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Neurospheres and Multipotent Astrocytic Stem Cells: Neural Progenitor Cells Rather Than Neural Stem Cells

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PAGE 1

NEUROSPHERES AND MULTIPOTENT ASTROCYTIC STEM CELLS: NEURAL PROGENITOR CELLS RATHER THAN NEURAL STEM CELLS By GREGORY PAUL MARSHALL, II 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 2005

PAGE 2

Copyright 2005 By Gregory Paul Marshall, II

PAGE 3

To my beautiful wife Kathleen, whose stead fast love and support made all of this possible

PAGE 4

ACKNOWLEDGMENTS I would like to thank Ed Scott, Dennis Steindler, and Eric Laywell for all of their guidance throughout my graduate career; my wonderful family for their love and support; my fellow lab rats for always being there for me; and the Gainesville Rugby Club for providing a welcoming diversion from my otherwise hectic life. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES.........................................................................................................viii ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION TO ADULT MURINE STEM CELLS..........................................1 Hematopoietic Stem Cells............................................................................................1 Hematopoiesis.......................................................................................................1 Isolation of the Adult Hematopoietic Stem Cell...................................................3 Determining Functional Engraftment....................................................................4 Irradiation of the Host Bone Marrow, the Niche of the HSC................................6 Plasticity of the Hematopoietic Stem Cell.............................................................7 Summary................................................................................................................9 Neural Stem Cells.........................................................................................................9 Neurogenesis and the Embryonic Neural Stem Cell.............................................9 The Adult Neural Stem Cell................................................................................10 Subependymal-Zone Neural Stem Cells.............................................................11 Multipotent Astrocytic Stem Cells......................................................................14 Potential Identifying Markers of the NSC...........................................................15 Transplantation of In Vitro Expanded NS and MASC........................................16 Adult NS and MASC: True Stem Cells?.............................................................17 Effects of Radiation on Adult Neurogenesis.......................................................18 Summary..............................................................................................................19 2 MATERIALS AND METHODS...............................................................................21 Reagents......................................................................................................................21 Avertin Mouse Anesthetic Solution....................................................................21 5-bromo-2-deoxy-uridine (BrdU) Solution.......................................................21 Epidermal Growth Factor (EGF) Stock Solution (1000x)..................................22 Basic Fibroblast Growth Factor (bFGF) Stock Solution (1000x).......................23 Immunocytochemistry Hybridization Buffer......................................................23 Neural Stem Cell (NSC) Culture Media..............................................................23 v

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4% Paraformaldehyde (100 mL).........................................................................24 Puromycin Stock Solution (1 mg/mL)................................................................24 Antibody List..............................................................................................................25 Primary Antibodies..............................................................................................25 Secondary Antibodies..........................................................................................25 Methods......................................................................................................................25 Cell Culture.........................................................................................................25 Isolation and culture of neurospheres...........................................................25 Isolation and culture of multipotent astrocytic stem cells............................26 Selection of gfp+ multipotent astrocytic stem cells by puromycin selection..................................................................................................27 Transplantation and Analysis..............................................................................27 Transplantation of gfp+ MASC and neurospheres into the lateral ventricles of adult C57BL/6 mice...........................................................27 Transplantation of gfp+ MASC and neurospheres into the lateral ventricles of neonatal C57BL/6 mice.....................................................27 Tissue immunohistochemistry......................................................................28 Analysis of engrafted gfp+ MASC and neurospheres into the brains of C57BL/6 mice.........................................................................................29 Analysis of engrafted gfp+ MASC into the olfactory bulb of C57BL/6 mice.........................................................................................................29 Immunohistochemical analysis of neurospheres and MASCs.....................29 Radiation Studies.................................................................................................30 Irradiation and bone marrow reconstitution.................................................30 Identification of proliferative cells by BrdU labeling..................................31 Blind analysis of the effects of lethal and sublethal irradiation on neurosphere yield....................................................................................32 3 LACK OF EVIDENCE FOR THE CLASSIFICATION OF NEUROSPHERES AND MULTIPOTENT ASTROCYTIC STEM CELLS AS TRUE STEM CELLS.34 Introduction.................................................................................................................34 Inability of Gfp+ Neural Stem Cells to Survive Re-isolation Following Transplantation into the Lateral Ventricles of C57BL/6 Mice..............................37 Gfp+ Neurospheres are not Generated from the Brains of Adult C57BL/6 Mice Transplanted with Gfp+ Neurospheres...................................................37 Gfp+ Neurospheres are not Generated from the Brains of Neonatal C57BL/6 Mice Transplanted with Gfp+ Neurospheres...................................................38 Gfp+ Multipotent Astrocytic Stem Cells are not Generated from the Brains of Neonatal C57BL/6 Mice Transplanted with Gfp+ Multipotent Astrocytic Stem Cells........................................................................................................41 MASC Isolated from the Brains of C57BL/6 Neonatal Mice Transplanted with Gfp MASC do not Survive Puromycin Treatment...................................43 Long Term Engraftment......................................................................................46 vi

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4 IONIZING RADIATION ENHANCES THE ENGRAFTMENT OF TRANSPLANTED IN VITRO DERIVED NEURAL STEM CELLS......................49 Introduction.................................................................................................................49 Effects of Lethal Irradiation on Subependymal Zone Neurogenesis..........................52 Lethal Irradiation Severely Depletes Migrating Neuroblasts in the RMS..........52 Analysis and Quantification of Neuroblast Depletion in the Subventricular Zone Following Lethal Irradiation, as Determined by BrdU Incorporation....54 Diminished Neurosphere Yield from Lethally Irradiated Mice..........................56 Lethal Irradiation Attenuates Engraftment of Transplanted Gfp+ Multipotent Astrocytic Stem Cells......................................................................................59 Effects of Sub-Lethal Irradiation on Subependymal Zone Neurogenesis..................62 Sublethal Irradiation Results in a Transient Decrease in the Number of Mitotic Subependymal Zone Neuroblasts........................................................62 Diminished Neurosphere Yield from Sublethally Irradiated Mice.....................63 Sublethal Irradiation Enhances Engraftment of Transplanted Gfp+ Multipotent Astrocytic Stem Cells...................................................................63 5 DISCUSSION AND CONCLUSIONS......................................................................69 LIST OF REFERENCES...................................................................................................78 BIOGRAPHICAL SKETCH.............................................................................................86 vii

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LIST OF FIGURES Figure page 1-1. Migratory neuroblasts express PSA-NCAM in the adult mouse rostral migratory stream.......................................................................................................................12 3-1. Gfp+ neurosphere derived cells are evident in the brains of host C57BL/6 mice three weeks following transplantation...............................................................................38 3-2. Gfp+ neurospheres are not capable of re-isolation following transplantation into the lateral ventricle of adult mice...................................................................................39 3-3. Gfp+ neurospheres display high levels of engraftment following transplantation into neonatal mice............................................................................................................40 3-4. Gfp+ neurospheres are not capable of re-isolation following transplantation into the lateral ventricle of neonatal mice.............................................................................41 3-5. Passage three gfp+ multipotent astrocytic stem cells contain astrocytes but are devoid of neurons.................................................................................................................42 3-6. Gfp+ multipotent astrocytic stem cells display high levels of engraftment following transplantation into neonatal mice............................................................................43 3-7. Gfp+ multipotent astrocytic stem cells are not present in the cultures of transplanted neonatal mice............................................................................................................44 3-8. Qualitative assessment of the effects of puromycin on multipotent astrocytic stem cells cultures derived from C57BL/6 and gfp+ neonatal mice................................45 3-9. Transplantation of puromycin selected multipotent astrocytic stem cells isolated from C57BL/6 mice transplanted with gfp+ multipotent astrocytic stem cells exhibit no gfp donor cell engraftment......................................................................45 3-10. Multipotent astrocytic stem cells isolated from the forebrains of mice transplanted with gfp+ multipotent astrocytic stem cells do not survive puromycin treatment...47 3-11. Donor-derived cells exist in the brains of gfp+ neurosphere transplanted animals 14 months after surgery............................................................................................48 viii

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4-1. Lethal irradiation drastically reduces the volume of PSA-NCAM and -III Tubulin positive migratory neuroblasts.................................................................................53 4-2. Reduction in the volume of -III Tubulin positive migratory neuroblasts persists months after lethal irradiation..................................................................................53 4-3. Region of analysis for quantification of mitotic neuroblast depletion in the subependymal zone of lethally irradiated mice........................................................55 4-4. Depletion of mitotic neuroblasts in the subependymal zone of lethally irradiated animals.....................................................................................................................56 4-5. Lethal irradiation significantly decreases the number of BrdU positive SEZ neuroblasts................................................................................................................57 4-6. Depletion of migratory neuroblasts in the rostral migratory stream positive for both BrdU and -III Tubulin in lethally irradiated mice..................................................58 4-7. Lethal irradiation significantly reduces the yield of neurospheres cultured from the adult subependymal zone.........................................................................................60 4-8. Neurosphere differentiation potential........................................................................60 4-9. Attenuation of gfp+ multipotent astrocytic stem cell engraftment by lethal irradiation.................................................................................................................61 4-10. Mitotic subependymal zone neuroblasts are transiently depleted following sublethal irradiation..................................................................................................64 4-11. Sublethal irradiation results in transient, recoverable depletion of mitotic subependymal zone neuroblasts...............................................................................65 4-12. Sublethal irradiation significantly reduces the generation of neurospheres cultured from adult subependymal zone................................................................................66 4-13. Gfp+ multipotent astrocytic stem cells transplanted into the lateral ventricles of control and sub-lethally irradiated mice produce donor-derived migratory cells in the host olfactory bulb..............................................................................................67 4-14. Sublethal irradiation enhances engraftment of donor-derived neuroblasts.............68 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy NEUROSPHERES AND MULTIPOTENT ASTROCYTIC STEM CELLS: NEURAL PROGENITOR CELLS RATHER THAN NEURAL STEM CELLS By Gregory Paul Marshall, II May, 2005 Chair: Edward W. Scott Major Department: Molecular Genetics and Microbiology Adult hematopoiesis is driven by the hematopoietic stem cell (HSC), a cell residing in the bone marrow that possesses unique, functional characteristics defining it as a true stem cell. Neural stem cells (NSCs) are reported to exist in the brains of adult mice in two well characterized regions: the subependymal zone (SEZ) of the lateral ventricles (LV) and the hippocampal dentate gyrus. Mitotic neuroblasts are continuously generated by the SEZ and migrate along the rostral migratory stream (RMS) toward the olfactory bulb, where they functionally integrate as interneurons. SEZ NSCs can be cultured under specific conditions to generate neurospheres (NS) or multipotent astrocytic stem cells (MASC), cell types that possess certain stem cell qualities in vitro. However, the in vivo stem cell characteristics possessed by cultured NSCs (specifically functional engraftment and serial transplantation) are not fully understood. x

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Green fluorescent protein (gfp) NS and MASC transplanted into the LV of both neonatal and adult C57BL/6 mice resulted in engraftment into the recipient brain. with donor-derived migratory neuroblasts visible in the RMS weeks after transplantation. Neither displayed the capacity to be re-isolated from adult or neonatal brains even under rigorous enrichment conditions, nor were they evident in the brains of secondary recipient mice, indicating that functional, long-term engraftment by the transplanted cells failed to occur. Exposure of adult C57BL/6 mice to lethal levels of ionizing radiation resulted in the ablation of both active hematopoiesis and SEZ neurogenesis, with subsequent transplantation of gfp MASC into the LV resulting in no observable engraftment. Exposure to sublethal levels of radiation resulted in a transient depletion of SEZ neurogenesis and moderately enhanced engraftment levels of transplanted gfp+ MASC, potentially providing a more ideal model system for NSC transplantation and analysis. In this study, both NS and MASC failed to meet the criteria of true stem cells as defined by the properties of the adult HSC. Thus it is possible that neurogenesis in the adult brain is provided not by an isolatable NSC but rather by neural progenitor cells derived from a migratory adult stem cell residing in the bone marrow. xi

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CHAPTER 1 INTRODUCTION TO ADULT MURINE STEM CELLS Stem cells are represented in a variety of organs in the adult mouse. Cells with stem-like properties have been reported to exist in the bone marrow (7), skeletal muscle (67), liver (13), pancreas (90), intestinal lining (68), skin (74) and the brain (2, 3, 44) representing all three of the primordial germ layers: endoderm, ectoderm and mesoderm. Stem cells in the adult mouse are believed to be developmental remnants of germinal cells that continue to renew the tissues of the organ in which they reside. Adult stem cells are characterized by the ability to continuously generate all cell types necessary to perpetuate the existence of their native organ; and the ability to asymmetrically divide, so that a duplicate stem cell is generated as well as a more lineage-committed daughter cell, allowing for cell generation to continue for the life of the animal. Hematopoietic Stem Cells Hematopoiesis The hematopoietic stem cell (HSC) is responsible for generating all the cells of the blood lineage for the life of the animal, a process known as hematopoiesis. During development, hematopoietic cells arise from the mesoderm after gastrulation and become organized into blood islands in the extra-embryonic yolk sac near embryonic Day 7.5 (E 7.5) in the mouse (31, 53). The first isolatable manifestation of the HSC appears in the intra-embryonic aorta-gonod-mesonephros (AGM) at E 10.5, resulting in the initial onset of definitive hematopoiesis in the developing animal (48, 54). Via waves of migratory HSCs from the 1

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2 AGM, hematopoiesis then shifts to the fetal liver (with the presence of HSCs in the AGM virtually eliminated by E 13). Migration of fetal HSCs is believed to be chemokine driven, with stromal derived factor 1 (SDF-1) and steel factor (SLF) as the likely agents of directional migration (11). Numbers of HSCs in the fetal liver begin to decrease around E 16, presumably as the migratory HSCs leave the liver to seed the developing bone marrow and spleen however, fetal liver hematopoiesis persists until shortly after birth; whereas in the adult mouse, the bone marrow becomes the sole region of hematopoiesis throughout the life of the animal. Three definitive characteristics define the HSC as a true stem cell: asymmetric division, pluripotency, and the ability to functionally reconstitute depleted bone marrow (69). Asymmetric cellular division is a process in which the HSC switches between generating an exact duplicate of itself and generating a more lineage committed daughter cell that allows the HSC to perpetuate its existence throughout the life of the animal. The HSC is capable, by generating lineage-specific hematopoietic progenitor cells (HPCs), of producing each of the numerous cell types present in the blood. This includes (but is not limited to) erythrocytes, macrophages, lymphocytes, T-cells, and B-cells. These two abilities together make up the third criteria: The HSC is capable of repopulating the bone marrow of a myeloablated mouse (a mouse that has been exposed to sufficient doses of radiation so that the bone marrow is depleted of viable cells, a lethal condition without treatment). The HSC can home to the injured bone marrow after intravenous transplantation, repopulating the niche and giving rise to all of the numerous cells within the hematopoietic lineage. This homing mechanism is believed to be similar to that observed

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3 during development, with an increase in the expression of SDF-1 after myeloablation of the bone marrow causing an increase in the expression of matrix metalloproteinase-9 (MMP-9). This results in the release of soluble Kit ligand, creating a diffusion gradient that the migratory c-kit+ HSC follows as it homes to the bone marrow (33). Asymmetric division of the now engrafted HSCs allows for donor-derived hematopoiesis to continue for the life of the mouse, deriving from the primary transplanted HSC, later-generated daughter HSCs and the progeny of the resultant progenitor cells. A fourth criterion has been proposed, in which isolated candidate HSCs must display the aforementioned capabilities and the ability to serially rescue a myloablated animal. In this paradigm, donor-derived HSCs can be re-isolated from the bone marrow of a mouse rescued by the transplantation and engraftment of a single HSC, and transplanted into a secondary myloablated host with the same rescue ability conferred to the animal (40). These four criteria set the gold standard for defining the HSC, a functional standard rather than a phenotypic one. Cell-surface markers unique to only the HSC have yet to be identified, but techniques exist that allow for the enrichment of cells extracted from the bone marrow for HSCs. Isolation of the Adult Hematopoietic Stem Cell The bone marrow is home to a variety of cell types, from stromal support cells and mesenchymal stem cells (MSC) to HSCs, HPCs, and their resultant progeny. After extracting the bone marrow, the MSC is removed by exploiting the ability of the MSC to adhere to adhesive substrates such as laminin. As the HSC lacks this ability, placement of the cell suspension onto an adhesive substrate allows the MSCs to adhere to the substrate, leaving only the cells of the hematopoietic lineage. Removal of terminal progeny is accomplished by using the surface markers unique to the selected cells: B220

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4 is expressed by B-cells, CD11b by macrophages, Ter119 by T-cells, and Gr-1 by granulocytes. Fluorescently labeled antibodies specific to these antigens allow for the selection of cells by a technique known as Fluorescence Activated Cell Sorting (FACS) in which cells can be selected on the basis of the presence (positive selection) or absence (negative selection) of definitive cell-surface markers. The FACS sorter is capable of distinguishing between cell types based on the particular fluorochrome expression pattern (and the specific antibody conjugated to that fluorochrome) present on treated cell populations. Exclusion of the terminal daughter cells by negative selections leaves the remaining cell population consisting of stem and progenitor cells. To further enrich for the HSC, two specific cell-surface markers (88) can be utilized: c-Kit and Sca-1. C-kit is the receptor for stem cell factor (SCF) also known as steel factor (SF), a cytokine shown to inhibit apoptosis in hematopoietic cells potentially giving HSCs the ability to perpetuate longer than terminally differentiated cells (1). Sca-1 (stem cell antigen), also known as lymphocyte activation protein-6a (Ly6-A), is a cell-surface receptor shown to play a role in HSC proliferation; the identity of its binding ligand has yet to be identified (36). Selecting cells positive for these two markers while depleted of terminally differentiated daughter cells results in a population of cells 1000to 2000-fold enriched for the presence of HSCs. While this process greatly increases the chances of the resulting cells being HSCs, the aforementioned functional properties of long-term bone marrow reconstitution must be ascertained before the isolated cell can be definitively labeled as a true HSC. Determining Functional Engraftment After enrichment of whole bone marrow for the presence of HSCs, an assay to ascertain the rescue capacity of the enriched cell population is critical for the cells

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5 isolated to be deemed HSCs. Mice exposed to levels of radiation sufficient to deplete the bone marrow of viable hematopoietic cells (lethal irradiation) are generated as recipients for candidate HSCs. Lethally irradiated mice (also known as myeloablated) will expire within a few days of exposure without intravenous transplantation of either whole bone marrow or HSCs, so survival of the irradiated mouse is the first indication of candidate cell types being HSCs. Duration of survival indicates the identity of the cell transplanted, as short-term HPCs provide transient protection from myeloablation for a period of only 3 to 4 mo (69). As previously mentioned, HSCs exist for the life of the animal and long-term survival of a myloablated mouse (longer than 6 mo) indicates engraftment of a true HSC. To ascertain that the engrafted HSC is responsible for the lethally irradiated animals hematopoiesis, transplanted HSCs need to differ in some fashion from the host animal. A common tool to meet this requirement is the gfp mouse; a transgenic animal with vast experimental capabilities. The gfp mouse contains a transgene that expresses gfp driven by the chicken beta actin promoter enhanced by a cytomegalovirus enhancer, resulting in ubiquitous expression of gfp in theoretically every cell of the mouse (32). Cells isolated from the gfp mouse do not initiate an immune response when transplanted into mice from the C57BL/6 line, as this line was used to generate the gfp transgenic animal. Furthermore, the expression of gfp continues in vitro after removal of tissue from the animal, allowing for in vitro manipulation and expansion. Myeloablated C57BL/6 mice transplanted with candidate HSCs isolated from the bone marrow of gfp mice result in a chimeric animal whose bone marrow and resultant progeny are gfp+. By sampling portions of the peripheral blood of transplanted mice, it

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6 is possible to determine which cell types are gfp+ and the percent chimerism contributed by the transplanted cells. Using fluorescent antibodies against the cell-surface markers present on terminal daughter cells listed earlier, FACS analysis allows researchers to determine if circulating progeny are gfp+ and thus the result of an engrafted gfp+ HSC (donor-derived). The presence of donor-derived progeny 6 mo or longer after transplantation indicates that the cell type transplanted has characteristics of an HSC. Serial transplantation provides definitive evidence that the transplanted cell was indeed an HSC. If a true HSC was transplanted into a myeloablated animal and engrafted into the bone marrow, resulting in long-term functional hematopoiesis; then it would stand to reason that the transplanted HSC underwent asymmetric division, producing additional HSCs. To determine this to be the case, the bone marrow from long-term reconstituted mice transplanted with gfp+ candidate HSC is extracted and the resulting cell suspension is again enriched for the presence of HSC. Should the isolated cells rescue a secondary myloablated mouse, and donor-derived progeny be observed in the peripheral blood 6 mo or longer after transplantation, it can then be stated that the cell type transplanted satisfied all the necessary criteria to be classified as an HSC. Irradiation of the Host Bone Marrow, the Niche of the HSC Transplantation of enriched bone marrow results in robust, long-term engraftment only when the host animal has first undergone myeloablation (18, 19, 20). Irradiation dosage is critical, as insufficient levels will not deplete the bone marrow; and over-exposure will result in mortal damage to internal organs. While lethal irradiation is sufficient to deplete the bone marrow of HSC and their progeny, it apparently does little to damage the support cells that make up the HSC niche. Niche (a term recently applied to stem cell biology) describes the cellular make-up of the microenvironment in which a

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7 particular stem cell is believed to reside, and the specific mitogens and cytokines needed to maintain the potency of the native stem cell. As previously mentioned the bone marrow contains cells of the hematopoietic lineage and also contains stromal support cells. In vitro studies indicate that osteoblasts, fibroblasts, and endothelial cells are capable of supporting HSCs and HPCs in culture. While this has yet to be confirmed in vivo, HSCs in the bone marrow are known to exist in close proximity to the endothelial vasculature and osteoblasts (87). The current lack of available information on the exact chemical and cellular components that comprise the microenvironment of the HSC niche has led to the inability to expand the HSC in vitro while retaining full potency and rescue ability (14). While it is possible for HSCs to exist in vitro and to differentiate into different hematopoietic cell types in certain culture conditions, the HSC has yet to be proven capable of successful expansion sufficient to daughter HSCs capable of repopulating the bone marrow of a myeloablated animal. Plasticity of the Hematopoietic Stem Cell Because hematopoiesis (and subsequently, the HSC) is the most robust system of cellular generation in the adult mammal, the HSC is arguably the stem cell with the most potential for regenerating damaged tissue. The concept of an adult stem cell transdifferentiating to produce cell types foreign to its native organ is a concept that has undergone tremendous scrutiny in recent years. At the same time, the concept of HSC transdifferentiation has elicited excitement in the field of regenerative medicine. The HSC has been reported to repopulate the bone marrow after transplantation into a myeloablated mouse, and also sometimes contribute to the brain (8, 17, 49), liver (41, 61, 76, 77), heart (34, 55, 56), muscle (21, 30), and blood vessels (28). HSCs isolated from

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8 mice expressing specific reporter genes were found to generate progeny that appeared morphologically identical to neighboring cells in these organs and that expressed the appropriate cell surface markers. The possibility that cellular fusion was the reason behind these cells appearing donor-derived weakened the argument for transdifferentiation (75, 86). Cell fusion is an extremely rare event, with neighboring cells merging to form a cell that expresses the characteristics of one or both cells involved. This fusion occurs at the cytoplasmic and nuclear level, with the resultant cell existing in higher than a diploid state, expressing the cell-surface markers of both contributing cells, and adopting the morphology of the surrounding tissue. Currently, for an investigator to disprove a fusion event and thereby support transdifferentiation, specific steps must be implemented in the experimental design. One such step is to transplant male donor cells into female host tissue with later analysis for the presence or absence of the y-chromosome in potentially transdifferentiated cells. Another such step is to use donor animals that constitutively express a reporter gene downstream (gfp for example) of a cre-recombinase gene, and host animals that constitutively express a second reporter gene (lacZ) immediately downstream of a stop codon flanked by lox-p sites. In the host animal, lacZ is turned off until such time as the upstream stop codon is removed. Should a fusion event occur, the cre-recombinase from the donor cells will excise the stop-codon of the host cell via the lox-p sites and allow the expression of lacZ along with the expression of gfp. Recent investigations involving the long-term (more than 6 mo) contribution of HSC-derived cells to the brain have yielded positive results, with the transdifferentiated neurons giving no indication of being the result of fusion (12, 84). The long-term design

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9 of these studies may be a crucial point, in that the HSC could require time to generate progeny capable of infiltrating the brains regions of stem cell activity and producing the observed cell types. Injury to the target region of engraftment as well as the regions of potential transdifferentiation may also be a critical requirement, as little evidence has been collected in non-injured recipients (83). Summary The HSC can very well be classified as the grandfather of adult stem cells. With almost 50 years of research dedicated to its identification and potential, the HSC has a nearly insurmountable head start on other adult stem cells. Much of what is known about stem cells (from their definitive phenotypes and capabilities, to their organs of residence) has been derived from HSC studies. Candidate stem-cell populations isolated from other tissues will need to meet the rigorous criteria set forth by the field of hematopoiesis before they can be deemed to be true stem cells. Neural Stem Cells Neurogenesis and the Embryonic Neural Stem Cell Development of the central nervous system begins after gastrulation, with induction of the neural plate from the ectoderm to eventually form the neural crest. As development continues, the structure of the brain emerges via an inside-out pattern of growth (the deepest layers of the brain are formed first and outer layers are then formed by migratory cells traveling and passing through previously generated layers). This pattern of migration points toward regions of persistent neurogenesis in the interior portion of the developing brain. While opinions still differ as to the existence of a single type of neural stem cell (NSC) that contributes to the formation of the entire brain, it is generally accepted that a

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10 population of neural stem cells exists during this time, dedicated to producing cells of the neural and glial lineage. These stem cells presumably generate additional stem cells and also populations of lineage-specific progenitor cells. Neural progenitors (sometimes called neuroblasts) generate the numerous manifestations of neurons such as interneurons and granule neurons while glial progenitors give rise to astrocytes and oligodendrocytes. Astrocytes comprise the majority of cell types found in the brain, offering mechanical and metabolic support to neurons. Oligodendrocytes are responsible for the production of the myelin (the axonal covering that speeds neuronal transmission). The Adult Neural Stem Cell It was believed for many years that while stem cells were required to promote developmental neurogenesis, the brain of the adult mammal was a static structure without the ability to repair or modify itself (abilities conferred by stem cells). The presence of newly generated cells indicated that neurogenesis occurred in the postnatal rat brain, but a lack of available techniques to positively identify these cells as neurons left the observations relatively ignored (2, 3). It is now generally accepted that adult neurogenesis is restricted to two regions: the subgranule layer of the hippocampal dentate gyrus (9, 25, 38) and the ventriclar subependymal zone (SEZ) (44, 47). During the middle 1990s, numerous reports emerged showing that (in the adult mammal) cells with stem-like properties can be isolated from the hippocampus, spinal cord, and SEZ (25, 44, 85). The method of isolation differs from that seen in the field of hematopoiesis in that no definitive markers for the NSC are currently available. Instead, candidate stem cells are isolated from regions of neurogenesis and cultured in defined media conditions to ascertain what (if any) stem cell properties they may possess.

