCs mimic the behavior of ne ural progenitor cells. Deng et al. (2006) more recently showed that, upon intraventricular in jection, murine BMSCs integrated into the postnatal neurogenic pathway of the RMS/olfact ory bulb system by migrating appropriately and differentiating into olfactory granule cells, supporting the c ontention that the bone marrow derived adult stem cell indeed possesses neural transdifferentia tion capability under the influence of environment cues from the brain. We found that although most of BMSCs remained in the injection site a small number of young canine BMSCs penetrated into the SVZ a nd assumed various morphologies resembling neurons or astrocytes. Immunostaining suggested that some of these cells in the SVZ express III-tubulin. We believe that migrati on of young canine BMSCs into the SVZ and differentiation toward the neuronal phenotype occurred in a specific way in response to microenvironmental cues provided by the SVZ. The SVZ forms adjacent to the lateral ventricle during embryogenesis and is known to contain multi potent neural stem cells (Doetsch et al.,

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87 1997; Lois and Alvarez-Buylla, 1993; Weiss et al ., 1996). The SVZ is also the area with active neurogenesis in the postnatal and adult brain (Altman, 1969; Altman and Das, 1966; Luskin, 1993). Our observation that migrating BMSCs were found in the RMS and also localized in the olfactory bulb further sugge sted that young canine BMSCs we re integrated into the RMS/olfactory bulb neurogenic system. The olf actory bulb is one destination of neural progenitors generated in the SVZ (Altman, 1969; Lois and Alvarez-Buylla, 1994). Little is currently known about what drives im planted BMSCs to differentiate into neural cells in vivo Postnatal brain development occurs and th is process is governed in part by various growth factors of which the expression leve ls are regulated preci sely in a controlled spatiotemporal manner. For example, levels of fibroblast growth factor 2 (FGF-2 or bFGF) expression increase dramatically during late embryonic and early postnatal stages of development in rodent brain (Caday et al., 1990; Ford-Perriss et al., 2001). FGF-2 is known to promote proliferation and self-renewal of neural stem cells derived from the neuroepithelium of the developing cortex (Gritti et al., 1996) and the adult SVZ (Bartle tt et al., 1995). FGF-2 is also a potent mitogenic factor for BMSCs and it is known that FGF-2 promotes self-renewal of BMSCs in vitro (Locklin et al., 1995; Oliv er et al., 1990; Tsutsumi et al., 2001). Therefore, high levels of FGF-2 present in the developing brain might have stimulated proliferation and more importantly the self-renewal capacity of canine BM SCs. It is also li kely that neurotrophic factors played a role in instructing BMSCs to differentiate into the neuronal phenotype. Neurotrophic factors for which BMSCs express r eceptors, such as NGF (Caneva et al., 1995) and BDNF (Li et al., 2007), might have induced neural differentiation of BMSCs when coupled with other cues present in the SVZ.

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88 What contributed to the observed differ ence between adult donor BMSCs and young donor BMSCs in their behavior in the mouse brain? It may simply be that bone marrow from young dogs contained a larger number of stem cells, which survived transpla ntation, than adult bone marrow. Alternatively, it could be that transd ifferentiated BMSCs were derived from a more primitive stem cell population present in the BM SC fraction of the young donors. The presence of a rare pluripotent stem cell population has be en reported in adult mouse (Jiang et al., 2002a) and human (Reyes et al., 2001) bone marrow and s hown to have transdifferentiation capacity. These cells can only be expanded at extremel y low cell density on fibronectin and in the presence of LIF and PDGF. Existence of a si milar cell population in adult bone marrow has not been reported for other species including canines. As we utilized the standard culture condition (without growth factors at standa rd cell density) to prepare all BMSCs for transplantation, cells with multipotent differentiation properties may not have been expanded from the adult bone marrow sample. Identification of cell populations with multipotential differentiation properties resident in canine bone marro w needs to be investigated. In conclusion, BMSCs isolated from young donors exhibited neuronal phenotype upon transplantation into the developing mouse br ain. Transplanted young canine BMSCs responded to instructive cues provided by the SVZ and migrated to the olfactory bulb via the RMS. These migratory and differentiation properties of young canine BMSCs resemble behavior of indigenous neural progenitors. Adult canine BM SCs failed to demonstrate a similar degree of migration and differentiation neither did canine fibroblasts used as a control. The results suggest that a primitive stem cell population with neural transdifferentiation capacity may exist in the BMSC compartment isolated from young dogs. Id entification of the re sponsible cell population

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89 in the young canine BMSCs and the development of selective culture tech niques may aid further understanding of the identity of neurogenic stem cell populations in the canine bone marrow.