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11 Subependymal-Zone Neural Stem Cells In the adult mouse, the SEZ is arguably the most robust region of neurogenesis, existing as a layer of cells adjacent to the ependyma running along the length of the wall of the lateral ventricles. Throughout the life of the animal, mitotic neuroblasts are generated in the SEZ and migrate along a well-defined glial structure known as the rostral migratory stream (RMS) towards the olfactory bulb (OB), where they integrate as interneurons (4, 45, 47, 78). Presumably these interneurons allow for the maintenance of olfactory sensation. Migration of the mitotic neuroblasts occurs in a rostral orientation until the RMS enters the OB proper, whereupon migration shifts to a tangential orientation. At this point, the neuroblasts adopt a neuronal morphology and integrate into the existing cytoarchitecture of the OB as granule or periglomerular interneurons. The migration of mitotic neuroblasts is rather rapid (taking 2-6 days to traverse 2-8 mm) and occurs via a unique form of chain migration (46). The migrating cells adhere to one another (presumably via the expression of a polysialylated form of the neural cell adhesion molecule, dubbed PSA-NCAM) and migrate at an estimated rate of 120 m/h through the RMS in chains that are devoid of glial cells (Figure 1-1). An estimated 30,000 new neurons are produced each day in the adult mouse, leading many to conclude that a self-renewing stem cell must reside in the SEZ in order for this rate to be sustained for the life of the animal (45). While the exact identity of the SEZ NSC has yet to be determined, numerous reports have shed light on its characteristics and function. A heterogeneous population of cells exists, replicate, and migrate adjacent to the ciliated ependymal layer, a layer of cells lining the walls of the lateral ventricle that was previously believed to either

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12 Figure 1-1. Migratory neuroblasts express PSA-NCAM in the adult mouse rostral migratory stream. Sagittal photographic montage (5x images) of migratory neuroblasts in a 6 week old C57BL/6 female migrating through the RMS to the OB from the subependymal zone of the LV. (*) denotes chains of PSA-NCAM positive (red) neuroblasts. OB = olfactory bulb, RMS = rostral migratory stream, LV = lateral ventricle. contain or consist of NSCs (37). Little is known of the specific cell types and factors that comprise the microenvironment of this NSC niche, although recent studies are helping to better understand the cellular make-up. It has recently been reported that the SEZ NSC is a type of slowly dividing astrocyte, termed the type B cell (15). The processes of type B cells form tube-like structures through which migratory neuroblasts (type A cells) travel toward the OB. Transit amplifying neural progenitor cells (referred to as type C cells) are interspersed among the type B cells, and it is this cell type that is believed to be responsible for the production of the type A cells. Experiments involving the injection of the anti-mitotic agent cytosine--D-arabinofuranoside (Ara-C) into the SEZ of adult rats showed that the cells initially eliminated were the faster-dividing progenitor cells (the type A and C cells), and that after removal of Ara-C the first cell to return was the type B cell (16). The type B

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13 cells then generated type C cells, that in turn generated type A cells. Expression of cell-surface markers of these three cell types are not unique enough to allow selective isolation or enrichment by FACS, as earlier shown with the HSC. The type B cell expresses the astrocytic markers glial fibrillary acidic protein (GFAP) and vimentin, while the type A cell expresses both the migratory marker PSA-NCAM and the pan-neuronal marker -III tubulin (27). Unfortunately, these markers are also expressed on a variety of terminally differentiated cells in the brain, ruling them out as potential unique markers for the isolation of the NSC. Interestingly, type C cells appear to exclusively express Nestin (a marker expressed by primitive neuroepethelium during development) indicating that this neural precursor is extremely primitive (27). Regardless of its expression profile, the type B cell is generally accepted to be the NSC of the adult SEZ. After surgical dissection, adult neural tissue from the SEZ can be dissociated into a single-cell suspension and cultured in the presence of mitogens; specifically, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). When cultured in the absence of adhesive substrates, cultured cells form spherical structures with phase contrast-bright boundaries known as neurospheres (NS): presumably clonal structures consisting of multi-lineage daughter cells in varying stages of maturation (22, 29, 39, 42, 62-65, 83). These NS are multipotent and capable of generating cell types from all three neural lineages upon induction of differentiation. The NS do not normally form in the absence of EGF or bFGF, validating the necessity of these mitogens in culture. NSCs during development express receptors to EGF and bFGF in a temporaland region-specific manner, varying as the embryo develops. Expression of bFGF seems crucial to normal development, as bFGF knock-out mice have fewer neurons and glia and reduced

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14 tissue volume (57, 81). This observation was supported by a study (73) in which antibodies designed to neutralize bFGF were injected into neonatal brains resulting in decreased DNA synthesis in multiple regions of the brain. Conversely, it was observed that intra-cerebral injections of bFGF results in increased neurogenesis (81). EGF also plays an important role in neural development: EGF knock-out mice exhibit defects in cortical neurogenesis, and injecting EGF into the brain has been shown to increase neurogenesis (79). In addition to their multipotency, another stem cell quality of cultured NS is that they are capable of in vitro expansion (64). After dissociation and secondary culture, primary NS yield numerous secondary spheres, with the newly generated NS retaining all of the pluripotent characteristics of the primary NS. Furthermore, the expansion capability of the NS is retained even after ten passages, indicating self-renewal of a stem cell by asymmetric division, although conclusions from conflicting reports would argue as to the exact number of passages the NS can undergo while retaining potency. What is not yet known is which cell in the isolated tissue gives rise to the NS (the sphere-forming cell), if the sphere-forming cell is indeed a true stem cell, or if every NS in culture is the product of an active NSC. While the results listed above identify the type B cell as the NSC in vivo, definitive isolation of the adult NSC and its subsequent in vitro analysis and expansion has yet to be conclusively accomplished. Multipotent Astrocytic Stem Cells In addition to the NS, another in vitro manifestation has recently been reported: the multipotent astrocytic stem cell (MASC) (43). MASC can be isolated from the cerebellum and forebrain of neonatal mice (up to 2 weeks of age), but are only attainable from the SEZ in adult mice. Dissociated SEZ tissue forms a monolayer of astrocytes

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15 after culture onto an adhesive substrate in the absence of EGF/bFGF; and a subpopulation of these astrocytes are believed to be MASC. The theory that the NSC is a form of astrocyte supports the belief that the MASC is an in vitro manifestation of the NSC (27). Furthermore, MASC are capable of generating NS upon culture in non-adhesive conditions in the presence of EGF and bFGF, with resulting NS capable of serial expansion and multi-lineage differentiation. Potential Identifying Markers of the NSC The HSC population can be enriched from the bone marrow cell population using a series of cell-surface markers, and recent reports indicate that similar protocols can be utilized to enrich the cell population of the SEZ for NSC. As described earlier, the SEZ NSC is potentially a form of astrocyte, and because GFAP is expressed by astrocytes GFAP has been proposed for use as one marker for the isolation of NSC (35). The inter-filament protein Nestin is expressed by developing neuroepithelium, and is believed to be expressed on adult NSCs, hinting at the developmental immaturity of these cells (63, 66). Lewis-X is a carbohydrate moiety expressed on the cell surface of embryonic stem cells, germinal zones in the developing mouse brain, and on some astrocytes; and was recently reported to be present on the surface of adult NS-forming SEZ cells (10). Finally, in the human model, purification of fetal brain using antibodies against CD133 (a marker purported to be on the surface of HSCs) greatly enriched for NS (80). While these protocols theoretically enrich for the presence of NSC (or at the very least, NS-forming cells), it remains to be seen if one or a combination of these markers will allow for the isolation of a pure population of NSCs. Furthermore, should these techniques make it possible for the in vitro enrichment of NSCs, protocols are needed to determine the in vivo potential possessed by the isolated and enriched NSC populations.

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16 Transplantation of In Vitro Expanded NS and MASC In addition to pluripotency and expansion, cultured NS and MASC also display the ability to survive transplantation back into the brain; an important ability, indicating that extended culture conditions have not adversely affected the cells. Integration capabilities are limited to the aforementioned regions of neurogenesis (glial cells are typically the only cell type observed following transplantation into non-neurogenic regions), and the age of the host animal is also critical. Neonatal mice (1-3 days post embryonic) are still undergoing active developmental neurogenesis and presumably offer an environment rich in extra-cellular cues for enhanced engraftment of transplanted NSC (26), while neurogenesis in the adult animal is dramatically lower and is limited to the SEZ and hippocampus. In vitro expanded hippocampal NSCs are capable of integrating into the dentate gyrus after transplantation (24), and also contribute to functional granule cells in the OB after transplantation into the SEZ (70). Labeled SEZ NS and MASC will survive transplantation to the SEZ, RMS or lateral ventricles, with donor-derived migratory neuroblasts present in these regions weeks following transplantation (80, 89). While the presence of donor-derived neurons and neuroblasts in the OB indicates engraftment, the degree of functionality is difficult to determine. The transplanted cells may appear to generate progeny similar to the surrounding cells, yet they do not always express the appropriate cell-surface markers. Furthermore, the engraftment is typically only manifested by production of cells of the neuronal lineage as opposed to the multi-lineage engraftment observed in HSC transplant studies. This would argue that a progenitor cell was transplanted rather than a NSC, although the limited neurogenesis in the SEZ could explain the mono-lineage nature of the newly generated daughter cells. It

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17 is also unknown if the transplanted cells truly engraft into the SEZ in a stable fashion and produce daughter progenitor cells that become evident in the OB as interneurons, or if the transplanted cells immediately enter into the RMS as neuroblasts and migrate to the OB (again a phenomenon more consistent with progenitor-cell activity). Adult NS and MASC: True Stem Cells? In the beginning of this chapter, four properties were listed as belonging to true stem cells: multipotency, asymmetric division, functional long term engraftment and serial transplantation. While it is believed that the endogenous NSC in vivo possesses these characteristics, little has been done to determine if candidate NSC or their in vitro manifestations (the NS and MASC), can be defined as true stem cells. Of the four properties, only multipotency has been conclusively shown to be a property of NS and MASC, although in vivo studies have indicated that their potential is limited to interneurons, granule neurons, and neuroblasts. While both NS and MASC are able to expand following in vitro passages, this ability is often limited to only four to six passages, though this may be due to deficiencies in the culture conditions rather than a deficiency on the part of either cell type. While donor derived cells are observed in host RMS and OB following transplantation of NS and MASC, their presence is transitory, often ceasing to exist a few months following transplantation. This is indicative of short-term engraftment of the transplanted cells, rather than long-term (assuming true engraftment has even occurred). To date, no data exists to determine if the observed donor-derived cells are the product of engrafted NSCs in the SEZ, or if the transplanted cells adopted the morphology of migratory neuroblasts immediately following transplantation.

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18 Finally, serial transplantability is a condition yet to be met by either the NS or the MASC, shedding doubt on the level of stem-ness possessed by either of these cell types. There is a distinct possibility that the NS and MASC are in reality neural progenitor cells, as they do not seem to possess all of the characteristics of a true stem cell. Effects of Radiation on Adult Neurogenesis In humans, whole body irradiation prior to bone marrow transplantation is a requirement for the treatment of many forms of leukemia to enhance the engraftment of the HSC following bone marrow transplantation, and recent investigations have focused on the effects this exposure may have on neurogenesis, particularly on the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). Throughout the lifespan of the mouse newly generated neuroblasts generated from subgranular NSC migrate into the granule cell layer and differentiate into functional granule neurons (9). These neurons functionally integrate into the DG, potentially playing a role in synaptic plasticity and learning. This region of neurogenesis appears to be extremely sensitive to x-irradiation in a dose dependent manner, with the numbers of neuroblasts and granule neurons significantly depleted by as little as 2Gy of exposure, with little to no recovery observed months after exposure (50, 59, 71). The death of these cells is reported to be apoptotic (60) and appears to be linked to an inflammatory response produced by the damaged tissue, as anti-inflammatory agents injected into the animals before and after irradiation protected the SGZ from cell loss (52). Exposure to high levels (10Gy) of focused x-rays results in near total, permanent ablation of mitotic cells in the SGZ and DG, with pronounced changes in both the vascular microenvironment and cell types as cells with a glial morphology become predominant following exposure (51).

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19 Relatively few studies have been performed to determine the effects radiation exposure may have on SEZ neurogenesis. Studies performed only in the rat model have shown a similar result to that observed in the SGZ and DG in the mouse. Mimicking the results observed in the hippocampus, radiation exposure adversely effected neurogenesis in the SEZ of rats in a dose dependent manner, with little to no recovery observed months following irradiation (72). Interestingly, low levels of focused x-irradiation (1-3Gy) resulted in an increase of Nestin positive cells in the SEZ in the weeks immediately following exposure, with levels eventually returning to normal (6). Researchers have hypothesized that the depletion observed in both the SGZ and SEZ is due in part to the elimination of NSCs and/or NPCs known to reside in these tissues. 10Gy of x-irradiation not only permanently depletes the SGZ of mitotic neuroblasts, but also negatively influences the microenvironment with surviving cells adopting a glial morphology. This has also been shown to render the niche unreceptive to transplanted non-irradiated NPCs, with a significant decrease in the number of transplanted NPCs adopting a neuronal morphology (51). It is currently believed that the brain is a relatively radio-sensitive organ compared to the bone marrow, as low doses of radiation only transiently lower the numbers of HSCs and their resultant progeny while similar doses permanently diminish neurogenesis in both the SEZ and SGZ. Summary While it is generally accepted that NSC driven neurogenesis persists in the adult mammal in the SEZ and SGZ, the exact identity of the NSC remains a mystery. Candidate NSCs have been extracted from SEZ and SGZ based upon expression of cell surface markers, and these cells can be cultured in the presence of EGF and bFGF to

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20 generate NS and MASC. Subsequent transplantation of NS and MASC back into regions of neurogenesis results in minimal engraftment by the donor cells with donor-derived progeny existing as neuroblasts and/or granule neurons in the host tissue, but it is not known whether the presence of donor-derived cells indicates functional engraftment or short-term contribution to the surrounding tissue. Exposure to radiation adversely affects neurogenesis in the adult rodent in a dose-dependent manner, indicating that the brain is a more radio-sensitive structure than is the bone marrow. Of the four criteria held by true stem cells (multipotency, asymmetric division, functional long-term engraftment, and serial transplantation) the in vitro manifestations of the NSC fulfill only one: multipotency. Protocols are needed to not only examine the ability of the NS and MASC to demonstrate robust, long-term engraftment, but also to ascertain their ability to survive serial transplantation before any claims can be made as to the levels of stem-ness possessed by these relatively uncharacterized cell types.

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CHAPTER 2 MATERIALS AND METHODS Reagents Avertin Mouse Anesthetic Solution Materials: Avertin (2-2-2 Tribromoethanol, Aldrich, St. Louis, MO; T4, 840-2), tert-amyl alcohol, 50 mL sterile polystyrene conical tube, 50 mL Steriflip disposable vacuum filtration system (0.22 m pore, Millipore, Billerica, MA; SCGP00525), phosphate buffered saline, aluminum foil, stir bar/plate. Protocol: Stock solution: Using the bottle in which the avertin was packaged, added 15.5 mL tert-amyl alcohol and magnetic stirrer and allowed to stir overnight. NOTE: As avertin is toxic if allowed to photo-oxidize, the stock bottle was kept tightly capped and wrapped in aluminum foil. Stable at room temperature for well over one year. Working solution (20 mg/mL): Combined 0.6 mL of the avertin stock and 39.4 mL of PBS in 50 mL conical. Agitated briefly to mix the two components, then filtered through the Steri-flip and wrapped the filtered solution in foil to exclude all light. Working solution is stable at 4C for several months. 5-bromo-2-deoxy-uridine (BrdU) Solution Materials: 5-bromo-2-deoxy-uridine (Roche, Florence, SC; 280-879), appropriate size sterile polystyrene conical tube, phosphate buffered saline, 37C water bath. 21

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22 Protocol: Calculated out the volume of BrdU required for all planned injections and measured out enough BrdU so that the final concentration was 10mg/mL. NOTE: BrdU is extremely mutagenic. Be sure that the powder is handled carefully. Combined the BrdU and PBS in the conical tube and warmed in the water bath for 15 minutes. Vigorous agitation is essential to ensure that all of the BrdU has entered into suspension. Made fresh for each experiment. Epidermal Growth Factor (EGF) Stock Solution (1000x) Materials: Recombinant Human Epidermal Growth Factor (R & D Systems, Minneapolis, MN; 236-EG, 200 g), Glacial Acetic Acid, Bovine Serum Albumin Fraction V heat shock (Roche Diagnostics, 03 116 999 001), 5 cc syringe w/ regular luer tip (Becton Dickinson, Franklin Lakes, NJ; 309603), Blunt Needle w/ Aluminum Hub (Monoject, Becton Dickinson, 202314), Sterile Syringe Filter, 0.45 m pore size (Corning Incorporated, Corning, NY; 31220), 15 mL and 50 mL polystyrene sterile conical tubes, 1.5 mL sterile micro centrifuge tubes, sterile 200 l pipette tips, distilled water, ice and ice bucket. Protocol: Generation of EGF suspension solution: 17.24 l of Acetic Acid was mixed with 30 mL of distilled water in a 50 mL conical tube to generate a final concentration of 10 mM. 0.030 g of BSA was added to solution (final concentration, 0.1% w/v) and mixed vigorously to re-suspend. Working on ice in a sterile laminar flow hood, 200 g of EGF was re-suspended in 10 mL of suspension solution in a sterile fashion then filter sterilized using the 5 mL syringe with blunt needle and syringe filter. Evenly distributed 500 L aliquots of the EGF solution and stored at 80C. Final concentration 20 ng/l, used at 1:1000.

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23 Basic Fibroblast Growth Factor (bFGF) Stock Solution (1000x) Materials: Recombinant-human-FGF (25g, R&D systems, 233-FB), Phosphate Buffered Saline, Bovine Serum Albumin Fraction V heat shock (Roche Diagnostics, 03 116 999 001), 1M Dithioerythritol (DTT) (Sigma, St. Louis, MO; D-8255), 5 cc syringe w/ regular luer tip (Becton Dickinson, 309603), Blunt Needle w/ Aluminum Hub (Monoject, 202314), Sterile Syringe Filter, 0.45 m pore size (Corning Incorporated, 431220), 15 mL polystyrene sterile conical tube, 1.5 mL sterile micro centrifuge tubes, sterile 200 l pipette tips, distilled water, ice and ice bucket. Protocol: Generation of bFGF suspension solution: 3 mL of PBS, 0.003 g BSA (0.1%) and 3 L of 1M DTT (final concentration of 1mM) were combined and mixed.. Working on ice in a sterilized laminar flow hood, 25g bFGF was combined in 2.5mL of suspension solution and filtered sterilized. Aliquots of 100 L were stored at -80C. Final concentration 10 ng/L, used at 1:1000. Immunocytochemistry Hybridization Buffer Materials: Fetal bovine serum, Phosphate Buffered Saline (PBS), Triton X-100 (Sigma, T-9284), 50 mL sterile polypropylene tube. Protocol: In sterile laminar flow hood, pipetted 5% of the final volume of FBS into 50 mL tube. Out of the hood, filled to full volume with PBS and pipetted 0.01% Triton X-100. Mixed and stored at 4C. Stable for 1 week. Neural Stem Cell (NSC) Culture Media Materials: DMEM/F12 w/ HEPES and L-Glutamine (Gibco BRL, Carlsbad, CA; 11330-032), fetal bovine serum (FBS), N2 supplement 100X (Gibco BRL, 17502-048), GlutaMAX-1 supplement 100x (Gibco BRL, 35050-061). Endothelial Growth Factor

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24 stock supplement (1000x), Fibroblast Growth Factor-beta stock supplement (1000x), 50 mL sterile polystyrene conical tube, 50 mL Steriflip disposable vacuum filtration system (0.22 m pore, Millipore, SCGP00525). Protocol: Based on the total volume required, the appropriate volumes of the above serums and supplements were added to DMEM/F12 to their working concentration and filter sterilized prior to use. 4% Paraformaldehyde (100mL) Materials: Paraformaldehyde (Sigma P-6148), 250 mL glass beaker, distilled water, 1N sodium hydroxide (NaOH), 1N hydrochloric acid (HCl), 3x PBS, glass Pasteur pipettes, aluminum foil, 100+mLglass bottle, filter paper, small funnel, stir bar/magnetic stirrer hot plate, pH meter. Protocol: 4 g (4% w/v) paraformaldehyde was mixed with 60 mL of distilled water heated to approximately 55C in a glass beaker and covered with aluminum foil while mixing for 10 minutes. Two drops of 1N NaOH was added to the solution followed by an additional 5 minutes of mixing, after which the solution became semi-clear. 30 mL of 3x PBS was added to the solution and the pH adjusted to 7.2 using 1N HCl and 1N NaOH. The solution was then filled to 100mL with distilled water, filtered through Whatman paper into clean glass bottle and stored at 4C. Solution is stable for approximately 1 week. Puromycin Stock Solution (1 mg/mL) Materials: Puromycin dihydrochloride from Streptomyces alboniger (Sigma P-8833), 5 cc syringe w/ regular luer tip (Becton Dickinson, 309603), Blunt Needle w/ Aluminum Hub (Monoject, 202314). Sterile Syringe Filter, 0.45 m pore size (Corning

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25 Incorporated, 431220), DMEM/F12 w/ HEPES and L-Glutamine (Gibco BRL, 11330-032), sterile polypropylene 1.5 mL tubes. Protocol: Working in a sterile laminar flow hood, 1 mg of puromycin was added to 1 mL of sterile DMEM/F12 in 1.5 mL sterile tube and agitated to dissolve the puromycin powder. Re-suspended solution was filter sterilized and stored at -20C. Antibody List Primary Antibodies Monoclonal anti--III tubulin: Promega, Madison, WI; G712A, dilution 1:1000 Polyclonal anti--III tubulin: Covance; Princeton, NJ; PRB-435P, dilution 1:5000 Monoclonal anti-polysialic acid neural cell adhesion molecule [PSA-NCAM]: Chemicon, Temecula, CA; MAB5324, dilution 1:100 Monoclonal anti-BrdU: Human Hybridoma Bank, Gainesville, FL; dilution 1:30 Polyclonal anti-glial fibrillary acidic protein [GFAP]: Shandon Immunon, Milford MA; 490740, dilution 1:100) Secondary Antibodies Rhodamine red-X, goat anti-mouse IgG: Molecular Probes, Eugene, OR; R-6393, dilution 1:500 Rhodamine red-X, goat anti-rabbit IgG: Molecular Probes, R-6394, dilution 1:500 Fluoroscein anti-rabbit IgG: Vector Labs, Burlingame, CA; FI-1000, dilution 1:500 Oregon green 514 goat anti-mouse IgG: Molecular Probes, O-6383, dilution 1:500 Methods Cell Culture Isolation and culture of neurospheres Neurosphere cultures were generated from WT, LI, and SLI animals, as described (42). Briefly, animals were anesthetized with isofluorane, cervically dislocated and decapitated. The brain was exposed, surgically removed, and then placed on an ice-cold sterile dissection board. A rectangular forebrain block containing the SEZ was obtained

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26 by removing the olfactory bulb, cerebellum, hippocampus, lateral portions of the striatum and lateral and dorsal cerebral cortex. The block was minced with a sterile scalpel, and placed in ice-cold PBS containing anti-biotic and anti-mycotic agents (Penicillin-Streptomycin, Gibco, 15140-122, and Fungizone Antimycotic, Gibco, 15295-017) for 10 minutes. Minced tissue was then centrifuged for 5 minutes at 1100 rpm at 4C, re-suspended in 3 mL 0.25% Trypsin plus EDTA (Gibco, 25200-056), then incubated at 37C for 5 minutes. After neutralizing the trypsin by the addition of 1 mL of fetal bovine serum, the tissue was triturated into a single cell suspension by pipetting through a series of descending diameter, fire-polished Pasteur pipettes. The cells were washed in DMEM/F-12 (Gibco, 11330-032) at 1100 rpm for 5 minutes at 4C, and re-suspended in neural growth medium. The cells were plated out in non-adhesive 6-well plates (Corning Costar, 3471) at a density of 1000 cells/cm 2 Cultures were supplemented with EGF and bFGF (20 ng/mL and 10 ng/mL, respectively), every second day. Isolation and culture of multipotent astrocytic stem cells Primary SEZ tissue was isolated from neonatal mice transgenic for green fluorescent protein (gfp+) at post-embryonic day 2 and dissociated to a single cell suspension in the same manner as listed above. Cells were plated onto tissue culture flasks at high density in neural growth medium devoid of EGF and bFGF. 3 days following the initial plating, non-adherent cells were removed and fresh media was applied. Cultures were passaged once the resulting astrocytes had formed a confluent monolayer, and cultures were deemed suitable for transplantation once they had undergone three passages (necessary for removal of contaminating neurons in the cultures).

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27 Selection of gfp+ multipotent astrocytic stem cells by puromycin selection MASC cultures derived from the SEZ of gfp and C57BL/6 neonatal and adult mice were prepared as described above. Once a confluent monolayer had been established, cells were passaged at 25% confluency with puromycin stock solution added to the neural growth medium at a final concentration of 1-3 g/mL. Cells were exposed to puromycin in the neural growth medium for nine days, with media replaced with fresh neural growth medium plus puromycin on the fifth day. Cells were analyzed after removal of puromycin by phase contrast microscopy. In order for surviving cells to generate a secondary confluent monolayer, cells were collected by trypsinization and re-plated at the highest possible density in puromycin-free neural growth medium. Transplantation and Analysis Transplantation of gfp+ MASC and neurospheres into the lateral ventricles of adult C57BL/6 mice Passage three gfp+ MASC or neurospheres were collected via trypsinization and re-suspended in 1mL of growth medium (see above). Once cell number was calculated, cells were re-suspended in a volume of growth media yielding 50,000 cells per L. Recipient mice were anesthetized with Avertin (see above) and the scalp surgically exposed. 100,000 cells (2 L) were stereotaxically injected into the lateral ventricle via a 5L Hamilton syringe attached to a 28 gauge needle at the following coordinates: A-P: -0.2, M-L: -1.2, H-D: -2.5. Transplanted animals were allowed to recover and placed back in general housing. Transplantation of gfp+ MASC and neurospheres into the lateral ventricles of neonatal C57BL/6 mice Passage three gfp+ MASC or neurospheres were collected via trypsinization and re-suspended in 1 mL of growth medium (see above). Once the cell number was

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28 calculated, cells were re-suspended in a volume of growth media yielding 100,000 cells per l. Recipient C57BL/6 neonatal mice (day post embryonic 1-3) were anesthetized by placement at -C for 5 minutes. Cells were transplanted in a volume of 1 L via a 5 L Hamilton syringe attached to a 28 gauge needle into the lateral ventricle using the bregma skull suture as a reference point. Following transplantation, neonatal mice were warmed to consciousness and returned to the mothers cage prior to return to general housing. Tissue immunohistochemistry Both wild type (WT), LI and SLI animals were given a lethal dose of the anesthetic Avertin before being perfused through the left ventricle with 4% paraformaldehyde (PFA) in PBS. Following perfusion, the brain was removed and post-fixed overnight by immersion in 4% PFA at 4C. Fixed brains were then serially sectioned through either the coronal or sagittal plane at 40 m using a Leica vibratome (model VT-1000-S) equipped with a sapphire blade. Tissue was prepared for immunohistochemistry by blocking at room temperature for 1 hour in PBS containing 10% fetal bovine serum and 0.01% Triton X-100. Primary antibodies were applied to the sections overnight with moderate agitation at 4C. Residual primary antibody was removed by three 5 minute washes (PBS plus 0.01% Triton X-100), and secondary antibodies were applied at room temperature for 50 minutes. Finally, sections were washed in PBS three times for 5 minute, mounted on positively charged glass slides (Fisherbrand Superfrost/Plus, 12-550-15), and allowed to dry for 15 minutes at 37C, before being cover-slipped in Vectashield (Vector Labs, H-1000) mounting medium. Sections were analyzed and photographed by fluorescence microscopy using either a Zeiss Axioplan 2 upright microscope (Carl Zeiss

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29 Inc, Thornwood, NY), Leica DMLB (Leica Microsystems AG, Wetzlar, Germany), or a Leica TCS SP2 AOBS spectral confocal microscope Analysis of engrafted gfp+ MASC and neurospheres into the brains of C57BL/6 mice Three or four weeks following transplantation, brains of transplanted animals were prepared for tissue immunohistochemistry as described above. The transplanted hemisphere was sectioned to through the sagittal plane (40 m thick) with every section containing the olfactory bulb collected in a serial fashion for subsequent analysis. Tissue sections were exposed to antibodies against -III tubulin as described above and mounted onto slides using the serial sectioning for visual reconstruction of the brain. All sections were analyzed at 20x magnification for the presence of gfp+ cells in the SEZ, RMS, and OB. Analysis of engrafted gfp+ MASC into the olfactory bulb of C57BL/6 mice Three weeks following transplantation, brains of transplanted animals were prepared for tissue immunohistochemistry as described above. For each brain, the transplanted hemisphere was sectioned through the sagittal plane (40m thick), with every section containing the olfactory bulb collected for analysis. Resulting sections were exposed to antibodies against -III tubulin as described above and mounted for analysis. Gfp+ migratory cells present in the olfactory bulb proper (that is, the region rostral of the descending horn of the rostral migratory stream) were scored as engrafted, with every section analyzed at 20x magnification for each animal. Immunohistochemical analysis of neurospheres and MASC NS were picked from their cultures using a handheld pipetter set at 2l and placed in DMEM/F-12 plus 5% FBS atop a laminin/poly-D-lysine coated, chambered culture

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30 slide (Becton/Dickinson, 352688). Spheres or aliquots of MASC were allowed to attach and differentiate for 2 days, at which time the media was removed and the cells were fixed by incubation in 4% PFA in PBS at room temperature for 30 minutes. After fixation, the cells were processed for immunolabeling with antibodies against -III tubulin and GFAP, as above. Radiation Studies Irradiation and bone marrow reconstitution Animals were placed in individual chambers of a plexi-glass container for irradiation. Lethal irradiation (LI) was induced by exposure to a Cs 137 source in a Gamma Cell 40 irradiator until 850 Rads had been obtained. This amount of radiation is sufficient to deplete the bone marrow of viable cells, while not inducing immediate death. Immediately following irradiation, LI mice were anesthetized with isoflurane (Aerrane, Baxter Deerfield, IL; NDC 10019-773-40) and a rescue dose of 1 x 10 6 whole bone marrow (WBM) cells was administered to each mouse via retro-orbital sinus (ROS) injection. WBM was isolated from the femurs of a sacrificed litter mate (anesthetized by exposure to isoflurane then sacrificed by cervical dislocation). Isolated WBM was then washed in 10 mL of phosphate buffered saline (PBS), and re-suspended in an appropriate volume of PBS after quantifying with a hemacytometer. A 32 gauge needle attached to a 1 mL insulin syringe was inserted into the ROS and the WBM was injected in a volume of 150 L. Animals were allowed to recover before being returned to conventional animal housing. For sublethal irradiation (SLI) studies, the animals were exposed to 450 Rads of ionizing radiation in the same fashion as listed above. No rescue dose of WBM is

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31 required for survival of the animal at this exposure, and animals were immediately returned to conventional housing. Identification of proliferative cells by BrdU labeling Three days, three weeks or 3 months following irradiation, WT, LI and SLI mice received 5-bromo-2-deoxyuridine (BrdU, Sigma, B-5002) three times a day for three days via intra-peritoneal (IP) injection (3 g/300 L per injection). The brains were fixed, removed, and sectioned as above. Sections were prepared for BrdU immunohistochemistry by first incubating in 2XSSC/Formamide solution (1:1) for 2 hours at 65C. After washing in 2xSSC for 5 minutes at room temperature, sections were then incubated in 2 N HCl for 30 minutes at 37C. Sections were washed in 0.1 M borate buffer for 10 minutes at room temperature prior to processing for immunolabeling with monoclonal anti-BrdU and polyclonal anti--III tubulin antibodies, as described above. Serial coronal sections were analyzed for BrdU positive cells in the SEZ using a blind study format (sections coded and scored by separate investigators). The region of the SEZ analyzed encompassed an area extending from the inferior tip of the lateral ventricle, superiorly along the wall of the lateral ventricle (approximately 5 cell bodies deep). This area continued to a point extending 700 m from the dorsolateral corner of the lateral ventricle. The defined region was carefully analyzed at a magnification of 40X, and at both focal planes of the tissue. To maintain consistency between sections, BrdU labeled cells were scored as positive, regardless of the intensity of the antibody fluorescence. Three adjacent sections per tissue were scored, using the location of the anterior commissure as a consistent landmark between sections. The cell numbers were collected,

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32 averaged, and placed into a graphical format using a Microsoft Excel spreadsheet. Statistical significance of values was determined by paired students t-test analysis with p-values less than 0.05 deemed to be significant. Blind analysis of the effects of lethal and sublethal irradiation on neurosphere yield In order to determine the effects of irradiation on NS generation, a blind paradigm was utilized to enable unbiased preparation and examination of the cultures from three WT and three LI mice (two months following lethal irradiation, age matched) and from four WT and 4 SLI mice (3 weeks following sub-lethal irradiation, age matched). Briefly, the animals were sacrificed and their brains removed by investigator A, who gave each brain an identifying number (1 through 8). The brains were then given to investigator B who removed the SEZ (as described above) from each brain in an identical fashion. The isolated SEZ tissue was then re-coded with a letter (A through H) by investigator C, who remained the only individual to know both the letter and number code. The tissue was then returned to investigator A for culture (as described above) and quantification. At 21 days in vitro, NS were collected, pelleted, and re-suspended in 2 mL of media. To determine NS yield, four 50 l aliquots from each culture were placed in a 12-well tissue culture plate. The aliquots were analyzed with a Nikon inverted phase microscope at both 4x and 10x magnifications. NS diameter was determined by use of the SPOT program (Diagnostic Instruments, Sterling Heights, MI). NS below 40 m in diameter were excluded as a means to avoid potential confusion with hypertrophied cells. Additionally, spherical aggregates that did not display the NS criteria of a tight phase contrast-bright perimeter were not counted. The total number of spheres per

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33 aliquot was determined, and the total yield and percent yield of each culture was then calculated from these numbers. Statistical significance of values was determined by paired students t-test analysis with p-values less than 0.05 deemed to be significant. At the conclusion of the analysis, the code was broken and the identity of the cultures was revealed.

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CHAPTER 3 LACK OF EVIDENCE FOR THE CLASSIFICATION OF NEUROSPHERES AND MULTIPOTENT ASTROCYTIC STEM CELLS AS TRUE STEM CELLS Introduction Adult stem cells (ASCs) have been the subject of numerous studies in recent years, many of which have focused on the isolation and characterization of candidate stem cell populations. ASCs are represented in a variety of organs in the adult mouse. Cells with stem-like properties have been reported to exist in the bone marrow (7), skeletal muscle (67), liver (13), pancreas (90), intestinal lining (68), skin (74) and the brain (2, 3, 44) representing all three of the primordial germ layers: endoderm, ectoderm and mesoderm. Perhaps the most robust region of stem cell activity in the adult animal is the bone marrow, in which the hematopoietic stem cell (HSC) vigorously replenishes the myeloid and lymphoid lineages of the hematopoietic system for the life of the animal. As isolation of the HSC has not yet been definitively accomplished, the use of a combination of antibodies against specific cell surface markers has emerged as a technique to highly enrich the heterogeneous cell population of the bone marrow for the presence of HSCs. Bone marrow derived cells (BMDCs) negative for the terminally differentiated markers B220, CD11b, and Ter119 but positive for stem cell antigen (Sca.1) and the receptor for steel factor (C-Kit) yield a population of cells enriched for HSCs. Yet functional reconstitution of the bone marrow must be established for the isolated population of cells to be defined as a HSC, making the definition of the HSC reliant on a function rather than identity. 34

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35 A single highly enriched BMDC transplanted into the peripheral blood of a myeloablated mouse that displays the ability to home to the injured bone marrow, re-populate the hematopoietic system, and allow for stable, long-term hematopoiesis to persist for the life of the animal can be labeled an HSC. The duration of the contributed hematopoiesis is critical, as this ability is indicative of the transplanted cell undergoing asymmetric division, a phenomenon where the stem cell shifts between generating an exact copy of itself and a more lineage committed daughter progenitor cell. This results in the presence of more HSCs than initially transplanted, with only a small population of these cells active at any given time allowing for long-term hematopoietic contribution. This also allows for a vital requirement for the definition of a HSC to be satisfied: serial transplantation. Work by Krause et al has indicated that a single transplanted HSC can not only reconstitute the bone marrow of a myeloablated mouse long-term, but also be re-isolated and transplanted into a secondary myeloablated host and confer the same radio-protective properties as in the initial transplant (40). From the extensive investigations into the HSC, a gold-standard definition of a stem cell has emerged: an isolatable cell capable of asymmetric division, multi-lineage long-term engraftment or contribution to the native tissue, and serial transplantability. Candidate stem cell populations isolated from other adult organs would need to meet these same rigorous criteria in order to be classified as true adult stem cell rather than a progenitor cell. Neurogenesis in the adult mammalian brain is currently believed to be restricted to the subependymal zone (SEZ) (44, 47) and the sub-granule cell layer of the hippocampus (HC) (9, 25, 38). Both regions have been shown to produce migratory daughter cells for the life of the animal, yet the levels of neurogenesis appear to decrease with age. In vivo

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36 the embryonic NSC has been proposed to contribute to all of the cell types in the developing brain, but in the adult brain NSCs in both the dentate gyrus and SEZ produce only one cell type: migratory neuroblasts, neural progenitor cells (NPCs) that differentiate into granule neurons and periglomerular interneuons. SEZ neuoroblasts migrate to the olfactory bulb (OB) via a well-defined glial pathway called the rostral migratory stream (RMS). Migratory neuroblasts divide and differentiate during migration, eventually functionally integrating into the OB neural circuitry as granule and periglomerular interneurons (4, 45, 47, 78). Hippocampal NSCs from the subgranule cell layer generate migratory neuroblasts that migrate a short distance through the granule cell layer to the mitral cell layer of the dentate gyrus, where functional integration as granule interneruons occurs (38). A relative newcomer to the field of stem cell biology, the SEZ NSC has displayed the potential to be cultured in vitro as both a spherical aggregate of clonal cells known as a neurospheres (NS) (22, 29, 39, 42, 62-65, 83) and as a monolayer of multipotent astrocytic stem cells (MASC) (43). Both manifestations display the stem cell properties in vitro of multi-potency and serial expansion. Upon induction of differentiation, NS and MASC generated NS are capable of producing neurons, astrocytes, and oligodendrocytes. They are also capable of increasing in number following repeated passages, although this expansion is not unlimited. Transplantation of gfp+ NS or MASC into regions of active neurogenesis in the adult brain results in donor-derived neuroblasts and neurons, while transplantation into terminally differentiated regions typically results in donor-derived glia, with few instances of neuronal contribution (23, 26, 80, 89). In SEZ transplantation, engraftment

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37 is often defined by the presence of donor-derived migratory neurons and periglomerular interneurons in the OB of the host animal. Contrary to HSC engraftment, NSC engraftment is transient, with the presence of migratory neuroblasts lasting only a few months, and mono-lineage as only neuroblasts and their resulting neurons are evident post transplantation (24, 69). Currently the HSC has not been shown to retain all of the aforementioned characteristics of a true stem cell in vitro, a shortcoming attributed to an incomplete reconstruction of the bone marrow microenvironment by the selected culture conditions. The resulting cell type displays pluripotency and the ability to expand following passages yet lacks the capacity to reconstitute the bone marrow of a myeloablated mouse, this cell type has been deemed a hematopoietic progenitor cell (HPC) rather than a true HSC. While both NS and MASC display the stem cell characteristics of pluripotency, culture expansion and functional engraftment following transplantation into regions of proven neurogenesis, little has been done to investigate the potential of the NSC to survive serial transplantation or to exhibit long-term engraftment. The characteristics of the NSC with respect to the better-understood HSC were examined to determine the level of stem-ness exhibited by the NSC. Inability of Gfp+ Neural Stem Cells to Survive Re-isolation Following Transplantation into the Lateral Ventricles of C57BL/6 Mice Gfp+ Neurospheres are not Generated from the Brains of Adult C57BL/6 Mice Transplanted with Gfp+ Neurospheres Gfp+ NS cultured from day post-embryonic (dpe) 3 gfp+ neonates were dissociated and transplanted (20,000 cells in 2L) into the lateral ventricles (LV) of three month old C57BL/6 females (n = 15, three independent experiments of 5 animals each) and allowed to engraft for 3 weeks, at which time the host animals were sacrificed. For each series,

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38 two animals were perfused via intra-cardiac puncture with paraformaldehyde and prepared for tissue immunohistochemistry to determine levels of engraftment by the transplanted NS. SEZ tissue was isolated from the remaining 3 animals and cultured for the generation of NS, with resulting NS analyzed for the presence of gfp. While a majority of the transplants resulted in some level of engraftment (Figure 3-1), no gfp+ NS were evident in any of the cultures, with the NS population consisting only of spheres similar in appearance to age matched control C57BL/6 NS (Figure 3-2). Figure 3-1. Gfp+ neurosphere derived cells are evident in the brains of host C57BL/6 mice three weeks following transplantation. Photographic montage of 10x images from four month C57BL/6 female transplanted with gfp+ NS three weeks prior to analysis. Arrow depicts the presence of gfp+ cells in the neural tissue surrounding the lateral ventricle (V). Red = -III tubulin, green = gfp. OB = olfactory bulb, RMS = rostral migratory stream. Gfp+ Neurospheres are not Generated from the Brains of Neonatal C57BL/6 Mice Transplanted with Gfp+ Neurospheres As the yield of engraftment following transplantation of gfp+ NS into the LV of adult mice is variable and minimal, the assay design was modified to use neonatal C57BL/6 mice as the recipient animal in hopes that the higher levels of engraftment in the neonatal model would allow for recovery of the now engrafted, transplanted cells. As previously mentioned, neurogenesis in the neonatal (dpe 1-3) mouse brain is

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39 Figure 3-2. Gfp+ neurospheres are not capable of re-isolation following transplantation into the lateral ventricle of adult mice. NS were cultured from the forebrain of adult C57BL/6 mice transplanted with gfp+ NS 3 weeks prior. Resultant spheres were more similar in fluorescence to C57BL/6 control NS than gfp+ control NS derived from age-matched gfp mice. A) Gfp+ control NS derived from gfp mice. B) C57BL/6 control NS. C) NS isolated from C57BL/6 mice transplanted with gfp+ NS 3 weeks earlier. All images at 10x magnification. more active than observed in the adult, theoretically providing higher levels of extra-cellular cues for differentiation and engraftment of transplanted NSCs. Gfp+ NS cultured from dpe 1-3 neonatal mice were dissociated and transplanted (75,000 cells, 1L) into the LV of dpe 1-3 C57BL/6 mice (n = 9) and allowed to engraft for one month. Immunohistochemical analysis of three transplanted animals indicated engraftment levels higher than that seen in the previous adult transplants, with donor-derived cells present in the SEZ, RMS, and OB (Figure 3-3). The morphology of the donor-derived cells in the OB are similar to granule neurons, indicating that the transplanted NS cells differentiated from an immature stem cell to that of a terminally differentiated neuron, presumably via a migratory neuroblast intermediate. The remaining 6 transplanted animals were sacrificed and cultured for the generation of NS, with generated NS analyzed for the expression of gfp as compared to control NS isolated from both gfp and C57BL/6 mice. NS cultures from the forebrain of the remaining transplanted animals yielded no gfp+ NS (Figure 3-4) with resultant spheres more similar to C57BL/6 control NS than gfp+ NS.

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40 Figure 3-3. Gfp+ neurospheres display high levels of engraftment following transplantation into neonatal mice. Four weeks post-transplantation, donor-derived cells from gfp+ NS are present in the SEZ, RMS, and OB of C57BL/6 recipient mice. A) Fluorescent microscopic analysis reveals the presence of engrafted donor-derived cells at the site of injection in the lateral ventricle (photographic montage, 10x light microscope images). B) Donor-derived cells in the OB display extensive integration of processes into the surrounding neural architecture of the OB (20x confocal image). C) Donor-derived cells are present in the SEZ of recipient animals and adopt a migratory appearance (20x confocal image). OB = olfactory bulb, RMS = rostral migratory stream, V = lateral ventricle. All images: red = -III tubulin, green = gfp

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41 Figure 3-4. Gfp+ neurospheres are not capable of re-isolation following transplantation into the lateral ventricle of neonatal mice. NS were cultured from the forebrain of adult C57BL/6 mice transplanted with gfp+ NS 4 weeks prior. Resultant spheres were more similar in fluorescence to C57BL/6 control NS than gfp+ control NS derived from age-matched gfp mice. A) Gfp+ control NS derived from gfp mice. B) C57BL/6 control NS. C) NS isolated from C57BL/6 mice transplanted with gfp+ NS 4 weeks earlier. All images at 10x magnification. Gfp+ Multipotent Astrocytic Stem Cells are not Generated from the Brains of Neonatal C57BL/6 Mice Transplanted with Gfp+ Multipotent Astrocytic Stem Cells While NS cultures are an accepted in vitro correlate to the NSC, the protocol for the generation and dissociation of NS results in low yield and extended culture conditions. Conversely, the MASC is relatively simple to culture and generates large cell numbers in a comparatively short time without the presence of mitogens required for NS growth. To this end, gfp+ MASC were utilized as the transplanted NSC to determine if a cell type that produces a high yield in culture may reveal the presence of gfp+ cells following transplantation and re-isolation. MASC were cultured from the SEZ of dpe1-3 gfp neonatal mice, and passaged three times prior to transplantation. This step is necessary for the removal of any contaminating neuronal cells that may persist in the initial days in vitro but will die off in the absence of EGF and bFGF. Prior to transplantation, aliquots of prospective MASC cultures were plated onto adherent chamber slides and allowed to attach. Attached cells

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42 were later stained for the presence of -III tubulin positive neurons and GFAP positive astrocytes (Figure 3-5). Cultures devoid of neurons were collected for transplantation. Figure 3-5. Passage three gfp+ multipotent astrocytic stem cells contain astrocytes but are devoid of neurons. Following three passages in vitro, gfp+ MASC were plated onto adherent chamber slides and analyzed for the presence of -III tubulin positive neurons and GFAP positive astrocytes. Slides analyzed at 20x magnification revealed confluent monolayers of GFAP expressing gfp+ cells with the morphology of astrocytes that contained no observable neurons. A) Gfp+ MASCs (green) stained for -III tubulin (red). B) Gfp+ MASCs (green) stained for GFAP (red). All images at 20x magnification. C57BL/6 neonatal mice (dpe 1-3, n = 50, 7 independent experiments) were transplanted with passage-3 gfp+ MASC (100,000 cells per transplant in 1L) into the LV and allowed to engraft for three weeks. In each series of transplants, animals analyzed for engraftment exhibited levels similar to those seen in previous NS transplants (Figure 3-6). Donor derived cells were present in the SEZ, RMS and OB, with cells in the OB adopting a granule neuron morphology. MASC cultures derived from the forebrain of the remaining animals did not appear to express gfp as compared to controls (Figure 3-7). MASC Isolated from the Brains of C57BL/6 Neonatal Mice Transplanted with Gfp MASC do not Survive Puromycin Treatment. MASC cultures derived from transplanted mice may contain gfp MASC, albeit in extremely low amounts. In an effort to enrich the resulting cultures for the presence of

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43 Figure 3-6. Gfp+ multipotent astrocytic stem cells display high levels of engraftment following transplantation into neonatal mice. Three weeks post-transplantation, donor-derived cells from gfp+ MASC are present in the SEZ, RMS, and OB. A) Fluorescent microscopic analysis reveals the presence of engrafted donor-derived cells at the site of injection in the lateral ventricle (photographic montage, 10x light microscope images). B) Donor-derived cells in the OB display extensive integration of processes into the surrounding neural architecture of the OB (20x confocal image). C) Donor-derived cells are present in the SEZ of recipient animals and adopt a migratory appearance (20x confocal image). OB = olfactory bulb, RMS = rostral migratory stream, V = lateral ventricle. All images: red = -III tubulin, green = gfp gfp positive cells, MASC monolayers were cultured in the presence of puromycin. Puromycin was the selective agent utilized during the generation of the gfp transgenic animal (32), and all cells in the gfp animal theoretically express the puromycin resistance

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44 gene along with gfp. Indeed, MASC cultures isolated from the forebrains of gfp neonatal mice (dpe1-3) survived in the presence of 1, 2, and 3 g/mL concentrations of puromycin. MASC isolated from the forebrains of control C57BL/6 neonatal mice failed to survive at any of the three concentrations (Figure 3-8). MASC cultures isolated from the brains of 18 animals transplanted with gfp+ MASC three weeks earlier were cultured in the presence of 2g/mL puromycin for nine days. Surviving cells were returned to normal culture conditions to generate confluent monolayers after which they were transplanted into the lateral ventricles of neonatal C57BL/6 mice (dpe 1-3, 100,000 cells, n = 5). Analysis of the brains of transplanted animals revealed the presence of auto-fluorescent cellular material in the RMS and walls of the LV, but the noticeable absence of gfp+ cells in the SEZ/RMS/OB of recipient mice three weeks following transplantation into the LV (Figure 3-9). Figure 3-7. Gfp+ multipotent astrocytic stem cells are not present in the cultures of transplanted neonatal mice. MASC were cultured from the forebrain of C57BL/6 mice transplanted with gfp+ MASC 3 weeks prior (n = 50, 7 independent experiments). Resultant cells did not express gfp as compared to age matched gfp+ MASC control cultures. A) Gfp+ MASC control culture. B and C) MASC cultures derived from C57BL/6 mice transplanted with gfp+ MASC 3 weeks earlier. All images at 20x magnification. To ensure that any cells surviving exposure to puromycin were indeed resistant and not merely remnants of puromycin-negative cells, MASC isolated from primary transplanted animals (n = 8) were treated with 2 g/mL puromycin for nine days. Surviving cells were collected and re-cultured to generate a secondary monolayer of

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45 Figure 3-8. Qualitative assessment of the effects of puromycin on multipotent astrocytic stem cells cultures derived from C57BL/6 and gfp+ neonatal mice. Passage 2 MASC cultures were grown in the presence of puromycin for nine days at 1, 2 and 3 g/mL puromycin. The above 10x phase-contrast images reveal that while all three concentrations of puromycin are lethal to C57BL/6 MASC, gfp MASC proliferate regardless of the concentration of the selective agent. Figure 3-9. Transplantation of puromycin selected multipotent astrocytic stem cells isolated from C57BL/6 mice transplanted with gfp+ multipotent astrocytic stem cells exhibit no gfp donor cell engraftment. Three weeks following transplantation, recipient animals display no evidence of gfp+ cells. Auto-fluorescent cellular material is present in the RMS and LV of transplanted animals, indicative of lipofusion or apoptotic cellular debris. A) RMS of transplanted animal, 10x gfp image. B) 10x image of RMS depicted in A, red = -III tubulin. C) Merged image of A and B. D) LV of transplanted animal, 10x gfp+ image. E) 10x image of LV depicted in D, red = -III tubulin. F) Merged image of D and E.