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90 CHAPTER 5 EFFECTS OF CANINE BONE MARROW STROMAL CELLS ON NEURITE EXTENSION FROM DORSAL ROOT GANGLI ON NEURONS IN VITRO Background and Introduction Bone m arrow stromal cells (BMSCs), also re ferred to as mesenchymal stem cells, have been known to play a key role in regulati ng hematopoiesis through interactions with hematopoietic stem cells within the bone marrow microenvironment (Dormady et al., 2001). More recent investigations have provided evidence indicati ng that BMSCs may also serve as a stem cell reservoir for mesodermal cells and thus participate in rege neration of mesodermal tissues (Pittenger et al., 1999). In addition, se veral lines of evidence suggest that bone marrow derived cells from rodents and humans may be cap able of transdifferen tiation and thereby have the capacity to generate cells in vitro (Deng et al., 2001; Sanch ez-Ramos et al., 2000; Woodbury et al., 2000) as well as in vivo (Azizi et al., 1998; C ogle et al., 2004; Deng et al., 2006; Lee et al., 2003; Mezey et al., 2003) expressing various neuronal markers. After transplantation of BMSCs, functional recovery has been reported in animal models involving ischemic (Chen et al., 2001; Lee et al., 2003; Li et al., 2002; Zhao et al., 2002) and traumatic lesions (Chopp et al., 2000; Hofstetter et al., 2002; Lee et al., 2003; Ohta et al., 2004) of the CNS. Although studies have shown the presence of tr ansplanted BMSCs in host CNS tissues (Arnhold et al., 2006; Aziz i et al., 1998; Kopen et al., 1999) which in some cases appear to have distinct neuronal features (Cogle et al., 2004), there is currently no definitive evidence that BMSCs significantly contribut e directly to neuronal replacemen t after CNS injuries (Ohta et al., 2004). Alternatively, BMSCs have been shown to have other tissue repair properties such as promoting remyelination (Akiyama et al., 2002b ) and production of various neurotrophic and angiogenic growth factors, includi ng nerve growth factor (NGF) (Auffray et al., 1996; Chen et al., 2005; Chen et al., 2002a; Chen et al., 2002b; Crig ler et al., 2006; Garcia et al., 2004; Li et al.,

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91 2002), glial cell-derived neurotroph ic factor (GDNF) (Chen et al ., 2005), brain-derived growth factor (BDNF) (Chen et al., 2002a; Chen et al., 2002b; Crigler et al., 2006; Li et al., 2002), vascular endothelial growth factor (VEGF) (C hen et al., 2002a; Chen et al., 2002b), hepatocyte growth factor (HGF) (Chen et al., 2002a; Chen et al., 2002b), and brain natriuretic peptide (BNP) (Song et al., 2004). Neurotrophic factors such as NGF and BDNF support survival of damaged neurons (DeKosky et al., 1994; Goss et al., 1998; Hammond et al., 1999; Lu et al., 2001), and it has been suggested that production of these factors is increased in ischemic brain lesion models after BMSC transplantation whic h in turn can lead to reduced apoptosis in the penumbra, proliferation of endogenous cells in the subventri cular zone, and functional recovery (Li et al., 2002). Vasoactive peptides such as VEGF (C hen et al., 2002a; Chen et al., 2002b) and BNP (Song et al., 2004) may also participate in the repa rative processes after CNS injury by inducing angiogenesis and reducing secondary edema after injury. BMSCs also appear to stimulate neuritic outgr owth which may likewise be attributed to their ability to secrete neurot rophic factors (Mocchetti and Wrathall, 1995), as well as extracellular matrix (ECM) and adhesion molecule s such as fibronectin and laminin (Grayson et al., 2004; Hofstetter et al., 2002), which are known to promote attachment and extension of axons. Through production of these molecules BMSCs are thought to provide permissive microenvironment for regenerating axons (Hofstetter et al., 2002). The importance of this ECM milieu in which axonal regrowth takes place is well recognized after earl y studies of peripheral nerve grafts into CNS lesions (David and Aguayo, 1981; Rich ardson et al., 1980). Initial clinical tria ls in humans using autologous BM SCs have already been reported (Park et al., 2005; Yoon et al., 2007 ), and the potential therapeutic value of BMSCs is certainly not limited to human applications. For example, dogs can sustain traumatic spinal cord injuries

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92 at relatively high incidence. A need thus ex ists for developing novel treatments and cell-based therapies for veterinary practice. Based on exis ting evidence, BMSCs are a reasonable candidate cell platform. Veterinary clinical applicati ons also afford an important animal-to-human translation opportunity, especially the pathology of clinical spinal cord injury (SCI) in dogs appears very similar to that documented in humans, as well as in cats and numerous examples of experimental SCI (Smith and Jeffery, 2006). Previous studies (Arinzeh et al., 2003; Bruder et al., 1998; Kadiyala et al., 1997; Volk et al., 2005) of canine BMSCs have focused on thei r osteogenic properties because of the wide clinical applicability. The oste ogenic property of canine BMSCs ha s also been exploited in the field of tissue engineering, particularly in an attempt to promote peri odontal tissue regeneration (Hasegawa et al., 2006). Card iomyogenic property of canin e BMSCs has been studied and shown to result in improvement of cardiac func tion in a chronic myocardial infarction model after transplantation of autologous BMSCs (B artunek et al., 2007). These studies have demonstrated proof of concept for using BMSCs with clinical benefits. However, our understanding of the basic biology of canine BMSC s is rather scarce and their potential use for treatment of neurol ogical diseases has not been studied. In the present study, we investigated the effects of canine BMSCs on neuritogenesis from dorsal root ganglion neurons (DRG) in vitro. Our results suggest that canine BMSCs promote neurite outgrowth from DRG neurons with de velopment of complex and highly branched neuritic arborizations. These effects were pronounced in co-cultures of BMSCs and DRG neurons, indicating that production of non-diffusible molecules might be the prime attribute of BMSCs in promoting neuritogenesis.

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93 Materials and Methods Preparation of Canine BMSCs and Fibroblasts Bone m arrow aspirates from three canine cadav ers, obtained from a local animal shelter, were used to isolate and culture canine BMSC s. Exact ages of these dogs were unknown; however, we collected from only those which ha d a complete set of adult dentures with minimum dental calculus depositi on, thus meeting inclusion criteria for young adults. The use of these animals was approved by the Institu tional Animal Care and Use Committee of University of Florida. Detailed protocols for bone marrow collection, ce ll isolation, and culture expansion have been described previously (Kamishina et al., 2006). Briefly, bone marrow aspirates were obtained from the iliac crest imme diately after euthanasia. Mononucleated cells were isolated on a Ficoll density gradient, wash ed in PBS, and suspended in culture medium consisting of DMEM (1g/L glucose) supplemen ted with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin G, 100 g/ml st reptomycin sulfate, and 0.25 g/ml amphotericin B). Homogeneous BMSC cultures were established in the third passage cultures. Third passage canine fibroblasts were used for comparative pur poses. BMSCs or fibroblasts were plated at a cell density of 8 103/cm2 on 18mm glass coverslips placed in 12-well culture pl ates for direct co-culture experiments or in 6-well plates to yield conditioned media. Immunocytochemical Analysis for Expression of Extracellular and Adhesion Molecules We evaluated whether canine BMSCs expre ss som e of the putative ECM and adhesion molecules that are known to fa vor neurite outgrowth. Thir d passage canine BMSCs and fibroblasts were grown on glass coverslips until confluency a nd immunostained as follows. Cells were fixed in 4% paraformaldehyde for 30 minutes at 25 C, washed three times in PBS, and permeabilized in 1.0% Triton X-100 in PBS for 10 minutes at 25 C. Non-specific binding was blocked with a blocking solution (1.0% BSA in PBS) for 30 minutes at 25 C. Cells were

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94 then incubated overnight at 4 C with primar y antibodies directed ag ainst following proteins; laminin (C4 and D18, both from the Developmental Studies Hybridoma Ba nk at the University of Iowa [DSHB]), E-cadherin (DSHB), N-cadhe rin (DSHB), neural cell adhesion molecule (NCAM; DSHB), and fibronectin (Sigma). The primary antibodies were removed, cells washed with PBS, and cells incubated for 1 hour in dark at 25 C with secondary antibodies. The secondary antibodies were Cy2 conjugated goat anti-mouse IgG1 (1:400) or Rhodamine-Red X conjugated goat anti-mouse IgG2a (1:400). Ce lls were also incubated without primary antibodies to control for non-specific staining by secondary antibodies. Cells were washed with PBS and mounted with a mounting medium. St ained cells were obser ved under a fluorescent microscope (Olympus BX50, Olympus Corpora tion, Tokyo Japan) with appropriate filters. Direct Co-culture of BMSCs and Dorsal Root Ganglion Neurons Rat fetal DRG neurons (Ca mbrex Corporation) we re plated on 1) monolayer of BMSCs, 2) monolayer of fibroblasts, or 3) laminin-coated (2 g/ml, Sigma) coversli ps at a cell density of 3.8 103/cm2 in the same medium as above. After 48 hours, cells were fixed and processed for immunocytochemistry as described above. The primary and secondary antibodies used were anti-neurofilament 200 (1:50, Sigma) and Alex a Fluor 350 (1:200, Invitr ogen), respectively. Cells were also incubated without primary antibodies to control for non-specific staining by secondary antibodies. Stained cells were obs erved under a fluorescent microscope equipped with a digital camera (Retiga 1300, QImaging, Surre y, BC Canada) and pictures of DRG neurons were taken from non-overlapping fields using a objective lens. These experiments were performed in triplicate. Culture of DRG Neurons in Conditioned M edium Third passage BMSCs or fibrobl asts were cultured in 6-we ll culture plates for 72 hours after which conditioned media were collected. In this experiment, 6-well plates were coated with