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46 MASC, at which time the cells were again cultured in 2 g/mL puromycin for nine days. Of an estimated 1 x 10 6 cells exposed to a secondary puromycin treatment, only four cells remained. None of the four cells analyzed expressed gfp, and had a punctate, unhealthy morphology compared to age matched gfp MASC cultures (Figure 3-10), indicating that no gfp cells exist in the secondary cultures of animals transplanted with gfp+ MASC. Long Term Engraftment As previously mentioned, long-term engraftment is an essential quality possessed by the HSC and for the NSC to be classified as a true stem cell it must display a similar capability. Three neonatal C57BL/6 (dpe1-3) were sacrificed 14 months following transplantation of gfp+ NS into the lateral ventricle. Analysis of sagittal sections of these animals revealed the existence of donor-derived cells in the olfactory bulb, parenchyma, and SEZ. These donor-derived cells appeared as either granule cells with extensive processes or as cells with an astrocytic morphology (Figure 3-11). Of particular interest was the noticeable lack of migratory neuroblasts in the RMS of these animals. The absence of these cells would argue that long-term contribution to the migratory neuroblast population was not provided by the transplanted NS, with only transient contribution to the RMS and OB interneurons provided by the transplanted cells.

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47 Figure 3-10. Multipotent astrocytic stem cells isolated from the forebrains of mice transplanted with gfp+ multipotent astrocytic stem cells do not survive puromycin treatment. Following isolation and culture, MASC from transplanted forebrains were treated with 2g/mL puromycin twice for a duration of nine days per treatment. Surviving cells were not gfp positive as compared to gfp controls. A) Gfp control MASC, 10x phase contrast image. B) Gfp control MASC, 10x image, green = gfp. C) Cell which survived puromycin selection, 10x phase contrast image. D) Same cell depicted in C, gfp filter. E) Cell which survived puromycin selection, 10x phase contrast image. F) Same cell depicted in E, gfp filter.

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48 Figure 3-11. Donor-derived cells exist in the brains of gfp+ neurosphere transplanted animals 14 months after surgery. Dissociated gfp+ NS were transplanted into the lateral ventricles of neonatal C57BL/6 mice, and allowed to engraft for 14 months. Donor-derived gfp+ cells were present in the olfactory bulb as granule neurons with extensive processes and in the walls surrounding the lateral ventricles and subependymal zone as astrocytic cells. No donor-derived cells were observed in the rostral migratory stream of these animals. A) Sagittal photographic montage of C57BL/6 mouse transplanted with gfp+ NS 14 months earlier (10x images). B) 20x image of donor-derived cell in the SEZ. C) 20x image of donor-derived cell in the parenchyma surrounding the lateral ventricle. D) and E) 40x images of donor-derived cells in the ob adopting the morphology of granule neurons. All images: red = -III Tubulin, green = gfp. OB = olfactory bulb, RMS = rostral migratory stream, SEZ = subependymal zone, V = lateral ventricle.

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CHAPTER 4 IONIZING RADIATION ENHANCES THE ENGRAFTMENT OF TRANSPLANTED IN VITRO DERIVED NEURAL STEM CELLS Introduction Neurogenesis in the adult rodent is limited to two well-characterized regions of the brain: the subgranular layer of the hippocampal dentate gyrus, and the subependymal zone (SEZ) (9, 25, 27, 38). The former produces neurons that functionally integrate into the granular cell layer of the hippocampus. The SEZ produces neuroblasts in the walls of the lateral ventricles that migrate along a defined pathway, known as the rostral migratory stream (RMS), to the olfactory bulb (OB) where they differentiate and functionally integrate into the existing cytoarchitecture as granule or periglomerular interneurons (45, 46, 47, 78). These migrating neuroblasts have been well characterized, and are known to be immunopositive for both the pan-neuronal marker -III-tubulin, and the polysialylated neuronal cell adhesion molecule (PSA-NCAM), an antigen restricted mainly to cells in the brain that are undergoing active migration (15). The number of newly generated neurons produced daily in the adult mouse has been estimated at 30,000, leading many to conclude that a self-renewing stem cell must reside in the SEZ in order for this rate to be sustained for the life of the animal (5). The cell type in the SEZ believed to be the NSC has been identified by Doetsch and colleagues as a slowly dividing astrocyte known as the type B cell, that generates the migratory neuroblasts (type A cells) via a transit amplifying intermediate precursor (type C cell) (4). 49

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50 Neural stem cells can be isolated from the adult SEZ and cultured in vitro to form spherical clones known as neurospheres (NS) (22, 29, 39, 42, 62-65, 83), that are capable of producing the major cell types of the neural lineage (neurons, astrocytes, and oligodendrocytes) upon differentiation. They are also capable of self-renewal, allowing them to increase in number following repeated passages in culture while retaining their multipotency, and have also shown the ability to integrate into host neural tissue upon transplantation into the host brain (80). It is these traits that have led many to believe that the NS-forming cell is the in vitro correlate of the in vivo NSC. Multipotent astrocytic stem cells (MASC) isolated from the SEZ of neonatal mice have also been identified as an in vitro correlate of the NSC, displaying the potential to not only generate multipotent NS but also to integrate into host neural tissue upon transplantation in much the same way as the NS (43, 89). The relatively simple culture conditions required in maintaining the MASC in vitro offer an attractive alternative to the more complicated conditions required by the NS. Transplantation of in vitro NSC (NS or MASC) derived from animals transgenic for the reporter gene encoding green fluorescent protein (gfp+) into the LV of adult control animals results in minimal engraftment into the host neural tissue, with few donor-derived neuroblasts present in the RMS or OB. Transplantation into neonatal mice, however, results in relatively robust levels of engraftment with comparatively high numbers of donor-derived neuroblasts and interneurons present in the OB weeks after transplantation. This presents a problem for research into adult neurogenesis, as the neonatal and adult brains are dramatically different with respects to active neurogenesis and results obtained from neonatal transplants may not be applicable to adult studies. A

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51 method for enhancing the engraftment potential of in vitro NSC into adult animals is crucial in order to better understand the receptiveness of the adult SEZ to transplanted cells and consequently the functional identity of the transplanted cells. Perhaps the most well characterized adult stem cell is the hematopoietic stem cell, which in addition to the aforementioned stem cell characteristics of pluripotency and self-renewal also has the ability to repopulate the entire hematopoietic system of a myeloablated animal. Ablation of endogenous HSCs by exposure to high doses of ionizing radiation is critical for facilitating maximum engraftment of transplanted HSCs, that cannot normally compete with the native HSC for access to the stem cell niche (18, 19, 20). This reconstitution ability is an essential component in the generally accepted classic definition of a HSC. Depletion of the NSC niche has been previously achieved with anti-mitotic agents (15) and both ionizing and x-irradiation (6, 51, 59, 60, 71, 72) yielding transient and long-term depletion of neurogenesis in the hippocampus and SEZ. Neurogenesis in the hippocampus can be attenuated by exposure to varying levels of x-irradiation, as seen by a decrease in the number of migrating granular neurons out of the subgranular layer (52, 59, 60, 71). Previous studies investigating the effects of on SEZ neurogenesis following focused exposure of x-irradiation to the brain of adult rats have reported ablation of of mitotic cells in the SEZ immediately following irradiation, with a dose dependent recovery of these cells occurring within 2 months of exposure (6, 72). With the exception of all but the lowest level of exposure, the observed recovery of neurogenesis remained well below control levels. Little has been done, however, to characterize the

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52 effects of a single, whole-body lethal or sub-lethal dose of ionizing radiation on SEZ neurogenesis and subsequent engraftment of transplanted in vitro derived NSC in the adult mouse. Effects of Lethal Irradiation on Subependymal Zone Neurogenesis Lethal Irradiation Severely Depletes Migrating Neuroblasts in the RMS Three weeks following lethal irradiation (LI), bone marrow reconstituted mice show a marked decrease in the number of -III tubulin positive neuroblasts in the RMS (n = 8, Figure 4-1 D). This decrease is variable; some animals were found to have few or no migrating neuroblasts, while others contained a number of pockets of neuroblasts, though always drastically fewer than that seen in un-treated littermates (Figure 4-1 B). To determine if this reduction is permanent, (as opposed to the transient reduction observed with anti-mitotic treatments) animals were analyzed 3 months after LI. The results indicate that the depletion of migratory neuroblasts persists even at this time point (Figure 4-2). To show that the decrease in the number of -III tubulin positive neuroblasts is not due merely to antigen down-regulation, sagittal sections of brain from LI mice were also stained for the presence of PSA-NCAM; a marker specific to migrating neuroblasts (46). Three weeks following LI, the number of migrating PSA-NCAM positive neuroblasts was severely depleted in the RMS of those mice (Figure 4-1 C) as compared to un-treated littermates (Figure 4-1 A), again with a small portion of migratory neuroblasts remaining in the RMS (* in Figure 4-1 C). These findings led to the conclusion that the exposure to x-irradiation resulted in permanent damage to the neuroblast-producing cell of the SEZ, but due to the occasional observed cluster of migratory neuroblasts, accurate quantification of this depletion was performed.

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53 Figure 4-1. Lethal irradiation drastically reduces the volume of PSA-NCAM and -III Tubulin positive migratory neuroblasts. Sagittal photographic montages of 5x light microscopic images. A and B) Untreated C57BL/6 female brain. C and D) C57BL/6 female, 3 weeks post-lethal irradiation. Note the absence of -III Tubulin positive migratory neuroblasts in the LI brain (D) compared to control (B) and the diminished volume of PSA-NCAM positive migratory neuroblasts (C) as compared to control (A). Red = PSA-NCAM, Green = -III Tubulin. OB = olfactory bulb, RMS = rostral migratory stream, LV = lateral ventricle. (*) denote migratory neuroblasts. Figure 4-2. Reduction in the volume of -III Tubulin positive migratory neuroblasts persists months after lethal irradiation. Sagittal photographic montages of 10x light microscopic images. Red = -III Tubulin. A) C57BL/6 female brain two months post-LI. B) C57BL/6 female brain three months post-LI. Note the absence of migratory neuroblasts in both images as compared to non-irradiated controls (Figure 4-1, B).

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54 Analysis and Quantification of Neuroblast Depletion in the Subventricular Zone Following Lethal Irradiation, as Determined by BrdU Incorporation In order to quantify the degree of neuroblast depletion, 5-bromo-2-deoxyuridine (BrdU) was used to label mitotic cells within the SEZ. Briefly, 3 weeks and 3 months following LI (n = 4 for each condition), treated and un-treated mice were given BrdU intra-peritoneally three times a day for three days allowing for the incorporation of BrdU into the DNA of dividing cells. SEZ neuroblasts were analyzed by the application of antibodies against -III tubulin and BrdU on serial coronal sections at the level of the anterior commisure (AC). BrdU positive cells were scored blindly, by analyzing three adjacent sections per brain (scorer was ignorant to the experimental condition of the animals analyzed). Scoring of adjacent sections was performed only on those sections where the AC existed as a spherical white-matter structure, directly inferior to the inferior most extent of the LV. The region of analysis was maintained throughout all animals scored (Figure 4-3). In all un-treated animals, the SEZ was observed to contain a robust layer of dividing neuroblasts (Figure 4-4 A). 3 weeks following LI in age matched mice this same region exhibited a significant decrease in neuroblasts activity (Figure 4-4 B). This depletion was observed out to 3 months post-LI (Figure 4-4 C). -III tubulin immunolabeling (green, Figure 4-4 insets) was used to confirm that the cells scored were in fact neuroblasts and not mitotic glial cells of the SEZ. Figure 4-5 is a graphical representation of the number of BrdU positive cells for each condition (4 mice per condition, 3 sections per animal). 3-weeks following LI, the volume of BrdU positive SEZ neuroblasts decreases to 40% of control. Students t-test confirmed that this difference was significant (p value = 0.003).

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55 Figure 4-3. Region of analysis for quantification of mitotic neuroblast depletion in the subependymal zone of lethally irradiated mice. Coronal photographic montage of 10x light microscopic images, with the white outline depicting the region of analysis as encompassing an area from the inferior, descending horn of the LV to the superior horn of the LV, and continuing 700m lateral from the superior horn while remaining approximately 5 cells in width. Inset: reprentative image of BrdU positive neuroblasts lining the wall of the lateral ventricle expressing -III tubulin. CC = corpus callosum, LV = lateral ventricle, ST = striatum, SP = septum, and AC = anterior commissure. Red = BrdU, Green = -III tubulin.

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56 Figure 4-4. Depletion of mitotic neuroblasts in the subependymal zone of lethally irradiated animals. The observable volume of BrdU positive (red) neuroblasts is severely diminished three weeks and three months post-LI as compared to non-irradiated controls. A) Non-irradiated control. B) 3 weeks post-LI. C) 3 months post-LI. All are coronal photographic montages of 10x light microscope images. All insets are 40x images of neuroblasts lining the wall of the ventricles, positive for both BrdU and -III tubulin (green) identifying them as neuronal. There is an approximate 87% decrease in the number of BrdU positive cells at the 3-month time point as compared to control, and this difference is also significant (p value = 0.002). The 68% decrease in the number of BrdU positive cells between 3 weeks and 3 months post LI was also significant (p value = 0.03), indicating that the observed depletion is permanent. The depletion of mitotic cells in the SEZ is also observed in the RMS, with a marked decrease in the visible BrdU/-III tubulin positive migratory neuroblasts one month following LI (Figure 4-6). Diminished Neurosphere Yield from Lethally Irradiated Mice As it is well accepted that the NS forming cell is the in vitro manifestation of the NSC (63), NS from both WT and LI mice were cultured in a blind-study format to determine if the stem cell pool in the SEZ was affected by exposure to LI.

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57 Figure 4-5. Lethal irradiation significantly decreases the number of BrdU positive SEZ neuroblasts. The total number of BrdU positive neuroblasts in three adjacent coronal sections of either wild-type or LI mice was tabulated and placed into the above graphical format. Three weeks following lethal irradiation (n=4), the number of BrdU positive neuroblasts decreased to 40% of those calculated to exist in the wild-type controls (n = 4, p value of 0.003). Three months following lethal irradiation, this decrease is slightly greater, at 13% of control (n = 4, p value of 0.002). Analysis by students t-test confirmed these values to be significant, as compared to non-irradiated controls.

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58 Figure 4-6. Depletion of migratory neuroblasts in the rostral migratory stream positive for both BrdU and -III Tubulin in lethally irradiated mice. Sagittal montages of 20x images reveal that the visible volume of -III tubulin/BrdU positive cells is severely diminished in LI mice as compared to non-irradiated controls. A) Non-irradiated control. B) LI mouse, one month post-LI. Animals were injected with BrdU according to the protocol described in materials and methods. Brains were sectioned and stained for BrdU (red) and -III tubulin (green). Ob = olfactory bulb, rms = rostral migratory stream, V = lateral ventricle.

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59 NS cultured from LI brains displayed a significant decrease in yield of approximately 77%, when compared to the non-irradiated control cultures (Figure 4-7, p value = 0.02). This decrease closely corresponds to the decreased levels of BrdU positive neuroblasts in vivo following lethal irradiation, further supporting the validity of this finding. It has been reported that the stem cell population of the SEZ is between approximately 0.02% and 1.0% of the total cells (62, 66, 82), as determined by NS yield from dissociated SEZ tissue, and the average yield of NS from the wild-type brains in the present study falls within this range (0.15%). The average yield of NS isolated from the lethally irradiated brains was significantly lower, at 0.03%, indicating that exposure to lethal doses of radiation depletes the number of NSC in the brain responsible for the generation of NS. Preliminary studies revealed no effect on the potential for differentiation was observed in NS isolated from LI animals one month following irradiation as compared to non-irradiated controls. The approximate diameter of the resultant NS was comparable to controls (Figure 4-8), and when placed on adhesive substrates in the absence of growth factors to induce differentiation NS from LI mice produced both astrocytes and neurons. LI appears to only affect the yield of NS isolated from irradiated animals, with the few spheres formed retaining a normal phenotype, although further studies are needed to verify that irradiation in no way affects NS differentiation potential. Lethal Irradiation Attenuates Engraftment of Transplanted Gfp+ Multipotent Astrocytic Stem Cells Three weeks after irradiation, LI animals were transplanted with gfp+ MASC (generated from neonatal forebrains) into the lateral ventricle and the cells allowed to engraft for three weeks.

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60 Figure 4-7. Lethal irradiation significantly reduces the yield of neurospheres cultured from the adult subependymal zone. SEZ tissue isolated in a blind-study format was cultured to generate NS from both wild-type adult mice and adult mice LI two months prior, with all tissues treated in identical fashion (n=3). The resulting NS yield was determined according to the protocol described in the methods section. The LI cultures averaged 77% fewer NS than did the identical cultures derived from control mice (significant decrease; p value of 0.02). Figure 4-8. Neurosphere differentiation potential. NS isolated from non-irradiated control animals are capable of differentiation into astrocytes and neurons upon attachment to adhesive substrates in the absence of EGF and bFGF. A) 10x phase contrast image of NS derived from non-irradiated control. B) 20x image of differentiated control NS stained for GFAP (red). C) 40x image of differentiated control NS stained for -III tubulin (red) and DAPI (blue).

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61 Figure 4-9. Attenuation of gfp+ multipotent astrocytic stem cell engraftment by lethal irradiation. Control animals exhibited normal engraftment, as evidenced by the presence of donor-derived migratory neuroblasts. A) Photographic montage of 10x images from a sagittal section of non-irradiated animal two weeks post transplant (red = -III tubulin, green = gfp). B and C) 40x images of donor-derived migratory neuroblasts in non-irradiated control. LI animals displayed no evidence of engraftment, with no donor-derived cells seen in either the SEZ, RMS, or OB. D) Photographic montage of 10x images from a sagittal section of LI animal two weeks post transplant. E) 20x image of -III tubulin positive migratory neuroblasts in the RMS. F) Same image as E, but with gfp filter, revealing the absence of donor-derived migratory cells. OB = olfactory bulb, RMS = rostral migratory stream, V = ventricle.

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62 Analysis of 40 m sagittal sections revealed the presence of donor-derived cells only in the corpus callosum, parenchyma and SEZ, but the absence of donor-derived migratory cells in the RMS or OB (n = 5), while non-irradiated controls were observed to contain a small but consistent number of donor-derived migratory neuroblasts in the OB (Figure 4-9). As the lethal dose of ionizing radiation inhibited functional engraftment of the transplanted cells (possibly by inducing irreparable damage to the radio-sensitive support cells of the SEZ) a lower exposure dose of 450 rads was assayed as a milder form of injury for the potential enhancement of gfp+ MASC engraftment. Effects of Sub-Lethal Irradiation on Subependymal Zone Neurogenesis Sublethal Irradiation Results in a Transient Decrease in the Number of Mitotic Subependymal Zone Neuroblasts. Unlike LI, SLI (450 rads) does not complete abolish hematopoiesis in the bone marrow of adult mice, and SLI animals to not require transplantation of bone marrow to survive exposure. Low levels of focused gamma irradiation (1 to 3Gy) have been shown to result in a transient increase in mitotic cell activity in the SEZ, with levels eventually diminishing in the weeks following in a dose dependent fashion when compared to untreated controls (6, 72). Contrary to the observations following LI, no decrease was observed in the overall volume of -III tubulin positive migratory neuroblasts in the RMS of irradiated animals 3 weeks following whole body exposure to 450rads (a dose equivalent to 4.5Gy). BrdU quantification assays revealed that mitotic cell activity in the SEZ was significantly decreased in the hours immediately following irradiation (Figure 4-10). The levels of

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63 mitotic cell activity returned to near normal at 3 weeks and eventually increased to above control levels at 6 weeks (Figure 4-11). Diminished Neurosphere Yield from Sublethally Irradiated Mice Blind-analysis of NS yield (same protocol as the LI study, n = 4) from SLI brains at 3 weeks revealed a 30% decrease in the number of NS as compared to non-irradiated controls (Figure 4-12, p = 0.032), with control NS yield again falling into the aforementioned acceptable range of 0.2 to 1.0% (0.12%). As in the LI studies, no observable effect was observed in preliminary studies of NS potential in SLI mice, although further studies are needed to verify this initial observation. Sublethal Irradiation Enhances Engraftment of Transplanted Gfp+ Multipotent Astrocytic Stem Cells Three weeks following irradiation, SLI animals were transplanted with gfp+ MASC into the lateral ventricle and the cells were allowed to engraft for three weeks. Analysis of 40 m sagittal sections revealed that in a portion of the irradiated animals a 4 fold higher number of gfp+ migratory cells were present in the OB as compared to non-irradiated controls (n = 14, Figure 4-13). Taken as a whole, the average number of gfp+ migratory cells in the OB of SLI animals was twice the average number seen in non-irradiated controls (significant value, p = 0.014) indicating that SLI significantly enhances the engraftment potential of transplanted gfp+ MASC, albeit in a variable fashion (Figure 4-14).

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64 Figure 4-10. Mitotic subependymal zone neuroblasts are transiently depleted following sub-lethal irradiation. 6 hours following irradiation, the SEZ is almost completely devoid of BrdU (red) cells as compared to non-irradiated controls. 3 weeks following irradiation, BrdU positive cells in the SEZ are near to the levels observed in controls. A) Non-irradiated control. B) 6 hr post-SLI. C) 3 weeks post-SLI. All are coronal photographic montages of 10x light microscope images. All insets are 40x images of neuroblasts lining the wall of the ventricles, positive for both BrdU and -III tubulin (green) identifying them as neuronal.

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65 Figure 4-11. Sublethal irradiation results in transient, recoverable depletion of mitotic subependymal zone neuroblasts. The total number of BrdU positive neuroblasts in three adjacent coronal sections of either control or SLI mice was tabulated and placed into the above graphical format for quantification of the number of BrdU positive SEZ neuroblasts calculated as percent of control. Six hours following SLI there are approximately 83% fewer BrdU positive neuroblasts than in non-irradiated controls (p = 0.001, n = 3). This depletion persists 24 (78%, p = 0.001, n =3) and 48 hours (90%, p = 0.001, n = 3) following SLI. Three weeks following irradiation the number of mitotic SEZ neuroblasts recovers to near control levels, and eventually increases to 13% above control levels at 6 weeks (p = 0.003, n = 3).

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66 Figure 4-12. Sublethal irradiation significantly reduces the generation of neurospheres cultured from adult subependymal zone. SEZ tissue was isolated in a blind format from both non-irradiated control adult mice and adult mice SLI three weeks prior and cultured to generate NS, with all tissues treated in identical fashion. The resulting NS yield was determined according to the protocol described in the methods section. The SLI cultures averaged 30% fewer NS than did the identical cultures derived from control mice (significant decrease; p = 0.032, n = 4).

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67 Figure 4-13. Gfp+ multipotent astrocytic stem cells transplanted into the lateral ventricles of control and sub-lethally irradiated mice produce donor-derived migratory cells in the host olfactory bulb. Age matched mice (SLI and non-irradiated controls) were sacrificed three weeks following intra-ventricle transplantation of passage 3 gfp+ MASC. 40 m sagittal sections of the transplanted hemisphere were collected in a serial fashion, stained for -III tubulin (red) and analyzed by light and confocal microscopy for the presence of gfp+ donor-derived cells (green). A and D): 10x photographic montages of control and SLI mice (respectively). Circles represent the presence of donor-derived migratory neuroblasts, with representative cells captured at 40x (insets). B and C) 63x confocal images of gfp+ migratory cells in the olfactory bulb of non-irradiated control mice. E and F) 63x confocal images of gfp+ migratory cells in the olfactory bulb of SLI mice. Scale bars = 40m.

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68 Figure 4-14. Sublethal irradiation enhances engraftment of donor-derived neuroblasts. Quantification of 14 transplanted animals per condition revealed the presence of approximately twice as many donor-derived migratory neuroblasts in the olfactory bulb proper of SLI animals as compared to non-irradiated controls (p = 0.014). Transplanted hemispheres were sectioned three-weeks following transplantation of passage 3 gfp+ MASC into the lateral ventricle. All sections containing the olfactory bulb were collected in a serial fashion and stained for the presence of -III tubulin.