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95 laminin (2 g/mL) and poly-D-lysine (30 g/mL) and DRG neurons were pl ated in each well at a cell density of 1.0 103/cm2. DRG neurons were also plated in culture media that were incubated for 72 hours at 37 C without conditi oning cells. DRG neurons were cultured in conditioned media for 48 hours afte r which cells were washed with PBS and fixed as described. DRG neurons were stained with SimplyBlue SafeStain (Invitrogen) for 8 hours at 25 C, washed three times in PBS, and examined under a phase contrast microscope as previously described (Price et al., 2006). Digital pict ures of DRG neurons were acqui red from non-overlapping fields using a objective lens. These expe riments were performed in duplicate. Measurements of Neurite Outgrowth Digital im ages were transferred into image analysis software (NIH ImageJ ver 1.37v) for neurite morphometric analyses. All neuronal pro cesses were considered neurites as axons and dendrites were not distinguishable from one anothe r. Primary neurites we re defined as processes directly emerging from the cell body which usua lly have a thicker diameter than branching neurites. All primary and branching neurites were manually traced on the digital images. The following parameters were measured as previously described (Chakrabortty et al., 2000); 1) total neurite length/neuron, 2) total primary neurit e length/neuron, 3) mean length of primary neurite/neuron, 4) mean number of primary neur ites/neuron, and 5) mean number of branching neurites/neuron. Data are presen ted as mean standard deviati on. Statistical differences among groups were tested by One-way ANOVA followed by Tukeys HSD with p-value set at .05 (SPSS). Results Expression of Extracellular Matrix Molecules After 72 hours of plating, third passage canine BMSCs for med a uniform monolayer consisting of homogenous polygonal cells. Imm unocytochemistry demonstrated that canine

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96 BMSCs produced and deposited copious amounts of fibronectin (Fig. 5-1A) and laminin (Fig. 51B). Expressions of E-cadherin, N-cadherin, and NCAM were not detected in our BMSC culture. Canine fibroblasts were stained against fibronectin but no expression was observed for laminin, E-cadherin, Ncadherin, and NCAM. Figure 5-1. Immunofluorescent pho tomicrographs of canine BMSCs. Strong expression of fibronectin (A) and laminin (B) on BMSCs was observed. Direct co-culture of DRG on BMSC Monolayer DRG neurons were plated on BMSCs, fibrobl asts, or lam inin-coated coverslips and allowed to extend their axons for 48 hours. It was clearly observed that DRG neurons extended longer axons on fibroblasts and BMSCs than on laminin (Fig 2). On laminin, the conformation of DRG neurons was simple, typically having only 1 or 2 unbranched primary neurites (Fig. 52A). In contrast, on a fibroblast monolayer, DRG neurons extended more and longer primary neurites with a few branching neurite processes emerging from the primary neurites (Fig. 5-2B). On a BMSC monolayer, DRG neurons showed a more elaborate appearance (Fig. 5-2C). Typically, DRGs on BMSCs extended three or more primary neurites from which numerous branching neurites developed. These features of neurite outgrowth on different substrates were refl ected quantitatively in neurite measurements. The total neurite length including all primary and branching neurites per single DRG neuron was significantly longe r on BMSCs (1596.5 388.1M) than those on fibroblasts (994.9 225.7M) and laminin (221.9 25.0M) (Fig. 5-3A). Similarly, the total

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97 Figure 5-2. Representative photomicrographs of DRG neurons cultured on three different substrates for 48 hours. DRG neuron s and neurites were indentified by immunostaining using neurofilament-200 antibody. (A) DRG neurons on laminin substrates extended short primary neurit es with few branching neurites. (B) Fibroblasts stimulated extens ion of primary neurites with a few branching neurites. (C) BMSCs further stimulated growth of pr imary neurites and formation of complex arborization of branching neur ites. Scale bar = 50 M. primary neurite length per DRG neuron on BMSCs (797.1 161.8M) was significantly longer than those on fibroblasts (560.9 184.8M) a nd laminin (212.4 23.9M) (Fig. 5-3B). The mean lengths of individual primary neurit es on fibroblasts (233.1 48.7M) and on BMSCs (217.8 35.1M) were longer than that of lami nin (117.5 17.4M) (Fig. 5-3C). The mean number of primary neurites was significantly higher on BMSCs (3.9 0.7) than on fibroblasts (2.4 0.2) and on laminin (1.9 0.3) (Fig. 5-3D). The mean number of branching neurites was also significantly higher on BMSCs (10.4 3.5) than on fibroblasts ( 4.3 1.4) and on laminin (0.2 0.0) (Fig. 5-3E). Statisti cal differences were found between all combinations of the three culture conditions in the total neurite length and total primary neurite length. The mean primary neurite lengths were statistically different between laminin and fibroblasts and between laminin and BMSCs. The mean numbers of primary ne urites and the mean numbers of branching neurites were statistically di fferent between laminin and BMSCs and between fibroblasts and BMSCs.