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CHAPTER 5 DISCUSSION AND CONCLUSIONS Neurogenesis in the subependymal zone (SEZ) of the adult mouse persists for the life of the animal, alluding to the existence of a persistent neural stem cell (NSC) pool in this region. While NSCs cannot yet be prospectively purified like the hematopoietic stem cell (HSC), they can be cultured under specific conditions to form clonal neurospheres (NS) and multipotent astrocytic stem cells (MASC); potential NSC manifestations with multipotent characteristics attributed to stem cells. The extensive investigation of the HSC has produced a gold-standard definition of a stem cell: a single cell that is capable of producing all cell types of a particular organ for the life of the animal via asymmetrical division in which both an exact duplicate of the stem cell and a lineage-committed progenitor daughter cell are generated. Additionally, the stem cell can reconstitute its native niche following transplantation, and this ability is retained following secondary transplantation, a phenomenon known as serial reconstitution. Functional transplantation of a stem cell and its subsequent reconstitution of the niche is a vital requirement, as the isolated cell can now be considered a useful tool for tissue repair. Candidate stem cells from other organs would need to meet the same criteria if they are to be classified as true stem cells. Both NS and MASC transgenic for gfp exhibited low levels of engraftment upon transplantation into the lateral ventricles of adult mice, with few donor derived migratory neuroblasts in the RMS observed in the weeks immediately following transplantation. Neither the NS nor the MASC displayed the ability to be re-isolated following 69

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70 transplantation into proven regions of neurogenesis, a limitation resulting in the inability of either cell types to survive subsequent serial transplantation. As neurological development continues shortly after birth and potentially provides an environment rich in extra cellular cues favored by NSCs, transplantation into the brains of neonatal mice was performed in an effort to enhance the engraftment of transplanted NS and MASC. While the levels of engraftment were visibly higher than those seen in adult transplants, neither cell type survived secondary isolation attempts, even with the use of selective protocols designed to enrich for gfp+ cells. Long-term analysis of transplanted animals revealed the existence of donor-derived granule neurons and astrocytic cells in the olfactory bulb, parenchyma and SEZ, but a noticeable absence of migratory neuroblasts in the rostral migratory stream (RMS) of the transplanted animals. This potentially alludes to transient engraftment as opposed to long-term engraftment. NSC engraftment could be defined in this case as long-term contribution to neuroblast pool in the adult animal following transplantation, with functionality exhibited by the existence of terminally differentiated donor-derived cells in the neural circuitry of the olfactory bulb. HSC engraftment into the bone marrow of myeloablated mice is evidenced by the robust generation of daughter cells representing both the myeloid and lymphoid lineages. The obvious evidence of functional engraftment by the transplanted HSC is that the myeloablated animal survives the previous lethal dose of radiation; the depleted bone marrow becomes repopulated by the transplanted HSC and its resultant progeny. Long-term (i.e. 3 months or longer) engraftment and hematopoietic contribution is a critical requirement in the definition of the HSC, as short-term, transient

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71 engraftment can be supplied by hematopoietic progenitor cells. The ability to survive serial-transplantation while retaining the capacity to provide long-term bone marrow reconstitution fulfills the final requirement for classification as a true stem cell. Currently, the liver hepatocyte is the only adult stem cell other than the HSC that has displayed the potential for serial, functional engraftment. In a liver repopulation assay, transplanted hepatocytes functionally contributed to the regenerating liver in a robust, serial fashion with contribution observed following the sixth transplantation (58). The results obtained reported here indicate that the in vitro derived NSCs were not capable of re-isolation following transplantation into proven regions of neurogenesis. Whether this is due to the cell population transplanted or the niche into which the cells were placed is not known. The failure observed in the adult model could be attributed to the decreased level of neurogenesis in the adult animal, resulting in fewer engrafted cells and subsequently fewer isolatable cells. However, because transplants into neonatal mice yielded the same results the cause likely lies in the cells transplanted rather than in the niche itself. Both NS and MASC cultures are heterogeneous in nature, with cells existing in varying stages of maturation. It is possible that the cell types observed to be multipotent and proliferative in culture are progenitor cells derived from a relatively small number of NSCs, implying that not every NS or MASC in culture is a NSC. Transplantation into the SEZ results in mono-potent engraftment with only neurons and neuroblasts observed in the RMS and olfactory bulb, an observation concurrent with progenitor cell activity. Stable engraftment by the NSC should result in an increase of donor-derived NSC via asymmetric division, allowing for subsequent re-isolation. While initial engraftment was observed in the weeks immediately following

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72 transplantation as evidenced by the presence of donor derived cells in the SEZ, RMS and olfactory bulb, the engraftment ultimately was found to be transitory. As it was not possible in the experiments performed to re-isolate a donor-derived NS or MASC, the data presented here would indicate that the in vitro manifestations of the NSC are not true stem cells, but rather a form of neural progenitor cell lacking the characteristics attributed to true stem cells and may not be a viable cell source for potential stem cell therapy of neurological injury and disease. It is entirely possible that the observed neurogenesis in the adult brain is not the result of an endogenous, isolatable NSC pool, but rather the product of migratory adult stem cells that undergo a phenotypic shift upon integration into neurogenic regions of the brain and subsequently give rise to more lineage committed neural progenitor cells. Observations supporting the contribution of the HSC to adult neurogenesis are as numerous as they are conflicting. Donor-derived microglia and astrocytes (17) and neurons (8, 49) have been reported to exist in the brains of animals exposed to lethal irradiation and reconstituted with HSC transgenic for reporter proteins (17), while a recent investigation has argued strongly for the existence of non-fusion product, donor-derived neurons in the hippocampus of humans following sex-mismatched bone marrow transplant (12). It has also been suggested that 0.5% of astrocytes in the adult brain are generated from a bone marrow derived cell (17), and it is generally accepted that the NSC of the SEZ (the type B cell) is a form of astrocyte (4, 5, 15). With this in mind, the proposal that adult neurogenesis is driven by an adult stem cell residing in the bone marrow may not be implausible.

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73 While the neonatal transplant model provides an ideal system for relatively robust engraftment of transplanted NS and MASC, it is impractical for the investigation of adult neurogenesis and adult NSC potential as the adult brain is more quiescent than the developing neonatal brain. A technique is necessary to render the neurogenic niche (in this case, the SEZ) more receptive to transplanted cells. The current tactic in the field of hematopoiesis to maximize engraftment of transplanted HSC is to first deplete the host bone marrow of HSC by exposure to lethal doses of radiation (myeloablation). This renders the hematopoietic stem cell niche more receptive to transplanted cells, allowing for stable long-term, multi-lineage engraftment. It was hypothesized that depletion of the native NSC pool by lethal irradiation would render the SEZ more receptive to transplanted cells and subsequently enhance the engraftment efficiency of the transplanted NSC. Because a single, high dose of ionizing radiation is sufficient to permanently deplete the bone marrow of viable HSC in adult mice, a similar result should be observed in the SEZ, with the pool of NSC being similarly depleted. The number of mitotic cells in the SEZ was decreased by approximately 60% three weeks following irradiation and this decrease was slightly lower three months later, at 87%, indicating a long-term, if not permanent depletion. The levels of migrating neuroblasts in the RMS reflected this decrease, with the chains decreasing in volume at both three week and three month time-points. If the NSC pool itself had been diminished by the radiation exposure, the number of NS cultured from those brains should reflect this decrease in the number of BrdU positive neuroblasts. In fact, it was observed that there were approximately 77% fewer

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74 NS isolated from the brains of LI adult mice that had been irradiated two months earlier. Preliminary observations indicated that only the yield of NS appeared to be affected, with the resulting spheres being similar in size and multipotency to control NS. In addition, the resulting cultures from control tissue supported earlier findings that the number of NSC in the SEZ is between 0.02 and 1.0% (62, 66, 82), with the average NS yield in cultures being approximately 0.15% of the SEZ tissue cultured. The percent yield of NS cultured from the SEZ of LI mice was diminished, at 0.03%. These observations lead to the conclusion that the SEZ is significantly depleted of NSC following LI, and that this depletion is long-term, if not permanent. The reason for this permanent depletion of neurogenesis is not entirely clear, although recent studies have indicated that the neuro-inflammatory response to injury in the hippocampus inhibits neurogenesis by disruption of normal stem cell function (52). While the most prevalent in vitro manifestation of the NSC is the NS, the MASC has recently emerged as an alternative to the NS. The NSC in vivo is generally accepted to be a slowly dividing astrocytic cell described as the type B cell (4) residing in the ciliated ependymal layer of the SEZ. Work by Laywell et al. demonstrates that by surgically dissecting the SEZ of neonatal animals and culturing the subsequently generated dissociated cells in neural media on adhesive substrates, a monolayer of astrocytes is produced that contains a sub-population of MASC capable of generating multipotent NS (43). Transplantation of gfp+ MASC into the lateral ventricles of both neonatal and adult C57BL/6 mice results in observable engraftment, with donor-derived cells present in not only the SEZ but also in the RMS and OB as migratory neuroblasts

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75 (89). Taken as a whole, these observations allude to the MASC being an acceptable in vitro manifestation of the NSC. Transplantation of gfp+ MASC into the lateral ventricles of LI mice three weeks following irradiation did not result in the expected increase of donor-derived migratory cells in OB. Non-irradiated controls exhibited normal engraftment levels while transplanted LI animals were observed to only contain donor-derived cells in the corpus callosum and SEZ. While it is unknown why the transplanted cells did not engraft normally into the neurogenic regions of LI animals, recent work has proposed that deleterious effects to the microvasculature occur in the dentate gyrus following 10Gy of focused irradiation rendering the region unreceptive to transplanted cells (51) and a similar phenomenon may occur here. As LI was not observed to enhance engraftment of transplanted MASC, a milder form of injury to the SEZ in the form of SLI (450rads) would assessed for enhanced engraftment of the transplanted cells. Recent studies in stem cell biology have revealed that injury to the candidate site of transplantation is critical for engraftment and/or contribution of the transplanted stem cell, as the recipient niche is normally relatively quiescent in a healthy adult animal and induction of injury often renders the niche more receptive to transplanted cells (83). Indeed, SLI animals transplanted with gfp+ MASC were observed to contain significantly more donor-derived migratory cells in the OB as compared to non-irradiated controls. It is important to note that although this increase was statistically significant it was highly variable with fully half of the transplanted SLI animals exhibiting levels of engraftment similar to controls. The reason for this

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76 variability is not known, although it could easily be attributed to the inherent inconsistency observed to occur in adult transplants. BrdU incorporation experiments to determine the mitotic cell levels at the time of transplantation revealed that while the numbers of mitotic SEZ neuroblasts were significantly depleted immediately following exposure, the levels returned to near normal by three weeks and were found to be significantly higher at six weeks. This observation was further supported by blind NS culture assays of SLI animals in which the observed yield of NS cultured from brains exposed to SLI 3 weeks earlier was significantly decreased by 30%. As was observed in the LI study, the NS yield from non-irradiated controls was again between 0.02 and 1% (0.12%). This mild injury to the brain may potentially enhance neurogenic activity and allow for increased engraftment by transplanted NSCs. Candidate NSC have recently been isolated from the brain utilizing antibodies that bind to unique cell surface antigens, such as CD15 and CD133 (10, 80). Isolated cells are then subjected to culture conditions that will produce NS, indicating that the cell population isolated contains NSCs. In the field of hematopoiesis, attempted in vitro manipulation and subsequent expansion of cultured HSC has been shown to result in decreased pluripotency and engraftment by the HSC, with only hematopoietic progenitor cells lacking the capacity for self-renewal being successfully expanded in vitro (14) in theory due to the incomplete reconstruction of the bone marrow microenvironment in vitro. Transplantation of non-cultured, primary HSC isolated from donor bone marrow still yields the most robust, long-term engraftment into the niche, and it is entirely possible that the in vitro manipulation of NSC results in a similar loss of multipotency

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77 and engraftment ability. Ideally, the NSC would be isolated directly from primary SEZ tissue prior to manipulation or transplantation, but in order for the NSC to be identified and isolated, a model system for robust engraftment is necessary for the analysis of the functional ability of the NSC. By utilizing the injury model described here, candidate cell populations isolated according to surface antigen expression could be transplanted to the injury-activated SEZ, followed by later analysis of the RMS for increased, long-term production of donor derived neuroblasts. The resulting observations would then allow for a definitive conclusion to be made as to if the isolated cell population expressed the characteristics of a true stem cell.

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BIOGRAPHICAL SKETCH Gregory Paul Marshall II was born on August 6 th 1976 in Gainesville, FL. After graduating from Bradford High School in Starke, FL in 1994, he attended St. Andrews Presbyterian College in Laurinburg, NC where he earned a Bachelor of Science degree in biology and met Kathleen Kelly, his future wife. After a two year employment with Dr. Susan Frost as head laboratory technician, Greg entered into the University of Floridas College of Medicine Interdisciplinary Program in Biomedical Sciences in the year 2000. After joining the laboratory of Dr. Edward Scott, Greg began his research into the characterization of neural stem cells. He has presented posters of his research at the Keystone Symposia in 2003 and at the Annual Society for Neuroscience meeting in 2004, and has recently submitted a first author article for publication in the journal Stem Cells. 86


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NEUROSPHERES AND MULTIPOTENT ASTROCYTIC STEM CELLS: NEURAL
PROGENITOR CELLS RATHER THAN NEURAL STEM CELLS

















By

GREGORY PAUL MARSHALL, II


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

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

By

Gregory Paul Marshall, II

































To my beautiful wife Kathleen, whose steadfast love and support made all of this
possible















ACKNOWLEDGMENTS

I would like to thank Ed Scott, Dennis Steindler, and Eric Laywell for all of their

guidance throughout my graduate career; my wonderful family for their love and support;

my fellow lab rats for always being there for me; and the Gainesville Rugby Club for

providing a welcoming diversion from my otherwise hectic life.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF FIGURE S ............................. ........ ........ ........... ................ viii

A B ST R A C T .......... ..... ...................................................................................... x

CHAPTER

1 INTRODUCTION TO ADULT MURINE STEM CELLS.............................. .... 1

H em atop oietic Stem C ells ................................................................. ..................... 1
H em atopoiesis ........................ ............. .......... ........... ............... .
Isolation of the Adult Hematopoietic Stem Cell ................................................3
D term inning Functional Engraftm ent ...................................................................4
Irradiation of the Host Bone Marrow, the Niche of the HSC.............................6
Plasticity of the H em atopoietic Stem Cell.................................... .....................7
Sum m ary ...................................................................................... .. ................... 9
N eural Stem C ells................................... ... ............................. 9
Neurogenesis and the Embryonic Neural Stem Cell ..........................................9
The A dult N eural Stem C ell ......................................... ............ ................... .10
Subependymal-Zone Neural Stem Cells ..... ..............................11
M ultipotent A strocytic Stem Cells ............................ ................................... 14
Potential Identifying M arkers of the N SC ..........................................................15
Transplantation of In Vitro Expanded NS and MASC ........................................16
Adult N S and M ASC: True Stem Cells? ........................................ ...............17
Effects of Radiation on Adult Neurogenesis.....................................................18
S u m m a ry ....................................................................................1 9

2 M ATERIALS AND M ETHOD S ........................................... ........................ 21

R agents ...........................................................................................2 1
Avertin M house Anesthetic Solution ....................................... ............... 21
5-brom o-2'-deoxy-uridine (BrdU) Solution...................................................21
Epidermal Growth Factor (EGF) Stock Solution (1000x) ................................22
Basic Fibroblast Growth Factor (bFGF) Stock Solution (1000x) .....................23
Immunocytochemistry Hybridization Buffer ............................................... 23
Neural Stem Cell (N SC) Culture M edia................................... ............... 23









4% Paraformaldehyde (100 mL) ............................ ....................... 24
Puromycin Stock Solution (1 m g/mL) ..................................... .................24
A n tib o d y L ist ................................................... ................. ................ 2 5
Primary Antibodies............. ............................ 25
Secondary Antibodies............. .............................. 25
M e th o d s ..............................................................................2 5
C ell C u ltu re ...............................................................2 5
Isolation and culture of neurospheres ............... .....................25
Isolation and culture of multipotent astrocytic stem cells............................26
Selection of gfp+ multipotent astrocytic stem cells by puromycin
selection n ..................................................................... 2 7
Transplantation and A analysis ........................................ ....... .................. 27
Transplantation of gfp+ MASC and neurospheres into the lateral
ventricles of adult C57BL/6 mice ............ ........... ..................27
Transplantation of gfp+ MASC and neurospheres into the lateral
ventricles of neonatal C57BL/6 mice ...............................................27
Tissue im m unohistochem istry................................................................... 28
Analysis of engrafted gfp+ MASC and neurospheres into the brains of
C 57B L /6 m ice............. .............. ....... ............ ...................... ........ 29
Analysis of engrafted gfp+ MASC into the olfactory bulb of C57BL/6
m ic e ..................... ..... ... ........ ................. ............... 2 9
Immunohistochemical analysis of neurospheres and MASCs ..................29
R radiation Studies.............. ........ .......................................... ............. 30
Irradiation and bone marrow reconstitution ............ .............................. 30
Identification of proliferative cells by BrdU labeling ...............................31
Blind analysis of the effects of lethal and sublethal irradiation on
neurosphere yield ............ .................................... ............ .... ..... 32

3 LACK OF EVIDENCE FOR THE CLASSIFICATION OF NEUROSPHERES
AND MULTIPOTENT ASTROCYTIC STEM CELLS AS TRUE STEM CELLS .34

Introduction .................. ........ ........................................................ .. 34
Inability of Gfp+ Neural Stem Cells to Survive Re-isolation Following
Transplantation into the Lateral Ventricles of C57BL/6 Mice..............................37
Gfp+ Neurospheres are not Generated from the Brains of Adult C57BL/6
Mice Transplanted with Gfp+ Neurospheres......................................37
Gfp+ Neurospheres are not Generated from the Brains of Neonatal C57BL/6
Mice Transplanted with Gfp+ Neurospheres.............. ......................38
Gfp+ Multipotent Astrocytic Stem Cells are not Generated from the Brains of
Neonatal C57BL/6 Mice Transplanted with Gfp+ Multipotent Astrocytic
Stem C ells .................. ................................ ...... ....... ... .. ................... .4 1
MASC Isolated from the Brains of C57BL/6 Neonatal Mice Transplanted
with Gfp MASC do not Survive Puromycin Treatment..............................43
Long Term Engraftm ent ......................................................... .............. 46









4 IONIZING RADIATION ENHANCES THE ENGRAFTMENT OF
TRANSPLANTED IN VITRO DERIVED NEURAL STEM CELLS..................... 49

In tro d u ctio n ................... .. ... .. ..... ....... ..... ... ............................... .. ............... 4 9
Effects of Lethal Irradiation on Subependymal Zone Neurogenesis..........................52
Lethal Irradiation Severely Depletes Migrating Neuroblasts in the RMS ..........52
Analysis and Quantification ofNeuroblast Depletion in the Subventricular
Zone Following Lethal Irradiation, as Determined by BrdU Incorporation ....54
Diminished Neurosphere Yield from Lethally Irradiated Mice ........................56
Lethal Irradiation Attenuates Engraftment of Transplanted Gfp+ Multipotent
A strocytic Stem C ells ........................ ...................... ............... ... 59
Effects of Sub-Lethal Irradiation on Subependymal Zone Neurogenesis ..................62
Sublethal Irradiation Results in a Transient Decrease in the Number of
Mitotic Subependymal Zone Neuroblasts............................ ............... 62
Diminished Neurosphere Yield from Sublethally Irradiated Mice ...................63
Sublethal Irradiation Enhances Engraftment of Transplanted Gfp+
M ultipotent A strocytic Stem Cells........................................ ............... 63

5 DISCUSSION AND CONCLUSIONS ........................................... ............... 69

L IST O F R E FE R E N C E S ............................................................................. ............. 78

BIO GRAPH ICAL SK ETCH .................................................. ............................... 86
















LIST OF FIGURES


Figure page

1-1. Migratory neuroblasts express PSA-NCAM in the adult mouse rostral migratory
store a m ............................................................................ 1 2

3-1. Gfp+ neurosphere derived cells are evident in the brains of host C57BL/6 mice three
weeks following transplantation................... ....... ............................ 38

3-2. Gfp+ neurospheres are not capable of re-isolation following transplantation into the
lateral ventricle of adult m ice........................................................ ............... 39

3-3. Gfp+ neurospheres display high levels of engraftment following transplantation into
n eo n atal m ice .................................................... ................ 4 0

3-4. Gfp+ neurospheres are not capable of re-isolation following transplantation into the
lateral ventricle of neonatal m ice .................................. ................ ................... 41

3-5. Passage three gfp+ multipotent astrocytic stem cells contain astrocytes but are devoid
o f n eu ro n s ..................................................................... ................ 4 2

3-6. Gfp+ multipotent astrocytic stem cells display high levels of engraftment following
transplantation into neonatal m ice....................................... ......................... 43

3-7. Gfp+ multipotent astrocytic stem cells are not present in the cultures of transplanted
n eo n atal m ice .................................................... ................ 4 4

3-8. Qualitative assessment of the effects of puromycin on multipotent astrocytic stem
cells cultures derived from C57BL/6 and gp+ neonatal mice...............................45

3-9. Transplantation of puromycin selected multipotent astrocytic stem cells isolated
from C57BL/6 mice transplanted with gp+ multipotent astrocytic stem cells
exhibit no gfp donor cell engraftm ent ........................................... ............... 45

3-10. Multipotent astrocytic stem cells isolated from the forebrains of mice transplanted
with gfp+ multipotent astrocytic stem cells do not survive puromycin treatment...47

3-11. Donor-derived cells exist in the brains of gfp+ neurosphere transplanted animals
14 m months after surgery ........................................................................ 48









4-1. Lethal irradiation drastically reduces the volume of PSA-NCAM and P-III Tubulin
positive m igratory neuroblasts ...................................................... .....................53

4-2. Reduction in the volume of 0-III Tubulin positive migratory neuroblasts persists
m months after lethal irradiation ............................................................................ 53

4-3. Region of analysis for quantification of mitotic neuroblast depletion in the
subependymal zone of lethally irradiated mice..................... ............................. 55

4-4. Depletion of mitotic neuroblasts in the subependymal zone of lethally irradiated
a n im a ls ........................................................................... 5 6

4-5. Lethal irradiation significantly decreases the number of BrdU positive SEZ
neuroblasts ............................................................... .... ..... ........ 57

4-6. Depletion of migratory neuroblasts in the rostral migratory stream positive for both
BrdU and P-III Tubulin in lethally irradiated mice ..................... ...............58

4-7. Lethal irradiation significantly reduces the yield of neurospheres cultured from the
adult subependym al zone. ............................................................. .....................60

4-8. N eurosphere differentiation potential ............................................. ............... 60

4-9. Attenuation ofgfp+ multipotent astrocytic stem cell engraftment by lethal
irra d iatio n ......................................................................... 6 1

4-10. Mitotic subependymal zone neuroblasts are transiently depleted following
su b leth al irradiation ................................................................. ........ ... ..... .64

4-11. Sublethal irradiation results in transient, recoverable depletion of mitotic
subependym al zone neuroblasts .................................... ............................. ........ 65

4-12. Sublethal irradiation significantly reduces the generation of neurospheres cultured
from adult subependym al zone ........................................... ......................... 66

4-13. Gfp+ multipotent astrocytic stem cells transplanted into the lateral ventricles of
control and sub-lethally irradiated mice produce donor-derived migratory cells in
the host olfactory bulb ............................................. .. ...... .. ........ .... 67

4-14. Sublethal irradiation enhances engraftment of donor-derived neuroblasts ............68















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

NEUROSPHERES AND MULTIPOTENT ASTROCYTIC STEM CELLS: NEURAL
PROGENITOR CELLS RATHER THAN NEURAL STEM CELLS

By

Gregory Paul Marshall, II

May, 2005

Chair: Edward W. Scott
Major Department: Molecular Genetics and Microbiology

Adult hematopoiesis is driven by the hematopoietic stem cell (HSC), a cell residing

in the bone marrow that possesses unique, functional characteristics defining it as a true

stem cell. Neural stem cells (NSCs) are reported to exist in the brains of adult mice in

two well characterized regions: the subependymal zone (SEZ) of the lateral ventricles

(LV) and the hippocampal dentate gyms. Mitotic neuroblasts are continuously generated

by the SEZ and migrate along the rostral migratory stream (RMS) toward the olfactory

bulb, where they functionally integrate as intemeurons. SEZ NSCs can be cultured under

specific conditions to generate neurospheres (NS) or multipotent astrocytic stem cells

(MASC), cell types that possess certain stem cell qualities in vitro. However, the in vivo

stem cell characteristics possessed by cultured NSCs (specifically functional engraftment

and serial transplantation) are not fully understood.









Green fluorescent protein (gfp) NS and MASC transplanted into the LV of both

neonatal and adult C57BL/6 mice resulted in engraftment into the recipient brain, with

donor-derived migratory neuroblasts visible in the RMS weeks after transplantation.

Neither displayed the capacity to be re-isolated from adult or neonatal brains even under

rigorous enrichment conditions, nor were they evident in the brains of secondary

recipient mice, indicating that functional, long-term engraftment by the transplanted cells

failed to occur.

Exposure of adult C57BL/6 mice to lethal levels of ionizing radiation resulted in

the ablation of both active hematopoiesis and SEZ neurogenesis, with subsequent

transplantation of gfp MASC into the LV resulting in no observable engraftment.

Exposure to sublethal levels of radiation resulted in a transient depletion of SEZ

neurogenesis and moderately enhanced engraftment levels of transplanted gfp+ MASC,

potentially providing a more ideal model system for NSC transplantation and analysis.

In this study, both NS and MASC failed to meet the criteria of true stem cells as

defined by the properties of the adult HSC. Thus it is possible that neurogenesis in the

adult brain is provided not by an isolatable NSC but rather by neural progenitor cells

derived from a migratory adult stem cell residing in the bone marrow.














CHAPTER 1
INTRODUCTION TO ADULT MURINE STEM CELLS

Stem cells are represented in a variety of organs in the adult mouse. Cells with

"stem-like" properties have been reported to exist in the bone marrow (7), skeletal muscle

(67), liver (13), pancreas (90), intestinal lining (68), skin (74) and the brain (2, 3, 44)

representing all three of the primordial germ layers: endoderm, ectoderm and mesoderm.

Stem cells in the adult mouse are believed to be developmental remnants of germinal

cells that continue to renew the tissues of the organ in which they reside. Adult stem

cells are characterized by the ability to continuously generate all cell types necessary to

perpetuate the existence of their native organ; and the ability to asymmetrically divide, so

that a duplicate stem cell is generated as well as a more lineage-committed daughter cell,

allowing for cell generation to continue for the life of the animal.

Hematopoietic Stem Cells

Hematopoiesis

The hematopoietic stem cell (HSC) is responsible for generating all the cells of the

blood lineage for the life of the animal, a process known as hematopoiesis. During

development, hematopoietic cells arise from the mesoderm after gastrulation and become

organized into blood islands in the extra-embryonic yolk sac near embryonic Day 7.5

(E 7.5) in the mouse (31, 53).

The first isolatable manifestation of the HSC appears in the intra-embryonic aorta-

gonod-mesonephros (AGM) at E 10.5, resulting in the initial onset of definitive

hematopoiesis in the developing animal (48, 54). Via waves of migratory HSCs from the









AGM, hematopoiesis then shifts to the fetal liver (with the presence of HSCs in the AGM

virtually eliminated by E 13). Migration of fetal HSCs is believed to be chemokine

driven, with stromal derived factor 1 (SDF-1) and steel factor (SLF) as the likely agents

of directional migration (11). Numbers of HSCs in the fetal liver begin to decrease

around E 16, presumably as the migratory HSCs leave the liver to seed the developing

bone marrow and spleen however, fetal liver hematopoiesis persists until shortly after

birth; whereas in the adult mouse, the bone marrow becomes the sole region of

hematopoiesis throughout the life of the animal.

Three definitive characteristics define the HSC as a true stem cell: asymmetric

division, pluripotency, and the ability to functionally reconstitute depleted bone marrow

(69). Asymmetric cellular division is a process in which the HSC switches between

generating an exact duplicate of itself and generating a more lineage committed daughter

cell that allows the HSC to perpetuate its existence throughout the life of the animal. The

HSC is capable, by generating lineage-specific hematopoietic progenitor cells (HPCs), of

producing each of the numerous cell types present in the blood. This includes (but is not

limited to) erythrocytes, macrophages, lymphocytes, T-cells, and B-cells. These two

abilities together make up the third criteria: The HSC is capable of repopulating the bone

marrow of a myeloablated mouse (a mouse that has been exposed to sufficient doses of

radiation so that the bone marrow is depleted of viable cells, a lethal condition without

treatment).

The HSC can home to the injured bone marrow after intravenous transplantation,

repopulating the niche and giving rise to all of the numerous cells within the

hematopoietic lineage. This homing mechanism is believed to be similar to that observed









during development, with an increase in the expression of SDF-1 after myeloablation of

the bone marrow causing an increase in the expression of matrix metalloproteinase-9

(MMP-9). This results in the release of soluble Kit ligand, creating a diffusion gradient

that the migratory c-kit+ HSC follows as it homes to the bone marrow (33). Asymmetric

division of the now engrafted HSCs allows for donor-derived hematopoiesis to continue

for the life of the mouse, deriving from the primary transplanted HSC, later-generated

daughter HSCs and the progeny of the resultant progenitor cells.

A fourth criterion has been proposed, in which isolated candidate HSCs must

display the aforementioned capabilities and the ability to serially rescue a myloablated

animal. In this paradigm, donor-derived HSCs can be re-isolated from the bone marrow

of a mouse rescued by the transplantation and engraftment of a single HSC, and

transplanted into a secondary myloablated host with the same rescue ability conferred to

the animal (40). These four criteria set the "gold standard" for defining the HSC, a

functional standard rather than a phenotypic one. Cell-surface markers unique to only the

HSC have yet to be identified, but techniques exist that allow for the enrichment of cells

extracted from the bone marrow for HSCs.

Isolation of the Adult Hematopoietic Stem Cell

The bone marrow is home to a variety of cell types, from stromal support cells and

mesenchymal stem cells (MSC) to HSCs, HPCs, and their resultant progeny. After

extracting the bone marrow, the MSC is removed by exploiting the ability of the MSC to

adhere to adhesive substrates such as laminin. As the HSC lacks this ability, placement

of the cell suspension onto an adhesive substrate allows the MSCs to adhere to the

substrate, leaving only the cells of the hematopoietic lineage. Removal of terminal

progeny is accomplished by using the surface markers unique to the selected cells: B220









is expressed by B-cells, CD1 lb by macrophages, Terl 19 by T-cells, and Gr-1 by

granulocytes. Fluorescently labeled antibodies specific to these antigens allow for the

selection of cells by a technique known as Fluorescence Activated Cell Sorting (FACS)

in which cells can be selected on the basis of the presence (positive selection) or absence

(negative selection) of definitive cell-surface markers. The FACS sorter is capable of

distinguishing between cell types based on the particular fluorochrome expression pattern

(and the specific antibody conjugated to that fluorochrome) present on treated cell

populations. Exclusion of the terminal daughter cells by negative selections leaves the

remaining cell population consisting of stem and progenitor cells.

To further enrich for the HSC, two specific cell-surface markers (88) can be

utilized: c-Kit and Sca-1. C-kit is the receptor for stem cell factor (SCF) also known as

steel factor (SF), a cytokine shown to inhibit apoptosis in hematopoietic cells potentially

giving HSCs the ability to perpetuate longer than terminally differentiated cells (1).

Sca-1 (stem cell antigen), also known as lymphocyte activation protein-6a (Ly6-A), is a

cell-surface receptor shown to play a role in HSC proliferation; the identity of its binding

ligand has yet to be identified (36). Selecting cells positive for these two markers while

depleted of terminally differentiated daughter cells results in a population of cells 1000-

to 2000-fold enriched for the presence of HSCs. While this process greatly increases the

chances of the resulting cells being HSCs, the aforementioned functional properties of

long-term bone marrow reconstitution must be ascertained before the isolated cell can be

definitively labeled as a true HSC.

Determining Functional Engraftment

After enrichment of whole bone marrow for the presence of HSCs, an assay to

ascertain the rescue capacity of the enriched cell population is critical for the cells









isolated to be deemed HSCs. Mice exposed to levels of radiation sufficient to deplete

the bone marrow of viable hematopoietic cells (lethal irradiation) are generated as

recipients for candidate HSCs. Lethally irradiated mice (also known as myeloablated)

will expire within a few days of exposure without intravenous transplantation of either

whole bone marrow or HSCs, so survival of the irradiated mouse is the first indication of

candidate cell types being HSCs. Duration of survival indicates the identity of the cell

transplanted, as short-term HPCs provide transient protection from myeloablation for a

period of only 3 to 4 mo (69). As previously mentioned, HSCs exist for the life of the

animal and long-term survival of a myloablated mouse (longer than 6 mo) indicates

engraftment of a true HSC.

To ascertain that the engrafted HSC is responsible for the lethally irradiated

animal's hematopoiesis, transplanted HSCs need to differ in some fashion from the host

animal. A common tool to meet this requirement is the gfp mouse; a transgenic animal

with vast experimental capabilities. The gfp mouse contains a transgene that expresses

gfp driven by the chicken beta actin promoter enhanced by a cytomegalovirus enhancer,

resulting in ubiquitous expression of gfp in theoretically every cell of the mouse (32).

Cells isolated from the gf mouse do not initiate an immune response when transplanted

into mice from the C57BL/6 line, as this line was used to generate the gfp transgenic

animal. Furthermore, the expression of gfp continues in vitro after removal of tissue from

the animal, allowing for in vitro manipulation and expansion.

Myeloablated C57BL/6 mice transplanted with candidate HSCs isolated from the

bone marrow of gfp mice result in a chimeric animal whose bone marrow and resultant

progeny are gfp+. By sampling portions of the peripheral blood of transplanted mice, it









is possible to determine which cell types are gfp+ and the percent chimerism contributed

by the transplanted cells. Using fluorescent antibodies against the cell-surface markers

present on terminal daughter cells listed earlier, FACS analysis allows researchers to

determine if circulating progeny are gfp+ and thus the result of an engrafted gfp+ HSC

(donor-derived). The presence of donor-derived progeny 6 mo or longer after

transplantation indicates that the cell type transplanted has characteristics of an HSC.

Serial transplantation provides definitive evidence that the transplanted cell was

indeed an HSC. If a true HSC was transplanted into a myeloablated animal and engrafted

into the bone marrow, resulting in long-term functional hematopoiesis; then it would

stand to reason that the transplanted HSC underwent asymmetric division, producing

additional HSCs. To determine this to be the case, the bone marrow from long-term

reconstituted mice transplanted with gfp+ candidate HSC is extracted and the resulting

cell suspension is again enriched for the presence of HSC. Should the isolated cells

rescue a secondary myloablated mouse, and donor-derived progeny be observed in the

peripheral blood 6 mo or longer after transplantation, it can then be stated that the cell

type transplanted satisfied all the necessary criteria to be classified as an HSC.

Irradiation of the Host Bone Marrow, the Niche of the HSC

Transplantation of enriched bone marrow results in robust, long-term engraftment

only when the host animal has first undergone myeloablation (18, 19, 20). Irradiation

dosage is critical, as insufficient levels will not deplete the bone marrow; and over-

exposure will result in mortal damage to internal organs. While lethal irradiation is

sufficient to deplete the bone marrow of HSC and their progeny, it apparently does little

to damage the support cells that make up the HSC niche. Niche (a term recently applied

to stem cell biology) describes the cellular make-up of the microenvironment in which a









particular stem cell is believed to reside, and the specific mitogens and cytokines needed

to maintain the potency of the native stem cell. As previously mentioned the bone

marrow contains cells of the hematopoietic lineage and also contains stromal support

cells. In vitro studies indicate that osteoblasts, fibroblasts, and endothelial cells are

capable of supporting HSCs and HPCs in culture. While this has yet to be confirmed in

vivo, HSCs in the bone marrow are known to exist in close proximity to the endothelial

vasculature and osteoblasts (87).

The current lack of available information on the exact chemical and cellular

components that comprise the microenvironment of the HSC niche has led to the inability

to expand the HSC in vitro while retaining full potency and rescue ability (14). While it

is possible for HSCs to exist in vitro and to differentiate into different hematopoietic cell

types in certain culture conditions, the HSC has yet to be proven capable of successful

expansion sufficient to daughter HSCs capable of repopulating the bone marrow of a

myeloablated animal.

Plasticity of the Hematopoietic Stem Cell

Because hematopoiesis (and subsequently, the HSC) is the most robust system of

cellular generation in the adult mammal, the HSC is arguably the stem cell with the most

potential for regenerating damaged tissue. The concept of an adult stem cell

transdifferentiating to produce cell types foreign to its native organ is a concept that has

undergone tremendous scrutiny in recent years. At the same time, the concept of HSC

transdifferentiation has elicited excitement in the field of regenerative medicine.

The HSC has been reported to repopulate the bone marrow after transplantation into a

myeloablated mouse, and also sometimes contribute to the brain (8, 17, 49), liver (41, 61,

76, 77), heart (34, 55, 56), muscle (21, 30), and blood vessels (28). HSCs isolated from









mice expressing specific reporter genes were found to generate progeny that appeared

morphologically identical to neighboring cells in these organs and that expressed the

appropriate cell surface markers. The possibility that cellular fusion was the reason

behind these cells appearing donor-derived weakened the argument for

transdifferentiation (75, 86).

Cell fusion is an extremely rare event, with neighboring cells merging to form a

cell that expresses the characteristics of one or both cells involved. This fusion occurs at

the cytoplasmic and nuclear level, with the resultant cell existing in higher than a diploid

state, expressing the cell-surface markers of both contributing cells, and adopting the

morphology of the surrounding tissue. Currently, for an investigator to disprove a fusion

event and thereby support transdifferentiation, specific steps must be implemented in the

experimental design. One such step is to transplant male donor cells into female host

tissue with later analysis for the presence or absence of the y-chromosome in potentially

transdifferentiated cells. Another such step is to use donor animals that constitutively

express a reporter gene downstream (gf for example) of a cre-recombinase gene, and

host animals that constitutively express a second reporter gene (lacZ) immediately

downstream of a stop codon flanked by lox-p sites. In the host animal, lacZ is turned off

until such time as the upstream stop codon is removed. Should a fusion event occur, the

cre-recombinase from the donor cells will excise the stop-codon of the host cell via the

lox-p sites and allow the expression of lacZ along with the expression ofgfp.

Recent investigations involving the long-term (more than 6 mo) contribution of

HSC-derived cells to the brain have yielded positive results, with the transdifferentiated

neurons giving no indication of being the result of fusion (12, 84). The long-term design









of these studies may be a crucial point, in that the HSC could require time to generate

progeny capable of infiltrating the brain's regions of stem cell activity and producing the

observed cell types. Injury to the target region of engraftment as well as the regions of

potential transdifferentiation may also be a critical requirement, as little evidence has

been collected in non-injured recipients (83).

Summary

The HSC can very well be classified as the grandfather of adult stem cells.

With almost 50 years of research dedicated to its identification and potential, the HSC

has a nearly insurmountable head start on other adult stem cells. Much of what is known

about stem cells (from their definitive phenotypes and capabilities, to their organs of

residence) has been derived from HSC studies. Candidate stem-cell populations isolated

from other tissues will need to meet the rigorous criteria set forth by the field of

hematopoiesis before they can be deemed to be true stem cells.

Neural Stem Cells

Neurogenesis and the Embryonic Neural Stem Cell

Development of the central nervous system begins after gastrulation, with induction

of the neural plate from the ectoderm to eventually form the neural crest. As

development continues, the structure of the brain emerges via an "inside-out" pattern of

growth (the deepest layers of the brain are formed first and outer layers are then formed

by migratory cells traveling and passing through previously generated layers).

This pattern of migration points toward regions of persistent neurogenesis in the interior

portion of the developing brain.

While opinions still differ as to the existence of a single type of neural stem cell

(NSC) that contributes to the formation of the entire brain, it is generally accepted that a









population of neural stem cells exists during this time, dedicated to producing cells of the

neural and glial lineage. These stem cells presumably generate additional stem cells and

also populations of lineage-specific progenitor cells. Neural progenitors (sometimes

called "neuroblasts") generate the numerous manifestations of neurons such as

interneurons and granule neurons while glial progenitors give rise to astrocytes and

oligodendrocytes. Astrocytes comprise the majority of cell types found in the brain,

offering mechanical and metabolic support to neurons. Oligodendrocytes are responsible

for the production of the myelin (the axonal covering that speeds neuronal transmission).

The Adult Neural Stem Cell

It was believed for many years that while stem cells were required to promote

developmental neurogenesis, the brain of the adult mammal was a static structure without

the ability to repair or modify itself (abilities conferred by stem cells). The presence of

newly generated cells indicated that neurogenesis occurred in the postnatal rat brain, but a

lack of available techniques to positively identify these cells as neurons left the

observations relatively ignored (2, 3).

It is now generally accepted that adult neurogenesis is restricted to two regions: the

subgranule layer of the hippocampal dentate gyms (9, 25, 38) and the ventriclar

subependymal zone (SEZ) (44, 47). During the middle 1990s, numerous reports

emerged showing that (in the adult mammal) cells with stem-like properties can be

isolated from the hippocampus, spinal cord, and SEZ (25, 44, 85). The method of

isolation differs from that seen in the field of hematopoiesis in that no definitive markers

for the NSC are currently available. Instead, candidate stem cells are isolated from

regions of neurogenesis and cultured in defined media conditions to ascertain what (if

any) stem cell properties they may possess.









Subependymal-Zone Neural Stem Cells

In the adult mouse, the SEZ is arguably the most robust region of neurogenesis,

existing as a layer of cells adjacent to the ependyma running along the length of the wall

of the lateral ventricles. Throughout the life of the animal, mitotic neuroblasts are

generated in the SEZ and migrate along a well-defined glial structure known as the rostral

migratory stream (RMS) towards the olfactory bulb (OB), where they integrate as

interneurons (4, 45, 47, 78). Presumably these interneurons allow for the maintenance of

olfactory sensation.

Migration of the mitotic neuroblasts occurs in a rostral orientation until the RMS

enters the OB proper, whereupon migration shifts to a tangential orientation. At this

point, the neuroblasts adopt a neuronal morphology and integrate into the existing

cytoarchitecture of the OB as granule or periglomerular interneurons. The migration of

mitotic neuroblasts is rather rapid (taking 2-6 days to traverse 2-8 mm) and occurs via a

unique form of chain migration (46). The migrating cells adhere to one another

(presumably via the expression of a polysialylated form of the neural cell adhesion

molecule, dubbed PSA-NCAM) and migrate at an estimated rate of 120 pm/h through the

RMS in chains that are devoid of glial cells (Figure 1-1). An estimated 30,000 new

neurons are produced each day in the adult mouse, leading many to conclude that a self-

renewing stem cell must reside in the SEZ in order for this rate to be sustained for the life

of the animal (45).

While the exact identity of the SEZ NSC has yet to be determined, numerous

reports have shed light on its characteristics and function. A heterogeneous population of

cells exists, replicate, and migrate adjacent to the ciliated ependymal layer, a layer of

cells lining the walls of the lateral ventricle that was previously believed to either


























Figure 1-1. Migratory neuroblasts express PSA-NCAM in the adult mouse rostral
migratory stream. Sagittal photographic montage (5x images) of migratory
neuroblasts in a 6 week old C57BL/6 female migrating through the RMS to
the OB from the subependymal zone of the LV. (*) denotes chains of
PSA-NCAM positive (red) neuroblasts. OB = olfactory bulb, RMS = rostral
migratory stream, LV = lateral ventricle.

contain or consist of NSCs (37). Little is known of the specific cell types and factors

that comprise the microenvironment of this NSC niche, although recent studies are

helping to better understand the cellular make-up. It has recently been reported that the

SEZ NSC is a type of slowly dividing astrocyte, termed the type B cell (15). The

processes of type B cells form tube-like structures through which migratory neuroblasts

(type A cells) travel toward the OB. Transit amplifying neural progenitor cells (referred

to as type C cells) are interspersed among the type B cells, and it is this cell type that is

believed to be responsible for the production of the type A cells.

Experiments involving the injection of the anti-mitotic agent

cytosine-P-D-arabinofuranoside (Ara-C) into the SEZ of adult rats showed that the cells

initially eliminated were the faster-dividing progenitor cells (the type A and C cells), and

that after removal of Ara-C the first cell to return was the type B cell (16). The type B









cells then generated type C cells, that in turn generated type A cells. Expression of

cell-surface markers of these three cell types are not unique enough to allow selective

isolation or enrichment by FACS, as earlier shown with the HSC. The type B cell

expresses the astrocytic markers glial fibrillary acidic protein (GFAP) and vimentin,

while the type A cell expresses both the migratory marker PSA-NCAM and the pan-

neuronal marker 0-III tubulin (27). Unfortunately, these markers are also expressed on a

variety of terminally differentiated cells in the brain, ruling them out as potential unique

markers for the isolation of the NSC. Interestingly, type C cells appear to exclusively

express Nestin (a marker expressed by primitive neuroepethelium during development)

indicating that this neural precursor is extremely primitive (27). Regardless of its

expression profile, the type B cell is generally accepted to be the NSC of the adult SEZ.

After surgical dissection, adult neural tissue from the SEZ can be dissociated into a

single-cell suspension and cultured in the presence of mitogens; specifically, epidermal

growth factor (EGF) and basic fibroblast growth factor (bFGF). When cultured in the

absence of adhesive substrates, cultured cells form spherical structures with phase

contrast-bright boundaries known as "neurospheres" (NS): presumably clonal structures

consisting of multi-lineage daughter cells in varying stages of maturation (22, 29, 39, 42,

62-65, 83). These NS are multipotent and capable of generating cell types from all three

neural lineages upon induction of differentiation. The NS do not normally form in the

absence of EGF or bFGF, validating the necessity of these mitogens in culture. NSCs

during development express receptors to EGF and bFGF in a temporal- and region-

specific manner, varying as the embryo develops. Expression ofbFGF seems crucial to

normal development, as bFGF knock-out mice have fewer neurons and glia and reduced









tissue volume (57, 81). This observation was supported by a study (73) in which

antibodies designed to neutralize bFGF were injected into neonatal brains resulting in

decreased DNA synthesis in multiple regions of the brain. Conversely, it was observed

that intra-cerebral injections of bFGF results in increased neurogenesis (81). EGF also

plays an important role in neural development: EGF knock-out mice exhibit defects in

cortical neurogenesis, and injecting EGF into the brain has been shown to increase

neurogenesis (79).

In addition to their multipotency, another stem cell quality of cultured NS is that

they are capable of in vitro expansion (64). After dissociation and secondary culture,

primary NS yield numerous secondary spheres, with the newly generated NS retaining all

of the pluripotent characteristics of the primary NS. Furthermore, the expansion

capability of the NS is retained even after ten passages, indicating self-renewal of a stem

cell by asymmetric division, although conclusions from conflicting reports would argue

as to the exact number of passages the NS can undergo while retaining potency.

What is not yet known is which cell in the isolated tissue gives rise to the NS (the

sphere-forming cell), if the sphere-forming cell is indeed a true stem cell, or if every NS

in culture is the product of an active NSC. While the results listed above identify the type

B cell as the NSC in vivo, definitive isolation of the adult NSC and its subsequent in vitro

analysis and expansion has yet to be conclusively accomplished.

Multipotent Astrocytic Stem Cells

In addition to the NS, another in vitro manifestation has recently been reported: the

multipotent astrocytic stem cell (MASC) (43). MASC can be isolated from the

cerebellum and forebrain of neonatal mice (up to 2 weeks of age), but are only attainable

from the SEZ in adult mice. Dissociated SEZ tissue forms a monolayer of astrocytes









after culture onto an adhesive substrate in the absence of EGF/bFGF; and a subpopulation

of these astrocytes are believed to be MASC. The theory that the NSC is a form of

astrocyte supports the belief that the MASC is an in vitro manifestation of the NSC (27).

Furthermore, MASC are capable of generating NS upon culture in non-adhesive

conditions in the presence of EGF and bFGF, with resulting NS capable of serial

expansion and multi-lineage differentiation.

Potential Identifying Markers of the NSC

The HSC population can be enriched from the bone marrow cell population using a

series of cell-surface markers, and recent reports indicate that similar protocols can be

utilized to enrich the cell population of the SEZ for NSC. As described earlier, the SEZ

NSC is potentially a form of astrocyte, and because GFAP is expressed by astrocytes

GFAP has been proposed for use as one marker for the isolation of NSC (35). The

inter-filament protein Nestin is expressed by developing neuroepithelium, and is believed

to be expressed on adult NSCs, hinting at the developmental immaturity of these cells

(63, 66). Lewis-X is a carbohydrate moiety expressed on the cell surface of embryonic

stem cells, germinal zones in the developing mouse brain, and on some astrocytes; and

was recently reported to be present on the surface of adult NS-forming SEZ cells (10).

Finally, in the human model, purification of fetal brain using antibodies against CD 133 (a

marker purported to be on the surface of HSCs) greatly enriched for NS (80).

While these protocols theoretically enrich for the presence of NSC (or at the very

least, NS-forming cells), it remains to be seen if one or a combination of these markers

will allow for the isolation of a pure population of NSCs. Furthermore, should these

techniques make it possible for the in vitro enrichment of NSCs, protocols are needed to

determine the in vivo potential possessed by the isolated and enriched NSC populations.









Transplantation of In Vitro Expanded NS and MASC

In addition to pluripotency and expansion, cultured NS and MASC also display the

ability to survive transplantation back into the brain; an important ability, indicating that

extended culture conditions have not adversely affected the cells. Integration

capabilities are limited to the aforementioned regions of neurogenesis gliall cells are

typically the only cell type observed following transplantation into non-neurogenic

regions), and the age of the host animal is also critical.

Neonatal mice (1-3 days post embryonic) are still undergoing active developmental

neurogenesis and presumably offer an environment rich in extra-cellular cues for

enhanced engraftment of transplanted NSC (26), while neurogenesis in the adult animal

is dramatically lower and is limited to the SEZ and hippocampus. In vitro expanded

hippocampal NSCs are capable of integrating into the dentate gyms after transplantation

(24), and also contribute to functional granule cells in the OB after transplantation into

the SEZ (70). Labeled SEZ NS and MASC will survive transplantation to the SEZ,

RMS or lateral ventricles, with donor-derived migratory neuroblasts present in these

regions weeks following transplantation (80, 89).

While the presence of donor-derived neurons and neuroblasts in the OB indicates

engraftment, the degree of functionality is difficult to determine. The transplanted cells

may appear to generate progeny similar to the surrounding cells, yet they do not always

express the appropriate cell-surface markers. Furthermore, the engraftment is typically

only manifested by production of cells of the neuronal lineage as opposed to the multi-

lineage engraftment observed in HSC transplant studies. This would argue that a

progenitor cell was transplanted rather than a NSC, although the limited neurogenesis in

the SEZ could explain the mono-lineage nature of the newly generated daughter cells. It









is also unknown if the transplanted cells truly engraft into the SEZ in a stable fashion and

produce daughter progenitor cells that become evident in the OB as interneurons, or if the

transplanted cells immediately enter into the RMS as neuroblasts and migrate to the OB

(again a phenomenon more consistent with progenitor-cell activity).

Adult NS and MASC: True Stem Cells?

In the beginning of this chapter, four properties were listed as belonging to true

stem cells: multipotency, asymmetric division, functional long term engraftment and

serial transplantation. While it is believed that the endogenous NSC in vivo possesses

these characteristics, little has been done to determine if candidate NSC or their in vitro

manifestations (the NS and MASC), can be defined as true stem cells.

Of the four properties, only multipotency has been conclusively shown to be a

property of NS and MASC, although in vivo studies have indicated that their potential is

limited to interneurons, granule neurons, and neuroblasts. While both NS and MASC

are able to expand following in vitro passages, this ability is often limited to only four to

six passages, though this may be due to deficiencies in the culture conditions rather than a

deficiency on the part of either cell type.

While donor derived cells are observed in host RMS and OB following

transplantation of NS and MASC, their presence is transitory, often ceasing to exist a few

months following transplantation. This is indicative of short-term engraftment of the

transplanted cells, rather than long-term (assuming true engraftment has even occurred).

To date, no data exists to determine if the observed donor-derived cells are the product of

engrafted NSCs in the SEZ, or if the transplanted cells adopted the morphology of

migratory neuroblasts immediately following transplantation.









Finally, serial transplantability is a condition yet to be met by either the NS or the

MASC, shedding doubt on the level of stem-ness possessed by either of these cell types.

There is a distinct possibility that the NS and MASC are in reality neural progenitor cells,

as they do not seem to possess all of the characteristics of a true stem cell.

Effects of Radiation on Adult Neurogenesis

In humans, whole body irradiation prior to bone marrow transplantation is a

requirement for the treatment of many forms of leukemia to enhance the engraftment of

the HSC following bone marrow transplantation, and recent investigations have focused

on the effects this exposure may have on neurogenesis, particularly on the subgranular

zone (SGZ) of the hippocampal dentate gyrus (DG). Throughout the lifespan of the

mouse newly generated neuroblasts generated from subgranular NSC migrate into the

granule cell layer and differentiate into functional granule neurons (9). These neurons

functionally integrate into the DG, potentially playing a role in synaptic plasticity and

learning. This region of neurogenesis appears to be extremely sensitive to x-irradiation in

a dose dependent manner, with the numbers of neuroblasts and granule neurons

significantly depleted by as little as 2Gy of exposure, with little to no recovery observed

months after exposure (50, 59, 71). The death of these cells is reported to be apoptotic

(60) and appears to be linked to an inflammatory response produced by the damaged

tissue, as anti-inflammatory agents injected into the animals before and after irradiation

protected the SGZ from cell loss (52). Exposure to high levels (10Gy) of focused x-rays

results in near total, permanent ablation of mitotic cells in the SGZ and DG, with

pronounced changes in both the vascular microenvironment and cell types as cells with a

glial morphology become predominant following exposure (51).









Relatively few studies have been performed to determine the effects radiation

exposure may have on SEZ neurogenesis. Studies performed only in the rat model have

shown a similar result to that observed in the SGZ and DG in the mouse. Mimicking the

results observed in the hippocampus, radiation exposure adversely effected neurogenesis

in the SEZ of rats in a dose dependent manner, with little to no recovery observed months

following irradiation (72). Interestingly, low levels of focused x-irradiation (1-3Gy)

resulted in an increase of Nestin positive cells in the SEZ in the weeks immediately

following exposure, with levels eventually returning to normal (6).

Researchers have hypothesized that the depletion observed in both the SGZ and

SEZ is due in part to the elimination of NSCs and/or NPCs known to reside in these

tissues. 10Gy of x-irradiation not only permanently depletes the SGZ of mitotic

neuroblasts, but also negatively influences the microenvironment with surviving cells

adopting a glial morphology.