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98 Figure 5-3. Quantitative neurite measurements of neurite outgrowth from DRG neurons cultured on laminin, fibroblasts, or BMSCs. Data are shown as the mean st andard deviation. a indicates a significant (p < 0.05) difference against laminin. b indicates a significant (p < 0.05) difference against fibroblasts. DRG Cultured in Conditioned Medium The effects of BMSCs on neuritogenesis m ay also be mediated by soluble factors secreted in the culture medium. When DRG neurons were cultured in conditioned media, we found that the patterns of neurite outgrowth among three groups were similar to those observed in direct coculture but the magnitude was much diminished. It was also noticed that there was a wide range in the observed measurements in all groups, partic ularly in the length m easurements. The total neurite lengths of DRG neurons were not statistically different between groups (control, 997.7 684.9 M; fibroblast, 1370.6 1096.4 M; BMSC, 1510.3 1253.0 M) (Fig. 5-4A). The total primary neurite length was longer in BMSC-conditioned media (749.0 468.2 M) compared to that in control media (490.0 393.8 M) but not to that in fibroblast-conditioned media (647.2 423.7 M) (Fig. 5-4B). The mean lengths of indi vidual primary neurites were not statistically different between groups (control, 217.5 102.3 M; fibroblast, 262.3 114.2 M; BMSC, 255.5 112.0 M) (Fig. 5-4C). The mean number of primary neurites was higher in BMSC-

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99 conditioned media (3.0 1.1) compared only to c ontrol media (2.3 0.9) but not to fibroblastconditioned media (2.7 1.6) (Fig. 5-4D). The mean numbers of branching neurites were not statistically different between groups (control, 6.4 3.5; fibr oblast, 7.5 6.6; BMSC, 7.9 8.4) (Fig. 5-4E). Figure 5-4. Quantitative neurite measurements of neurite outgrowth from DRG neurons cultured in control, fibroblast-conditioned, or BMSC-conditioned media. a indicates a significant (p < 0.05) difference against laminin. Conclusion and Discussion Bone m arrow cells are suggested to participate in adult neurogenesis by transdifferentiation (Cogle et al., 2004; Mezey et al., 2003). Several studies claim that BMSCs can also generate neural cells in vivo (Azizi et al., 1998; Deng et al., 2006; Lee et al., 2003). Transplantation of BMSCs has been shown to promote functional recovery in various CNS injuries models (Chen et al., 2001; Hofstetter et al., 2002; Li et al., 2002; Mahmood et al., 2005; Wu et al., 2007; Zhao et al., 2002). However, precise mechanisms responsible for functional recovery have not been clearl y demonstrated. Our data suggest that canine BMSCs have the ability to promote axonal regrowth from DRG neurons in vitro and that the effects are most

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100 prominent via direct contact with neurons. Th e ability of BMSCs to produce ECM molecules such as fibronectin and laminin appeared to be the prime attribute for neurite outgrowthpromoting effects of canine BMSCs. In the present study, direct co-cultures showed that DRG neurons extended substantially longer and more complex neurites on BMSCs than on laminin or fibroblasts. The total neurite length of DRG neurons on BMSCs was about 1. 5 times and 7 times longer than those on fibroblasts and laminin, respectively. The total primary neurite length was also significantly longer on BMSCs than on fibroblasts or laminin; the difference between BMSCs and fibroblasts was due to the increased number of primary neur ites on BMSCs and not to the increased length of individual primary neurites. The mean number of branching ne urites was also significantly higher on BMSCs compared to other substrates. These findings suggest that BMSCs promote neurite outgrowth from DRG neurons by stimula ting emergence of both primary and branching neurites. We speculate that neurite outgrowth-promoti ng effects of BMSCs are primarily mediated by the production of ECM and adhesion molecule s. Our results of immunocytochemistry demonstrated that canine BMSCs produce fibr onectin and laminin similar to human BMSCs (Grayson et al., 2004; Hofstetter et al., 2002). Fibronectin and lami nin are expressed by a variety of cell types and known to enhance neurite exte nsion (Kimura et al., 2004; Orr and Smith, 1988; Smith and Orr, 1987). A previous in vitro study demonstrated that these ECM molecules promote adhesion of neurons and neurite extension and the effects are most potent when used in combination (Orr and Smith, 1988). Our observation that fibroblasts al so produce fibronectin but not laminin points to a signifi cant involvement of laminin in promoting neurite outgrowth. This view is in line with earlier studies indi cating that cell surface f actors associated with