This has also been shown to render the niche unreceptive to transplanted non-

irradiated NPCs, with a significant decrease in the number of transplanted NPCs adopting

a neuronal morphology (51). It is currently believed that the brain is a relatively radio-

sensitive organ compared to the bone marrow, as low doses of radiation only transiently

lower the numbers of HSCs and their resultant progeny while similar doses permanently

diminish neurogenesis in both the SEZ and SGZ.

Summary

While it is generally accepted that NSC driven neurogenesis persists in the adult

mammal in the SEZ and SGZ, the exact identity of the NSC remains a mystery.

Candidate NSCs have been extracted from SEZ and SGZ based upon expression of cell

surface markers, and these cells can be cultured in the presence of EGF and bFGF to









generate NS and MASC. Subsequent transplantation of NS and MASC back into regions

of neurogenesis results in minimal engraftment by the donor cells with donor-derived

progeny existing as neuroblasts and/or granule neurons in the host tissue, but it is not

known whether the presence of donor-derived cells indicates functional engraftment or

short-term contribution to the surrounding tissue. Exposure to radiation adversely affects

neurogenesis in the adult rodent in a dose-dependent manner, indicating that the brain is a

more radio-sensitive structure than is the bone marrow. Of the four criteria held by true

stem cells (multipotency, asymmetric division, functional long-term engraftment, and

serial transplantation) the in vitro manifestations of the NSC fulfill only one:

multipotency. Protocols are needed to not only examine the ability of the NS and MASC

to demonstrate robust, long-term engraftment, but also to ascertain their ability to survive

serial transplantation before any claims can be made as to the levels of stem-ness

possessed by these relatively uncharacterized cell types.















CHAPTER 2
MATERIALS AND METHODS

Reagents

Avertin Mouse Anesthetic Solution

Materials: Avertin (2-2-2 Tribromoethanol, Aldrich, St. Louis, MO; T4, 840-2),

tert-amyl alcohol, 50 mL sterile polystyrene conical tube, 50 mL "Steriflip" disposable

vacuum filtration system (0.22 |tm pore, Millipore, Billerica, MA; SCGP00525),

phosphate buffered saline, aluminum foil, stir bar/plate.

Protocol: Stock solution: Using the bottle in which the avertin was packaged,

added 15.5 mL tert-amyl alcohol and magnetic stirrer and allowed to stir overnight.

NOTE: As avertin is toxic if allowed to photo-oxidize, the stock bottle was kept tightly

capped and wrapped in aluminum foil. Stable at room temperature for well over one

year.

Working solution (20 mg/mL): Combined 0.6 mL of the avertin stock and 39.4

mL ofPBS in 50 mL conical. Agitated briefly to mix the two components, then filtered

through the Steri-flip and wrapped the filtered solution in foil to exclude all light.

Working solution is stable at 40C for several months.

5-bromo-2'-deoxy-uridine (BrdU) Solution

Materials: 5-bromo-2'-deoxy-uridine (Roche, Florence, SC; 280-879),

appropriate size sterile polystyrene conical tube, phosphate buffered saline, 370C water

bath.









Protocol: Calculated out the volume of BrdU required for all planned injections

and measured out enough BrdU so that the final concentration was 10mg/mL.

NOTE: BrdU is extremely mutagenic. Be sure that the powder is handled carefully.

Combined the BrdU and PBS in the conical tube and warmed in the water bath for 15

minutes. Vigorous agitation is essential to ensure that all of the BrdU has entered into

suspension. Made fresh for each experiment.

Epidermal Growth Factor (EGF) Stock Solution (1000x)

Materials: Recombinant Human Epidermal Growth Factor (R & D Systems,

Minneapolis, MN; 236-EG, 200 [tg), Glacial Acetic Acid, Bovine Serum Albumin

Fraction V heat shock (Roche Diagnostics, 03 116 999 001), 5 cc syringe w/ regular luer

tip (Becton Dickinson, Franklin Lakes, NJ; 309603), Blunt Needle w/ Aluminum Hub

(Monoject, Becton Dickinson, 202314), Sterile Syringe Filter, 0.45 |tm pore size

(Corning Incorporated, Corning, NY; 31220), 15 mL and 50 mL polystyrene sterile

conical tubes, 1.5 mL sterile micro centrifuge tubes, sterile 200 [tl pipette tips, distilled

water, ice and ice bucket.

Protocol: Generation of EGF suspension solution: 17.24 [tl of Acetic Acid was

mixed with 30 mL of distilled water in a 50 mL conical tube to generate a final

concentration of 10 mM. 0.030 g ofBSA was added to solution (final concentration,

0.1% w/v) and mixed vigorously to re-suspend.

Working on ice in a sterile laminar flow hood, 200 [g of EGF was re-suspended in

10 mL of suspension solution in a sterile fashion then filter sterilized using the 5 mL

syringe with blunt needle and syringe filter. Evenly distributed 500 [L aliquots of the

EGF solution and stored at 800C. Final concentration 20 ng/[tl, used at 1:1000.









Basic Fibroblast Growth Factor (bFGF) Stock Solution (1000x)

Materials: Recombinant-human-FGF (25 ig, R&D systems, 233-FB), Phosphate

Buffered Saline, Bovine Serum Albumin Fraction V heat shock (Roche Diagnostics, 03

116 999 001), 1M Dithioerythritol (DTT) (Sigma, St. Louis, MO; D-8255), 5 cc syringe

w/ regular luer tip (Becton Dickinson, 309603), Blunt Needle w/ Aluminum Hub

(Monoject, 202314), Sterile Syringe Filter, 0.45 |tm pore size (Corning Incorporated,

431220), 15 mL polystyrene sterile conical tube, 1.5 mL sterile micro centrifuge tubes,

sterile 200 [tl pipette tips, distilled water, ice and ice bucket.

Protocol: Generation of bFGF suspension solution: 3 mL of PBS, 0.003 g BSA

(0.1%) and 3 ptL of 1M DTT (final concentration of ImM) were combined and mixed..

Working on ice in a sterilized laminar flow hood, 25[g bFGF was combined in 2.5mL of

suspension solution and filtered sterilized. Aliquots of 100 ptL were stored at -800C.

Final concentration 10 ng/[tL, used at 1:1000.

Immunocytochemistry Hybridization Buffer

Materials: Fetal bovine serum, Phosphate Buffered Saline (PBS), Triton X-100

(Sigma, T-9284), 50 mL sterile polypropylene tube.

Protocol: In sterile laminar flow hood, pipetted 5% of the final volume of FBS

into 50 mL tube. Out of the hood, filled to full volume with PBS and pipetted 0.01%

Triton X-100. Mixed and stored at 40C. Stable for 1 week.

Neural Stem Cell (NSC) Culture Media

Materials: DMEM/F 12 w/ HEPES and L-Glutamine (Gibco BRL, Carlsbad, CA;

11330-032), fetal bovine serum (FBS), N2 supplement 100X (Gibco BRL, 17502-048),

GlutaMAX-1 supplement 100x (Gibco BRL, 35050-061). Endothelial Growth Factor









stock supplement (1000x), Fibroblast Growth Factor-beta stock supplement (1000x), 50

mL sterile polystyrene conical tube, 50 mL "Steriflip" disposable vacuum filtration

system (0.22 |tm pore, Millipore, SCGP00525).

Protocol: Based on the total volume required, the appropriate volumes of the

above serums and supplements were added to DMEM/F 12 to their working concentration

and filter sterilized prior to use.

4% Paraformaldehyde (100mL)

Materials: Paraformaldehyde (Sigma P-6148), 250 mL glass beaker, distilled

water, IN sodium hydroxide (NaOH), IN hydrochloric acid (HC1), 3x PBS, glass Pasteur

pipettes, aluminum foil, 100+mLglass bottle, filter paper, small funnel, stir bar/magnetic

stirrer hot plate, pH meter.

Protocol: 4 g (4% w/v) paraformaldehyde was mixed with 60 mL of distilled

water heated to approximately 55C in a glass beaker and covered with aluminum foil

while mixing for 10 minutes. Two drops of IN NaOH was added to the solution

followed by an additional 5 minutes of mixing, after which the solution became semi-

clear. 30 mL of 3x PBS was added to the solution and the pH adjusted to 7.2 using IN

HC1 and IN NaOH. The solution was then filled to 100mL with distilled water, filtered

through Whatman paper into clean glass bottle and stored at 40C. Solution is stable for

approximately 1 week.

Puromycin Stock Solution (1 mg/mL)

Materials: Puromycin dihydrochloride from Streptomyces alboniger (Sigma

P-8833), 5 cc syringe w/ regular luer tip (Becton Dickinson, 309603), Blunt Needle w/

Aluminum Hub (Monoject, 202314). Sterile Syringe Filter, 0.45 |tm pore size (Corning









Incorporated, 431220), DMEM/F12 w/ HEPES and L-Glutamine (Gibco BRL,

11330-032), sterile polypropylene 1.5 mL tubes.

Protocol: Working in a sterile laminar flow hood, 1 mg of puromycin was added

to 1 mL of sterile DMEM/F12 in 1.5 mL sterile tube and agitated to dissolve the

puromycin powder. Re-suspended solution was filter sterilized and stored at -200C.

Antibody List

Primary Antibodies

* Monoclonal anti-P-III tubulin: Promega, Madison, WI; G712A, dilution 1:1000
* Polyclonal anti-P-III tubulin: Covance; Princeton, NJ; PRB-435P, dilution
1:5000
* Monoclonal anti-polysialic acid neural cell adhesion molecule [PSA-NCAM]:
Chemicon, Temecula, CA; MAB5324, dilution 1:100
* Monoclonal anti-BrdU: Human Hybridoma Bank, Gainesville, FL; dilution 1:30
* Polyclonal anti-glial fibrillary acidic protein [GFAP]: Shandon Immunon,
Milford MA; 490740, dilution 1:100)

Secondary Antibodies

* Rhodamine red-X, goat anti-mouse IgG: Molecular Probes, Eugene, OR; R-
6393, dilution 1:500
* Rhodamine red-X, goat anti-rabbit IgG: Molecular Probes, R-6394, dilution
1:500
* Fluoroscein anti-rabbit IgG: Vector Labs, Burlingame, CA; FI-1000, dilution
1:500
* Oregon green 514 goat anti-mouse IgG: Molecular Probes, 0-6383, dilution
1:500

Methods

Cell Culture

Isolation and culture of neurospheres

Neurosphere cultures were generated from WT, LI, and SLI animals, as described

(42). Briefly, animals were anesthetized with isofluorane, cervically dislocated and

decapitated. The brain was exposed, surgically removed, and then placed on an ice-cold

sterile dissection board. A rectangular forebrain block containing the SEZ was obtained









by removing the olfactory bulb, cerebellum, hippocampus, lateral portions of the striatum

and lateral and dorsal cerebral cortex. The block was minced with a sterile scalpel, and

placed in ice-cold PBS containing anti-biotic and anti-mycotic agents (Penicillin-

Streptomycin, Gibco, 15140-122, and Fungizone Antimycotic, Gibco, 15295-017) for 10

minutes. Minced tissue was then centrifuged for 5 minutes at 1100 rpm at 40C, re-

suspended in 3 mL 0.25% Trypsin plus EDTA (Gibco, 25200-056), then incubated at

37C for 5 minutes. After neutralizing the trypsin by the addition of 1 mL of fetal bovine

serum, the tissue was triturated into a single cell suspension by pipetting through a series

of descending diameter, fire-polished Pasteur pipettes. The cells were washed in

DMEM/F-12 (Gibco, 11330-032) at 1100 rpm for 5 minutes at 40C, and re-suspended in

neural growth medium. The cells were plated out in non-adhesive 6-well plates (Coming

Costar, 3471) at a density of 1000 cells/cm2. Cultures were supplemented with EGF and

bFGF (20 ng/mL and 10 ng/mL, respectively), every second day.

Isolation and culture of multipotent astrocytic stem cells

Primary SEZ tissue was isolated from neonatal mice transgenic for green

fluorescent protein (gfp+) at post-embryonic day 2 and dissociated to a single cell

suspension in the same manner as listed above. Cells were plated onto tissue culture

flasks at high density in neural growth medium devoid of EGF and bFGF. 3 days

following the initial plating, non-adherent cells were removed and fresh media was

applied. Cultures were passage once the resulting astrocytes had formed a confluent

monolayer, and cultures were deemed suitable for transplantation once they had

undergone three passages (necessary for removal of contaminating neurons in the

cultures).









Selection ofgfp+ multipotent astrocytic stem cells by puromycin selection

MASC cultures derived from the SEZ of gfp and C57BL/6 neonatal and adult mice

were prepared as described above. Once a confluent monolayer had been established,

cells were passage at 25% confluency with puromycin stock solution added to the neural

growth medium at a final concentration of 1-3 [g/mL. Cells were exposed to puromycin

in the neural growth medium for nine days, with media replaced with fresh neural growth

medium plus puromycin on the fifth day. Cells were analyzed after removal of

puromycin by phase contrast microscopy. In order for surviving cells to generate a

secondary confluent monolayer, cells were collected by trypsinization and re-plated at the

highest possible density in puromycin-free neural growth medium.

Transplantation and Analysis

Transplantation ofgfp+ MASC and neurospheres into the lateral ventricles of adult
C57BL/6 mice

Passage three gfp+ MASC or neurospheres were collected via trypsinization and

re-suspended in ImL of growth medium (see above). Once cell number was calculated,

cells were re-suspended in a volume of growth media yielding 50,000 cells per pL.

Recipient mice were anesthetized with Avertin (see above) and the scalp surgically

exposed. 100,000 cells (2 pL) were stereotaxically injected into the lateral ventricle via a

5[L Hamilton syringe attached to a 28 gauge needle at the following coordinates: A-P: -

0.2, M-L: -1.2, H-D: -2.5. Transplanted animals were allowed to recover and placed back

in general housing.

Transplantation of gfp+ MASC and neurospheres into the lateral ventricles of
neonatal C57BL/6 mice

Passage three gf+ MASC or neurospheres were collected via trypsinization and

re-suspended in 1 mL of growth medium (see above). Once the cell number was









calculated, cells were re-suspended in a volume of growth media yielding 100,000 cells

per tl. Recipient C57BL/6 neonatal mice (day post embryonic 1-3) were anesthetized by

placement at -20C for 5 minutes. Cells were transplanted in a volume of 1 [iL via a 5 [iL

Hamilton syringe attached to a 28 gauge needle into the lateral ventricle using the bregma

skull suture as a reference point. Following transplantation, neonatal mice were warmed

to consciousness and returned to the mother's cage prior to return to general housing.

Tissue immunohistochemistry

Both wild type (WT), LI and SLI animals were given a lethal dose of the anesthetic

Avertin before being perfused through the left ventricle with 4% paraformaldehyde

(PFA) in PBS. Following perfusion, the brain was removed and post-fixed overnight by

immersion in 4% PFA at 40C. Fixed brains were then serially sectioned through either

the coronal or sagittal plane at 40 [m using a Leica vibratome (model VT-1000-S)

equipped with a sapphire blade. Tissue was prepared for immunohistochemistry by

blocking at room temperature for 1 hour in PBS containing 10% fetal bovine serum and

0.01% Triton X-100. Primary antibodies were applied to the sections overnight with

moderate agitation at 40C. Residual primary antibody was removed by three 5 minute

washes (PBS plus 0.01% Triton X-100), and secondary antibodies were applied at room

temperature for 50 minutes. Finally, sections were washed in PBS three times for 5

minute, mounted on positively charged glass slides (Fisherbrand Superfrost/Plus, 12-550-

15), and allowed to dry for 15 minutes at 37C, before being cover-slipped in Vectashield

(Vector Labs, H-1000) mounting medium. Sections were analyzed and photographed by

fluorescence microscopy using either a Zeiss Axioplan 2 upright microscope (Carl Zeiss









Inc, Thornwood, NY), Leica DMLB (Leica Microsystems AG, Wetzlar, Germany), or a

Leica TCS SP2 AOBS spectral confocal microscope.

Analysis of engrafted gfp+ MASC and neurospheres into the brains of C57BL/6
mice

Three or four weeks following transplantation, brains of transplanted animals were

prepared for tissue immunohistochemistry as described above. The transplanted

hemisphere was sectioned to through the sagittal plane (40 |tm thick) with every section

containing the olfactory bulb collected in a serial fashion for subsequent analysis.

Tissue sections were exposed to antibodies against P-III tubulin as described above and

mounted onto slides using the serial sectioning for visual reconstruction of the brain. All

sections were analyzed at 20x magnification for the presence of gfp+ cells in the SEZ,

RMS, and OB.

Analysis of engrafted gfp+ MASC into the olfactory bulb of C57BL/6 mice

Three weeks following transplantation, brains of transplanted animals were

prepared for tissue immunohistochemistry as described above. For each brain, the

transplanted hemisphere was sectioned through the sagittal plane (40[tm thick), with

every section containing the olfactory bulb collected for analysis.

Resulting sections were exposed to antibodies against P-III tubulin as described above

and mounted for analysis. Gfp+ migratory cells present in the olfactory bulb proper (that

is, the region rostral of the descending horn of the rostral migratory stream) were scored

as engraftedd", with every section analyzed at 20x magnification for each animal.

Immunohistochemical analysis of neurospheres and MASC

NS were picked from their cultures using a handheld pipetter set at 2[tl and placed

in DMEM/F-12 plus 5% FBS atop a laminin/poly-D-lysine coated, chambered culture









slide (Becton/Dickinson, 352688). Spheres or aliquots of MASC were allowed to attach

and differentiate for 2 days, at which time the media was removed and the cells were

fixed by incubation in 4% PFA in PBS at room temperature for 30 minutes. After

fixation, the cells were processed for immunolabeling with antibodies against P-III

tubulin and GFAP, as above.

Radiation Studies

Irradiation and bone marrow reconstitution

Animals were placed in individual chambers of a plexi-glass container for

irradiation. Lethal irradiation (LI) was induced by exposure to a Cs137 source in a

Gamma Cell 40 irradiator until 850 Rads had been obtained. This amount of radiation is

sufficient to deplete the bone marrow of viable cells, while not inducing immediate death.

Immediately following irradiation, LI mice were anesthetized with isoflurane (Aerrane,

Baxter Deerfield, IL; NDC 10019-773-40) and a "rescue dose" of 1 x 106 whole bone

marrow (WBM) cells was administered to each mouse via retro-orbital sinus (ROS)

injection. WBM was isolated from the femurs of a sacrificed litter mate (anesthetized by

exposure to isoflurane then sacrificed by cervical dislocation). Isolated WBM was then

washed in 10 mL of phosphate buffered saline (PBS), and re-suspended in an appropriate

volume of PBS after quantifying with a hemacytometer. A 32 gauge needle attached to a

1 mL insulin syringe was inserted into the ROS and the WBM was injected in a volume

of 150 pL. Animals were allowed to recover before being returned to conventional

animal housing.

For sublethal irradiation (SLI) studies, the animals were exposed to 450 Rads of

ionizing radiation in the same fashion as listed above. No rescue dose of WBM is









required for survival of the animal at this exposure, and animals were immediately

returned to conventional housing.

Identification of proliferative cells by BrdU labeling

Three days, three weeks or 3 months following irradiation, WT, LI and SLI mice

received 5-bromo-2'-deoxyuridine (BrdU, Sigma, B-5002) three times a day for three

days via intra-peritoneal (IP) injection (3 .tg/300 [iL per injection). The brains were

fixed, removed, and sectioned as above. Sections were prepared for BrdU

immunohistochemistry by first incubating in 2XSSC/Formamide solution (1:1) for 2

hours at 650C. After washing in 2xSSC for 5 minutes at room temperature, sections were

then incubated in 2 N HC1 for 30 minutes at 37C. Sections were washed in 0.1 M borate

buffer for 10 minutes at room temperature prior to processing for immunolabeling with

monoclonal anti-BrdU and polyclonal anti-P-III tubulin antibodies, as described above.

Serial coronal sections were analyzed for BrdU positive cells in the SEZ using a blind

study format (sections coded and scored by separate investigators). The region of the

SEZ analyzed encompassed an area extending from the inferior tip of the lateral ventricle,

superiorly along the wall of the lateral ventricle (approximately 5 cell bodies deep).

This area continued to a point extending 700 [m from the dorsolateral corer of the

lateral ventricle.

The defined region was carefully analyzed at a magnification of 40X, and at both

focal planes of the tissue. To maintain consistency between sections, BrdU labeled cells

were scored as "positive", regardless of the intensity of the antibody fluorescence.

Three adjacent sections per tissue were scored, using the location of the anterior

commissure as a consistent landmark between sections. The cell numbers were collected,









averaged, and placed into a graphical format using a Microsoft Excel spreadsheet.

Statistical significance of values was determined by paired student's t-test analysis with

p-values less than 0.05 deemed to be significant.

Blind analysis of the effects of lethal and sublethal irradiation on neurosphere yield

In order to determine the effects of irradiation on NS generation, a blind paradigm

was utilized to enable unbiased preparation and examination of the cultures from three

WT and three LI mice (two months following lethal irradiation, age matched) and from

four WT and 4 SLI mice (3 weeks following sub-lethal irradiation, age matched).

Briefly, the animals were sacrificed and their brains removed by investigator A,

who gave each brain an identifying number (1 through 8). The brains were then given to

investigator B who removed the SEZ (as described above) from each brain in an identical

fashion. The isolated SEZ tissue was then re-coded with a letter (A through H) by

investigator C, who remained the only individual to know both the letter and number

code. The tissue was then returned to investigator A for culture (as described above) and

quantification. At 21 days in vitro, NS were collected, pelleted, and re-suspended in 2

mL of media.

To determine NS yield, four 50 [tl aliquots from each culture were placed in a 12-

well tissue culture plate. The aliquots were analyzed with a Nikon inverted phase

microscope at both 4x and 10x magnifications. NS diameter was determined by use of

the "SPOT" program (Diagnostic Instruments, Sterling Heights, MI). NS below 40 xtm

in diameter were excluded as a means to avoid potential confusion with hypertrophied

cells. Additionally, spherical aggregates that did not display the NS criteria of a tight

phase contrast-bright perimeter were not counted. The total number of spheres per









aliquot was determined, and the total yield and percent yield of each culture was then

calculated from these numbers. Statistical significance of values was determined by

paired student's t-test analysis withp-values less than 0.05 deemed to be significant. At

the conclusion of the analysis, the code was broken and the identity of the cultures was

revealed.














CHAPTER 3
LACK OF EVIDENCE FOR THE CLASSIFICATION OF NEUROSPHERES AND
MULTIPOTENT ASTROCYTIC STEM CELLS AS TRUE STEM CELLS

Introduction

Adult stem cells (ASCs) have been the subject of numerous studies in recent years,

many of which have focused on the isolation and characterization of candidate stem cell

populations. ASCs are represented in a variety of organs in the adult mouse. Cells with

stem-like properties have been reported to exist in the bone marrow (7), skeletal muscle

(67), liver (13), pancreas (90), intestinal lining (68), skin (74) and the brain (2, 3, 44)

representing all three of the primordial germ layers: endoderm, ectoderm and mesoderm.

Perhaps the most robust region of stem cell activity in the adult animal is the bone

marrow, in which the hematopoietic stem cell (HSC) vigorously replenishes the myeloid

and lymphoid lineages of the hematopoietic system for the life of the animal. As

isolation of the HSC has not yet been definitively accomplished, the use of a combination

of antibodies against specific cell surface markers has emerged as a technique to highly

enrich the heterogeneous cell population of the bone marrow for the presence of HSCs.

Bone marrow derived cells (BMDCs) negative for the terminally differentiated markers

B220, CD1 lb, and Terl 19 but positive for stem cell antigen (Sca. 1) and the receptor for

steel factor (C-Kit) yield a population of cells enriched for HSCs. Yet functional

reconstitution of the bone marrow must be established for the isolated population of cells

to be defined as a HSC, making the definition of the HSC reliant on a function rather than

identity.









A single highly enriched BMDC transplanted into the peripheral blood of a

myeloablated mouse that displays the ability to home to the injured bone marrow, re-

populate the hematopoietic system, and allow for stable, long-term hematopoiesis to

persist for the life of the animal can be labeled an HSC. The duration of the contributed

hematopoiesis is critical, as this ability is indicative of the transplanted cell undergoing

asymmetric division, a phenomenon where the stem cell shifts between generating an

exact copy of itself and a more lineage committed daughter progenitor cell. This results

in the presence of more HSCs than initially transplanted, with only a small population of

these cells active at any given time allowing for long-term hematopoietic contribution.

This also allows for a vital requirement for the definition of a HSC to be satisfied: serial

transplantation. Work by Krause et al has indicated that a single transplanted HSC can

not only reconstitute the bone marrow of a myeloablated mouse long-term, but also be re-

isolated and transplanted into a secondary myeloablated host and confer the same radio-

protective properties as in the initial transplant (40). From the extensive investigations

into the HSC, a gold-standard definition of a stem cell has emerged: an isolatable cell

capable of asymmetric division, multi-lineage long-term engraftment or contribution to

the native tissue, and serial transplantability. Candidate stem cell populations isolated

from other adult organs would need to meet these same rigorous criteria in order to be

classified as true adult stem cell rather than a progenitor cell.

Neurogenesis in the adult mammalian brain is currently believed to be restricted to

the subependymal zone (SEZ) (44, 47) and the sub-granule cell layer of the hippocampus

(HC) (9, 25, 38). Both regions have been shown to produce migratory daughter cells for

the life of the animal, yet the levels of neurogenesis appear to decrease with age. In vivo









the embryonic NSC has been proposed to contribute to all of the cell types in the

developing brain, but in the adult brain NSCs in both the dentate gyms and SEZ produce

only one cell type: migratory neuroblasts, neural progenitor cells (NPCs) that

differentiate into granule neurons and periglomerular intemeuons. SEZ neuoroblasts

migrate to the olfactory bulb (OB) via a well-defined glial pathway called the rostral

migratory stream (RMS). Migratory neuroblasts divide and differentiate during

migration, eventually functionally integrating into the OB neural circuitry as granule and

periglomerular interneurons (4, 45, 47, 78). Hippocampal NSCs from the subgranule

cell layer generate migratory neuroblasts that migrate a short distance through the granule

cell layer to the mitral cell layer of the dentate gyms, where functional integration as

granule intemeruons occurs (38).