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101 astrocytes and Schwann cells but not with fibr oblasts are primarily re sponsible for inducing active neurite outgrowth (Fallon, 198 5a; Fallon, 1985b). Nevertheless, in vivo studies showed that fibronectin may provide a scaffold for invading cellular elements including macrophages and Schwann cells which in tern stimulate axonal regeneration at the site of spinal cord injury (King et al., 2003; King et al., 2006 ). In addition, we examined whether canine BMSCs express other adhesion molecules that are known to stimulate neurit e extension, including E-cadherin (Oblander et al., 2007), N-cadheri n (Puch et al., 2001; Schense et al., 2000), and neural cell adhesion molecule NCAM (Crigler et al., 2006; Ki mura et al., 2004). However, these molecules were not expressed on canine BMSCs under our culture condition. A recent in vitro study showed that human BMSCs pr omote neurite extension from DRG explants over nerve-inhibitory molecules such as neural proteoglycans, myelin associated glycoprotein, and Nogo-A (Wright et al., 2007). In the study, BMSCs reduced the inhibitory effects of these molecules and thereby permitted extension of DRG neurites. The authors described that DRG explants extended neurites when co-cultured with BMSCs that acted as cellular bridges and also towed neurites over the nerve-inhibito ry molecules. In contrast, BMSC-conditioned medium stimulated neurite extension over nerve-permissive substrate (i.e., type I collagen) but not over ne rve-inhibitory substrates. Ther efore, axon-BMSC interactions appear to be important for BMSCs to promote ne urite extension, particular ly in injured nervous tissues where nerve-inhibitory molecules are pres ent. Another possibility is that BMSCs may degrade nerve-inhibitory molecules thereby allo wing regenerating axons to grow in and across the CNS injury site. Human BMSCs have been shown to produce membrane type I matrix metalloproteinase and matrix metalloproteina se 2 (Son et al., 2006) which degrade nerve-

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102 inhibitory molecules (d'Ortho et al., 1997; Fosa ng et al., 1992; Passi et al., 1999). Whether canine BMSCs hold similar enzymatic prope rties remained to be investigated. Although our results suggest that ECM molecu les are the prime contributors in promoting neurite outgrowth, involvement of soluble factors cannot be enti rely excluded. In fact, some parameters (i.e., the total primary neurit e length/neuron and th e number of primary neurites/neuron) were significantly greater in BMSC-conditioned media compared to control media. The candidate factors re sponsible for these changes might be neurotrophic factors (e.g., NGF, BDNF, and NT-3) since production of an arra y of neurotrophic fact ors has been shown in human BMSCs (Auffray et al., 1996; Chen et al ., 2002a; Chen et al., 2002b; Crigler et al., 2006; Li et al., 2002). However, there were some un certainties present in our study, including the cross-species reactivity of can ine neurotrophic factors to rat derived neurons and local concentrations of soluble factor s secreted in the conditioned medi a. In addition, a soluble form of laminin has been shown to have effects on neurite outgrowth (Kohno et al., 2005), indicating the possibility of laminin to play dual roles as an ECM molecule a nd a soluble factor. Investigations into identification and quantit ation of candidate neurite outgrowth-promoting soluble factors produced by canine BMSCs are cu rrently underway in our laboratory, using a range of neutralizing antibodies. In conclusion, we demonstrated that canin e BMSCs have the ability to promote neurite extension and branching from DRG neurons in vitro, primarily through production of ECM molecules. Because of their accessibility BM SCs may represent the most promising source for cellular treatment of CNS injuries. In order to translate our findings to clinical application of canine BMSCs, further investigations are needed to address several questions. In particular, although DRG neurons have both CNS and PNS components in vivo effects of canine BMSCs

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103 on pure CNS neurites need to be elucidated. Effects of BMSCs on neuritogenesis are also donordependent (Neuhuber et al., 2005) and there is even a considerable variation in the ability to produce neurotrophic factors among different populations of BMSCs from the same donor (Crigler et al., 2006). These previous reports highlight the need for thor ough characterization of canine BMSCs to facilitate the development and re finement of prospective cellular based therapy for dogs with CNS injuries. Finally, xenotransplantation studies in rodent models of CNS injury will allow assessment of procedural as well as bi ological safety associated with transplantation of canine BMSCs and behavioral benefits in vivo

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104 CHAPTER 6 SUMMARY AND CONCLUSION This dissertation presents a se ries of studies conducted to understand the biology of canine BMSCs. Chapter 2 repr esents an initial effort to systematically define the growth kinetics, phenotypic profile, and in vitro differentiation properties of canine BMSCs. It was also our goal to explore the potential of canine BMSCs for ce ll therapy for CNS disorders. Consequently, in vitro and in vivo neural differentiation prope rties of canine BMSCs were presented in chapter 3 and 4, respectively. Finally, in chapter 5, the effects of BMSCs on neurite extension were described. We showed that canine BMSCs share many characteristics with BMSCs of humans and rodents. First, canine primary BMSCs can be read ily isolated by use of their adhesive properties to culture plastic and expanded as CFU-F under th e standard culture condition. From classical CFU-F assay, we estimated the frequency of canine BMSCs to be approximately 0.0042% (1 BMSC in every 2.38 104 mononucleated cells). Passaged ca nine BMSCs grow as monolayer and the growth kinetics are dependent on the initial cell density with the low cell density producing the maximal fold increase of cell expa nsion. Second, the cell surface marker profile of canine BMSCs evaluated by flow cytometry is similar to human and rodent counterparts. Flow cytometry also revealed that expanded cel l populations did not cont ain a significant degree of contamination of other cell lineages. Third, similar to human and rodent BMSCs, canine BMSCs consist of heterogeneous cell populations with varying degrees of differentiation capacities. We found that canine BMSCs do contain a population of cells with the ability to differentiate into osteoblasts and adipocytes in vitro However, we speculate that the standard culture method does not support expansion of multipot ent stem cells present in the canine BMSC

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105 fraction as passaged canine BMSCs demonstrated limited differentiation along osteoblastic and adipogenic pathways. Interestingly, canine BMSCs spontaneously express neurona nd astrocyte-specific proteins in vitro. The significance of this finding is unknown but it may suggest that canine BMSCs are not only undifferentiated but also multi-differentiated as has been suggested for human BMSCs. Elevation of intracellular cycl ic AMP levels caused ra pid transformation of canine BMSCs into neuron-like morphology, but the morphological change per se was interpreted as a result of cytoskeletal shrinka ge rather than genuine neurite extension. Our xenotransplantation study suggested that BMSCs isolated from young canine donors may have the capacity of neural transdiffere ntiation in the neonatal mouse brain. Young donor BMSCs injected into the lateral ventricle of ne onatal mouse brains migrated into the SVZ and assumed neuronal or astrocytic morphology. A sma ll number of BMSCs further migrated in the RMS and reached the olfactory bulb where they expressed neuron-specific marker. Cell fusion events did not contribute to these observati ons of young donor BMSCs. Adult canine BMSCs, however, did not exhibit a similar degree of migra tion and differentiation. The results imply that neurogenic stem cells are present in the BM SC compartment of young dogs, whereas, in the adult canine BMSC compartment, cells of similar properties either do not ex ist or are lost during ex vivo expansion. Finally, we evaluated the ability of canine BMSCs in promoting neurite outgrowth from DRG neurons in vitro When DRGs neurons were co-cul tured with canine BMSCs, neurite outgrowth was strongly promoted. This observati on was attributed to the stimulatory effects of canine BMSCs on development of primary and bran ching neurites from DRG neurons. When DRG neurons were cultured in BMSC-conditioned medium, the magnitude of neurite outgrowth

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106 was suppressed compared to direct co-culture. We showed that canine BMSCs express copious amounts of laminin and fibronectin, two poten t neurite extension-pr omoting proteins. Consequently, canine BMSCs were thought to stimulate neurite outg rowth primarily via production of ECM molecules. In summary, canine BMSCs are easily accessible and expandable in vitro ; thus, represent a promising source for various cell therapies. Howeve r, there are still a lot of works to be done in order to better understand the biology of canine BM SCs. Future investig ations should emphasize the development of culture techniques that allow maximum expansion of multipotent canine BMSCs. In spite of controversies surroundi ng neural transdiffere ntiation of bone marrow derived cells, canine bone marrow (at least from young donors) is likely to contain neurogenic cells. Upon revealing the identity of these cells and refining culture techniques, it may become possible to isolate and expand similar cell populations from adult canine bone marrow. We showed that, with the current standard culture technique, canine BMSCs have the ability to promote neurite outgrowth from DRG neurons. It is thus warrant ed to investigate in vivo effects of canine BMSCs in rodent models of CNS injury. Ultimately, BMSC transplantation in spontaneous CNS injury in dogs will provide valuable insi ghts into the safety as well as clinical efficacy associated with this procedure.

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110 Chen, X., Li, Y., Wang, L., Katakowski, M., Zha ng, L., Chen, J., Xu, Y., Gautam, S. C., Chopp, M., 2002b. Ischemic rat brain extracts induce human marrow stromal cell growth factor production. Neuropathology. 22, 275-9. Chen, X., Wang, X. D., Chen, G., Lin, W. W ., Yao, J., Gu, X. S., 2006. Study of in vivo differentiation of rat bone marrow stromal cells into schwann cell-like cells. Microsurgery. 26, 111-5. Chopp, M., Li, Y., 2002. Treatment of neural injury with marrow stromal cells. Lancet Neurol. 1 92-100. Chopp, M., Zhang, X. H., Li, Y., Wang, L., Chen, J., Lu, D., Lu, M., Rosenblum, M., 2000. Spinal cord injury in rat: treatment w ith bone marrow stromal cell transplantation. Neuroreport. 11 3001-5. Cogle, C. R., Yachnis, A. T., Laywell, E. D., Zander, D. S., Wingard, J. R., Steindler, D. A., Scott, E. W., 2004. Bone marrow transdifferen tiation in brain after transplantation: a retrospective study. Lancet. 363 1432-7. Colter, D. C., Class, R., DiGirolamo, C. M., Prockop, D. J., 2000. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A. 97, 3213-8. Crigler, L., Robey, R. C., Asawachaichar n, A., Gaupp, D., Phinney, D. G., 2006. Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis. Exp Neurol. 198 54-64. David, S., Aguayo, A. J., 1981. Axonal elongation into peripheral nervous sy stem "bridges" after central nervous system injury in adult rats. Science. 214, 931-3. DeKosky, S. T., Goss, J. R., Miller, P. D., Styren, S. D., Kochanek, P. M., Marion, D., 1994. Upregulation of nerve growth factor fo llowing cortical trauma. Exp Neurol. 130 173-7. Deng, J., Petersen, B. E., Steindler, D. A., Jorg ensen, M. L., Laywell, E. D., 2006. Mesenchymal stem cells spontaneously expr ess neural proteins in cultu re and are neurogenic after transplantation. Stem Cells. 24, 1054-64. Deng, W., Obrocka, M., Fischer, I., Prockop, D. J., 2001. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun. 282 148-52. Dezawa, M., Ishikawa, H., Itokazu, Y., Yoshihara, T., Hoshino, M., Takeda, S., Ide, C., Nabeshima, Y., 2005. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science. 309 314-7.

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111 Dezawa, M., Kanno, H., Hoshino, M., Cho, H., Matsumoto, N., Itokazu, Y., Tajima, N., Yamada, H., Sawada, H., Ishikawa, H., Mimura, T., Kitada, M., Suzuki, Y., Ide, C., 2004. Specific induction of neuronal cells from bone marrow stromal cells and application for autologous tran splantation. J Clin Invest. 113 1701-10. Dezawa, M., Takahashi, I., Esaki, M., Takano, M., Sawada, H., 2001. Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. Eur J Neurosci. 14 1771-6. Diefenderfer, D. L., Osyczka, A. M., Gari no, J. P., Leboy, P. S., 2003a. Regulation of BMPinduced transcription in cu ltured human bone marrow stroma l cells. J Bone Joint Surg Am. 85-A Suppl 3 19-28. Diefenderfer, D. L., Osyczka, A. M., Reilly, G. C., Leboy, P. S., 2003b. BMP responsiveness in human mesenchymal stem cells. Co nnect Tissue Res. 44 Suppl 1 305-11. Digirolamo, C. M., Stokes, D., Colter, D., Phinney, D. G., Class, R., Prockop, D. J., 1999. Propagation and senescence of human marrow stromal cells in culture: a simple colonyforming assay identifies samples with the grea test potential to propagate and differentiate. Br J Haematol. 107 275-81. Doetsch, F., Garcia-Verdugo, J. M., Alvarez-B uylla, A., 1997. Cellular composition and threedimensional organization of the subventricu lar germinal zone in the adult mammalian brain. J Neurosci. 17, 5046-61. Dormady, S. P., Bashayan, O., Dougherty, R., Zhang, X. M., Basch, R. S., 2001. Immortalized multipotential mesenchymal cells and th e hematopoietic microenvironment. J Hematother Stem Cell Res. 10 125-40. d'Ortho, M. P., Will, H., Atkinson, S., Butler, G ., Messent, A., Gavrilovic, J., Smith, B., Timpl, R., Zardi, L., Murphy, G., 1997. Membrane-t ype matrix metalloproteinases 1 and 2 exhibit broad-spectrum pr oteolytic capacities comparable to many matrix metalloproteinases. Eur J Biochem. 250 751-7. Einhorn, T. A., 2003. Clinical applications of recombinant human BMPs: early experience and future development. J Bone Joint Surg Am. 85-A Suppl 3 82-8. Fallon, J. R., 1985a. Neurite guidance by non-neuronal cells in culture: prefer ential outgrowth of peripheral neurites on glial as compared to nonglial cell surfaces. J Neurosci. 5 3169-77. Fallon, J. R., 1985b. Preferential outgrowth of central nervous system neur ites on astrocytes and Schwann cells as compared with nong lial cells in vitro. J Cell Biol. 100, 198-207. Ford-Perriss, M., Abud, H., Murphy, M., 2001. Fibroblast growth factors in the developing central nervous system. Clin Exp Pharmacol Physiol. 28 493-503.

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127 BIOGRAPHICAL SKETCH Hiroaki Kam ishina was born on December 1, 1971, in Oita, Japan. He received his Bachelor of Veterinary Medica l Sciences degree from Raku no Gakuen University, Japan, in March 1996. He then worked in a small animal pr actice for four years. After that, he came to the University of Florida where he received a Master of Science in veterinary medical sciences in August 2003 and a Ph.D. in veterinary medical sciences in December 2007.