A relative newcomer to the field of stem cell biology, the SEZ NSC has displayed

the potential to be cultured in vitro as both a spherical aggregate of clonal cells known as

a neurospheres (NS) (22, 29, 39, 42, 62-65, 83) and as a monolayer of multipotent

astrocytic stem cells (MASC) (43). Both manifestations display the stem cell properties

in vitro of multi-potency and serial expansion. Upon induction of differentiation, NS and

MASC generated NS are capable of producing neurons, astrocytes, and oligodendrocytes.

They are also capable of increasing in number following repeated passages, although this

expansion is not unlimited.

Transplantation of gp+ NS or MASC into regions of active neurogenesis in the

adult brain results in donor-derived neuroblasts and neurons, while transplantation into

terminally differentiated regions typically results in donor-derived glia, with few

instances of neuronal contribution (23, 26, 80, 89). In SEZ transplantation, engraftment









is often defined by the presence of donor-derived migratory neurons and periglomerular

interneurons in the OB of the host animal. Contrary to HSC engraftment, NSC

engraftment is transient, with the presence of migratory neuroblasts lasting only a few

months, and mono-lineage as only neuroblasts and their resulting neurons are evident

post transplantation (24, 69).

Currently the HSC has not been shown to retain all of the aforementioned

characteristics of a true stem cell in vitro, a shortcoming attributed to an incomplete

reconstruction of the bone marrow microenvironment by the selected culture conditions.

The resulting cell type displays pluripotency and the ability to expand following passages

yet lacks the capacity to reconstitute the bone marrow of a myeloablated mouse, this cell

type has been deemed a hematopoietic progenitor cell (HPC) rather than a true HSC.

While both NS and MASC display the stem cell characteristics of pluripotency,

culture expansion and functional engraftment following transplantation into regions of

proven neurogenesis, little has been done to investigate the potential of the NSC to

survive serial transplantation or to exhibit long-term engraftment. The characteristics of

the NSC with respect to the better-understood HSC were examined to determine the level

of "stem-ness" exhibited by the NSC.

Inability of Gfp+ Neural Stem Cells to Survive Re-isolation Following
Transplantation into the Lateral Ventricles of C57BL/6 Mice

Gfp+ Neurospheres are not Generated from the Brains of Adult C57BL/6 Mice
Transplanted with Gfp+ Neurospheres

Gfp+ NS cultured from day post-embryonic (dpe) 3 gfp+ neonates were dissociated

and transplanted (20,000 cells in 2[tL) into the lateral ventricles (LV) of three month old

C57BL/6 females (n = 15, three independent experiments of 5 animals each) and allowed

to engraft for 3 weeks, at which time the host animals were sacrificed. For each series,









two animals were perfused via intra-cardiac puncture with paraformaldehyde and

prepared for tissue immunohistochemistry to determine levels of engraftment by the

transplanted NS. SEZ tissue was isolated from the remaining 3 animals and cultured for

the generation of NS, with resulting NS analyzed for the presence ofgfp. While a

majority of the transplants resulted in some level of engraftment (Figure 3-1), no gfp+ NS

were evident in any of the cultures, with the NS population consisting only of spheres

similar in appearance to age matched control C57BL/6 NS (Figure 3-2).















Figure 3-1. Gfp+ neurosphere derived cells are evident in the brains of host C57BL/6
mice three weeks following transplantation. Photographic montage of 10x
images from four month C57BL/6 female transplanted with gfp+ NS three
weeks prior to analysis. Arrow depicts the presence of gf+ cells in the neural
tissue surrounding the lateral ventricle (V). Red = P-III tubulin, green = gf.
OB = olfactory bulb, RMS = rostral migratory stream.

Gfp+ Neurospheres are not Generated from the Brains of Neonatal C57BL/6 Mice
Transplanted with Gfp+ Neurospheres

As the yield of engraftment following transplantation of gfp+ NS into the LV of

adult mice is variable and minimal, the assay design was modified to use neonatal

C57BL/6 mice as the recipient animal in hopes that the higher levels of engraftment in

the neonatal model would allow for recovery of the now engrafted, transplanted cells.

As previously mentioned, neurogenesis in the neonatal (dpe 1-3) mouse brain is

















Figure 3-2. Gfp+ neurospheres are not capable of re-isolation following transplantation
into the lateral ventricle of adult mice. NS were cultured from the forebrain of
adult C57BL/6 mice transplanted with gfp+ NS 3 weeks prior. Resultant
spheres were more similar in fluorescence to C57BL/6 control NS than gfp+
control NS derived from age-matched gfp mice. A) Gfp+ control NS derived
from gfp mice. B) C57BL/6 control NS. C) NS isolated from C57BL/6 mice
transplanted with gfp+ NS 3 weeks earlier. All images at 10x magnification.

more active than observed in the adult, theoretically providing higher levels of extra-

cellular cues for differentiation and engraftment of transplanted NSCs. Gfp+ NS

cultured from dpe 1-3 neonatal mice were dissociated and transplanted (75,000 cells,

1 tL) into the LV of dpe 1-3 C57BL/6 mice (n = 9) and allowed to engraft for one month.

Immunohistochemical analysis of three transplanted animals indicated engraftment levels

higher than that seen in the previous adult transplants, with donor-derived cells present in

the SEZ, RMS, and OB (Figure 3-3). The morphology of the donor-derived cells in the

OB are similar to granule neurons, indicating that the transplanted NS cells differentiated

from an immature stem cell to that of a terminally differentiated neuron, presumably via a

migratory neuroblast intermediate.

The remaining 6 transplanted animals were sacrificed and cultured for the

generation of NS, with generated NS analyzed for the expression of gfp as compared to

control NS isolated from both gfp and C57BL/6 mice. NS cultures from the forebrain of

the remaining transplanted animals yielded no gfp+ NS (Figure 3-4) with resultant

spheres more similar to C57BL/6 control NS than gfp+ NS.










































Figure 3-3. Gfp+ neurospheres display high levels of engraftment following
transplantation into neonatal mice. Four weeks post-transplantation, donor-
derived cells from gfp+ NS are present in the SEZ, RMS, and OB of C57BL/6
recipient mice. A) Fluorescent microscopic analysis reveals the presence of
engrafted donor-derived cells at the site of injection in the lateral ventricle
(photographic montage, 10x light microscope images). B) Donor-derived
cells in the OB display extensive integration of processes into the surrounding
neural architecture of the OB (20x confocal image). C) Donor-derived cells
are present in the SEZ of recipient animals and adopt a migratory appearance
(20x confocal image). OB = olfactory bulb, RMS = rostral migratory stream,
V = lateral ventricle. All images: red = P-III tubulin, green = gfp


















Figure 3-4. Gfp+ neurospheres are not capable of re-isolation following transplantation
into the lateral ventricle of neonatal mice. NS were cultured from the
forebrain of adult C57BL/6 mice transplanted with gfp+ NS 4 weeks prior.
Resultant spheres were more similar in fluorescence to C57BL/6 control NS
than gfp+ control NS derived from age-matched gfp mice. A) Gfp+ control
NS derived from gfp mice. B) C57BL/6 control NS. C) NS isolated from
C57BL/6 mice transplanted with gfp+ NS 4 weeks earlier. All images at 10x
magnification.

Gfp+ Multipotent Astrocytic Stem Cells are not Generated from the Brains of
Neonatal C57BL/6 Mice Transplanted with Gfp+ Multipotent Astrocytic Stem
Cells

While NS cultures are an accepted in vitro correlate to the NSC, the protocol for the

generation and dissociation of NS results in low yield and extended culture conditions.

Conversely, the MASC is relatively simple to culture and generates large cell numbers in

a comparatively short time without the presence of mitogens required for NS growth. To

this end, gfp+ MASC were utilized as the transplanted NSC to determine if a cell type

that produces a high yield in culture may reveal the presence of gfp+ cells following

transplantation and re-isolation.

MASC were cultured from the SEZ of dpel-3 gfp neonatal mice, and passage

three times prior to transplantation. This step is necessary for the removal of any

contaminating neuronal cells that may persist in the initial days in vitro but will die off in

the absence of EGF and bFGF. Prior to transplantation, aliquots of prospective MASC

cultures were plated onto adherent chamber slides and allowed to attach. Attached cells









were later stained for the presence of 0-III tubulin positive neurons and GFAP positive

astrocytes (Figure 3-5). Cultures devoid of neurons were collected for transplantation.












Figure 3-5. Passage three gfp+ multipotent astrocytic stem cells contain astrocytes but are
devoid of neurons. Following three passages in vitro, gfp+ MASC were
plated onto adherent chamber slides and analyzed for the presence of 0-III
tubulin positive neurons and GFAP positive astrocytes. Slides analyzed at
20x magnification revealed confluent monolayers of GFAP expressing gfp+
cells with the morphology of astrocytes that contained no observable neurons.
A) Gfp+ MASCs (green) stained for P-III tubulin (red). B) Gfp+ MASCs
(green) stained for GFAP (red). All images at 20x magnification.

C57BL/6 neonatal mice (dpe 1-3, n = 50, 7 independent experiments) were

transplanted with passage-3 gfp+ MASC (100,000 cells per transplant in 1ItL) into the

LV and allowed to engraft for three weeks. In each series of transplants, animals

analyzed for engraftment exhibited levels similar to those seen in previous NS transplants

(Figure 3-6). Donor derived cells were present in the SEZ, RMS and OB, with cells in

the OB adopting a granule neuron morphology. MASC cultures derived from the

forebrain of the remaining animals did not appear to express gfp as compared to controls

(Figure 3-7).

MASC Isolated from the Brains of C57BL/6 Neonatal Mice Transplanted with Gfp
MASC do not Survive Puromycin Treatment.

MASC cultures derived from transplanted mice may contain gfp MASC, albeit in

extremely low amounts. In an effort to enrich the resulting cultures for the presence of









































Figure 3-6. Gjp+ multipotent astrocytic stem cells display high levels of engrattment
following transplantation into neonatal mice. Three weeks post-
transplantation, donor-derived cells from gfp+ MASC are present in the SEZ,
RMS, and OB. A) Fluorescent microscopic analysis reveals the presence of
engrafted donor-derived cells at the site of injection in the lateral ventricle
(photographic montage, 10x light microscope images). B) Donor-derived
cells in the OB display extensive integration of processes into the surrounding
neural architecture of the OB (20x confocal image). C) Donor-derived cells
are present in the SEZ of recipient animals and adopt a migratory appearance
(20x confocal image). OB = olfactory bulb, RMS = rostral migratory stream,
V = lateral ventricle. All images: red = P-III tubulin, green = gfp


gfp positive cells, MASC monolayers were cultured in the presence of puromycin.

Puromycin was the selective agent utilized during the generation of the gfp transgenic

animal (32), and all cells in the gfp animal theoretically express the puromycin resistance









gene along with gfp. Indeed, MASC cultures isolated from the forebrains of gfp neonatal

mice (dpel-3) survived in the presence of 1, 2, and 3 [g/mL concentrations of

puromycin. MASC isolated from the forebrains of control C57BL/6 neonatal mice failed

to survive at any of the three concentrations (Figure 3-8).

MASC cultures isolated from the brains of 18 animals transplanted with gfp+

MASC three weeks earlier were cultured in the presence of 2[g/mL puromycin for nine

days. Surviving cells were returned to normal culture conditions to generate confluent

monolayers after which they were transplanted into the lateral ventricles of neonatal

C57BL/6 mice (dpe 1-3, 100,000 cells, n = 5). Analysis of the brains of transplanted

animals revealed the presence of auto-fluorescent cellular material in the RMS and walls

of the LV, but the noticeable absence ofgfp+ cells in the SEZ/RMS/OB of recipient mice

three weeks following transplantation into the LV (Figure 3-9).









Figure 3-7. Gfp+ multipotent astrocytic stem cells are not present in the cultures of
transplanted neonatal mice. MASC were cultured from the forebrain of
C57BL/6 mice transplanted with gfp+ MASC 3 weeks prior (n = 50, 7
independent experiments). Resultant cells did not express gfp as compared to
age matched gfp+ MASC control cultures. A) Gfp+ MASC control culture.
B and C) MASC cultures derived from C57BL/6 mice transplanted with gfp+
MASC 3 weeks earlier. All images at 20x magnification.

To ensure that any cells surviving exposure to puromycin were indeed resistant and

not merely remnants of puromycin-negative cells, MASC isolated from primary

transplanted animals (n = 8) were treated with 2 [g/mL puromycin for nine days.

Surviving cells were collected and re-cultured to generate a secondary monolayer of










2 ug/ml Puro


C57BL/6







GFP


Figure 3-8. Qualitative assessment of the effects of puromycin on multipotent astrocytic
stem cells cultures derived from C57BL/6 and gfp+ neonatal mice. Passage 2
MASC cultures were grown in the presence of puromycin for nine days at 1, 2
and 3 [g/mL puromycin. The above 10x phase-contrast images reveal that
while all three concentrations of puromycin are lethal to C57BL/6 MASC, gfp
MASC proliferate regardless of the concentration of the selective agent.

















Figure 3-9. Transplantation of puromycin selected multipotent astrocytic stem cells
isolated from C57BL/6 mice transplanted with gp+ multipotent astrocytic
stem cells exhibit no gfp donor cell engraftment. Three weeks following
transplantation, recipient animals display no evidence of gp+ cells. Auto-
fluorescent cellular material is present in the RMS and LV of transplanted
animals, indicative of lipofusion or apoptotic cellular debris. A) RMS of
transplanted animal, 10x gfp image. B) 10x image of RMS depicted in A, red
= 0-III tubulin. C) Merged image of A and B. D) LV of transplanted animal,
10x gfp+ image. E) 10x image of LV depicted in D, red = P-III tubulin. F)
Merged image of D and E.


1 ug/ml Puro


3 ug/ml Puro









MASC, at which time the cells were again cultured in 2 [g/mL puromycin for nine days.

Of an estimated 1 x 106 cells exposed to a secondary puromycin treatment, only four cells

remained. None of the four cells analyzed expressed gfp, and had a punctate, unhealthy

morphology compared to age matched gfp MASC cultures (Figure 3-10), indicating that

no gfp cells exist in the secondary cultures of animals transplanted with gfp+ MASC.

Long Term Engraftment

As previously mentioned, long-term engraftment is an essential quality possessed

by the HSC and for the NSC to be classified as a true stem cell it must display a similar

capability. Three neonatal C57BL/6 (dpel-3) were sacrificed 14 months following

transplantation of gfp+ NS into the lateral ventricle. Analysis of sagittal sections of these

animals revealed the existence of donor-derived cells in the olfactory bulb, parenchyma,

and SEZ. These donor-derived cells appeared as either granule cells with extensive

processes or as cells with an astrocytic morphology (Figure 3-11). Of particular interest

was the noticeable lack of migratory neuroblasts in the RMS of these animals. The

absence of these cells would argue that long-term contribution to the migratory

neuroblast population was not provided by the transplanted NS, with only transient

contribution to the RMS and OB intemeurons provided by the transplanted cells.










































Figure 3-10. Multipotent astrocytic stem cells isolated from the forebrains of mice
transplanted with gfp+ multipotent astrocytic stem cells do not survive
puromycin treatment. Following isolation and culture, MASC from
transplanted forebrains were treated with 2[g/mL puromycin twice for a
duration of nine days per treatment. Surviving cells were not gfp positive as
compared to gfp controls. A) Gfp control MASC, 10x phase contrast image.
B) Gfp control MASC, 10x image, green = gfp. C) Cell which survived
puromycin selection, 10x phase contrast image. D) Same cell depicted in C,
gfp filter. E) Cell which survived puromycin selection, 10x phase contrast
image. F) Same cell depicted in E, gfp filter.













































Figure 3-11. Donor-derived cells exist in the brains of gfp neurosphere transplanted
animals 14 months after surgery. Dissociated gfp+ NS were transplanted into
the lateral ventricles of neonatal C57BL/6 mice, and allowed to engraft for 14
months. Donor-derived gfp+ cells were present in the olfactory bulb as
granule neurons with extensive processes and in the walls surrounding the
lateral ventricles and subependymal zone as astrocytic cells. No donor-
derived cells were observed in the rostral migratory stream of these animals.
A) Sagittal photographic montage of C57BL/6 mouse transplanted with gfp+
NS 14 months earlier (10x images). B) 20x image of donor-derived cell in the
SEZ. C) 20x image of donor-derived cell in the parenchyma surrounding the
lateral ventricle. D) and E) 40x images of donor-derived cells in the ob
adopting the morphology of granule neurons. All images: red = P-III
Tubulin, green = gfp. OB = olfactory bulb, RMS = rostral migratory stream,
SEZ = subependymal zone, V = lateral ventricle.














CHAPTER 4
IONIZING RADIATION ENHANCES THE ENGRAFTMENT OF TRANSPLANTED
IN VITRO DERIVED NEURAL STEM CELLS

Introduction

Neurogenesis in the adult rodent is limited to two well-characterized regions of the

brain: the subgranular layer of the hippocampal dentate gyms, and the subependymal

zone (SEZ) (9, 25, 27, 38). The former produces neurons that functionally integrate into

the granular cell layer of the hippocampus. The SEZ produces neuroblasts in the walls of

the lateral ventricles that migrate along a defined pathway, known as the rostral migratory

stream (RMS), to the olfactory bulb (OB) where they differentiate and functionally

integrate into the existing cytoarchitecture as granule or periglomerular interneurons (45,

46, 47, 78).

These migrating neuroblasts have been well characterized, and are known to be

immunopositive for both the pan-neuronal marker P-III-tubulin, and the polysialylated

neuronal cell adhesion molecule (PSA-NCAM), an antigen restricted mainly to cells in

the brain that are undergoing active migration (15). The number of newly generated

neurons produced daily in the adult mouse has been estimated at 30,000, leading many to

conclude that a self-renewing stem cell must reside in the SEZ in order for this rate to be

sustained for the life of the animal (5). The cell type in the SEZ believed to be the NSC

has been identified by Doetsch and colleagues as a slowly dividing astrocyte known as

the type B cell, that generates the migratory neuroblasts (type A cells) via a transit

amplifying intermediate precursor (type C cell) (4).









Neural stem cells can be isolated from the adult SEZ and cultured in vitro to form

spherical clones known as neurospheres (NS) (22, 29, 39, 42, 62-65, 83), that are capable

of producing the major cell types of the neural lineage (neurons, astrocytes, and

oligodendrocytes) upon differentiation. They are also capable of self-renewal, allowing

them to increase in number following repeated passages in culture while retaining their

multipotency, and have also shown the ability to integrate into host neural tissue upon

transplantation into the host brain (80). It is these traits that have led many to believe that

the NS-forming cell is the in vitro correlate of the in vivo NSC. Multipotent astrocytic

stem cells (MASC) isolated from the SEZ of neonatal mice have also been identified as

an in vitro correlate of the NSC, displaying the potential to not only generate multipotent

NS but also to integrate into host neural tissue upon transplantation in much the same

way as the NS (43, 89). The relatively simple culture conditions required in maintaining

the MASC in vitro offer an attractive alternative to the more complicated conditions

required by the NS.

Transplantation of in vitro NSC (NS or MASC) derived from animals transgenic

for the reporter gene encoding green fluorescent protein (gfp+) into the LV of adult

control animals results in minimal engraftment into the host neural tissue, with few

donor-derived neuroblasts present in the RMS or OB. Transplantation into neonatal

mice, however, results in relatively robust levels of engraftment with comparatively high

numbers of donor-derived neuroblasts and interneurons present in the OB weeks after

transplantation. This presents a problem for research into adult neurogenesis, as the

neonatal and adult brains are dramatically different with respects to active neurogenesis

and results obtained from neonatal transplants may not be applicable to adult studies. A









method for enhancing the engraftment potential of in vitro NSC into adult animals is

crucial in order to better understand the receptiveness of the adult SEZ to transplanted

cells and consequently the functional identity of the transplanted cells.

Perhaps the most well characterized adult stem cell is the hematopoietic stem cell,

which in addition to the aforementioned stem cell characteristics of pluripotency and self-

renewal also has the ability to repopulate the entire hematopoietic system of a

myeloablated animal. Ablation of endogenous HSCs by exposure to high doses of

ionizing radiation is critical for facilitating maximum engraftment of transplanted HSCs,

that cannot normally compete with the native HSC for access to the stem cell niche (18,

19, 20). This reconstitution ability is an essential component in the generally accepted

classic definition of a HSC.

Depletion of the NSC niche has been previously achieved with anti-mitotic agents

(15) and both ionizing and x-irradiation (6, 51, 59, 60, 71, 72) yielding transient and

long-term depletion of neurogenesis in the hippocampus and SEZ. Neurogenesis in the

hippocampus can be attenuated by exposure to varying levels of x-irradiation, as seen by

a decrease in the number of migrating granular neurons out of the subgranular layer (52,

59, 60, 71).

Previous studies investigating the effects of on SEZ neurogenesis following

focused exposure of x-irradiation to the brain of adult rats have reported ablation of of

mitotic cells in the SEZ immediately following irradiation, with a dose dependent

recovery of these cells occurring within 2 months of exposure (6, 72). With the

exception of all but the lowest level of exposure, the observed recovery of neurogenesis

remained well below control levels. Little has been done, however, to characterize the









effects of a single, whole-body lethal or sub-lethal dose of ionizing radiation on SEZ

neurogenesis and subsequent engraftment of transplanted in vitro derived NSC in the

adult mouse.

Effects of Lethal Irradiation on Subependymal Zone Neurogenesis

Lethal Irradiation Severely Depletes Migrating Neuroblasts in the RMS

Three weeks following lethal irradiation (LI), bone marrow reconstituted mice

show a marked decrease in the number of P-III tubulin positive neuroblasts in the RMS

(n = 8, Figure 4-1 D). This decrease is variable; some animals were found to have few

or no migrating neuroblasts, while others contained a number of pockets of neuroblasts,

though always drastically fewer than that seen in un-treated littermates (Figure 4-1 B).

To determine if this reduction is permanent, (as opposed to the transient reduction

observed with anti-mitotic treatments) animals were analyzed 3 months after LI. The

results indicate that the depletion of migratory neuroblasts persists even at this time point

(Figure 4-2). To show that the decrease in the number of 0-III tubulin positive

neuroblasts is not due merely to antigen down-regulation, sagittal sections of brain from

LI mice were also stained for the presence ofPSA-NCAM; a marker specific to migrating

neuroblasts (46). Three weeks following LI, the number of migrating PSA-NCAM

positive neuroblasts was severely depleted in the RMS of those mice (Figure 4-1 C) as

compared to un-treated littermates (Figure 4-1 A), again with a small portion of

migratory neuroblasts remaining in the RMS (* in Figure 4-1 C). These findings led to

the conclusion that the exposure to x-irradiation resulted in permanent damage to the

neuroblast-producing cell of the SEZ, but due to the occasional observed cluster of

migratory neuroblasts, accurate quantification of this depletion was performed.




























Figure 4-1. Lethal irradiation drastically reduces the volume of PSA-NCAM and P-III
Tubulin positive migratory neuroblasts. Sagittal photographic montages of 5x
light microscopic images. A and B) Untreated C57BL/6 female brain. C and
D) C57BL/6 female, 3 weeks post-lethal irradiation. Note the absence of 0-III
Tubulin positive migratory neuroblasts in the LI brain (D) compared to
control (B) and the diminished volume of PSA-NCAM positive migratory
neuroblasts (C) as compared to control (A). Red = PSA-NCAM, Green = 3-
III Tubulin. OB = olfactory bulb, RMS = rostral migratory stream, LV =
lateral ventricle. (*) denote migratory neuroblasts.











Figure 4-2. Reduction in the volume of 0-III Tubulin positive migratory neuroblasts
persists months after lethal irradiation. Sagittal photographic montages of 10x
light microscopic images. Red = P-III Tubulin. A) C57BL/6 female brain
two months post-LI. B) C57BL/6 female brain three months post-LI. Note
the absence of migratory neuroblasts in both images as compared to non-
irradiated controls (Figure 4-1, B).









Analysis and Quantification of Neuroblast Depletion in the Subventricular Zone
Following Lethal Irradiation, as Determined by BrdU Incorporation

In order to quantify the degree of neuroblast depletion, 5-bromo-2'-deoxyuridine

(BrdU) was used to label mitotic cells within the SEZ. Briefly, 3 weeks and 3 months

following LI (n = 4 for each condition), treated and un-treated mice were given BrdU

intra-peritoneally three times a day for three days allowing for the incorporation of BrdU

into the DNA of dividing cells. SEZ neuroblasts were analyzed by the application of

antibodies against P-III tubulin and BrdU on serial coronal sections at the level of the

anterior commisure (AC). BrdU positive cells were scored blindly, by analyzing three

adjacent sections per brain (scorer was ignorant to the experimental condition of the

animals analyzed). Scoring of adjacent sections was performed only on those sections

where the AC existed as a spherical white-matter structure, directly inferior to the inferior

most extent of the LV. The region of analysis was maintained throughout all animals

scored (Figure 4-3).

In all un-treated animals, the SEZ was observed to contain a robust layer of

dividing neuroblasts (Figure 4-4 A). 3 weeks following LI in age matched mice this

same region exhibited a significant decrease in neuroblasts activity (Figure 4-4 B). This

depletion was observed out to 3 months post-LI (Figure 4-4 C). P-III tubulin

immunolabeling (green, Figure 4-4 insets) was used to confirm that the cells scored were

in fact neuroblasts and not mitotic glial cells of the SEZ. .

Figure 4-5 is a graphical representation of the number of BrdU positive cells for

each condition (4 mice per condition, 3 sections per animal). 3-weeks following LI, the

volume of BrdU positive SEZ neuroblasts decreases to 40% of control. Student's t-test

confirmed that this difference was significant (p value = 0.003).












































Figure 4-3. Region of analysis for quantification of mitotic neuroblast depletion in the
subependymal zone of lethally irradiated mice. Coronal photographic
montage of 10x light microscopic images, with the white outline depicting the
region of analysis as encompassing an area from the inferior, descending horn
of the LV to the superior horn of the LV, and continuing 700[tm lateral from
the superior horn while remaining approximately 5 cells in width. Inset:
reprentative image of BrdU positive neuroblasts lining the wall of the lateral
ventricle expressing P-III tubulin. CC = corpus callosum, LV = lateral
ventricle, ST = striatum, SP = septum, and AC = anterior commissure. Red =
BrdU, Green = P-III tubulin.


























Figure 4-4. Depletion of mitotic neuroblasts in the subependymal zone of lethally
irradiated animals. The observable volume of BrdU positive (red) neuroblasts
is severely diminished three weeks and three months post-LI as compared to
non-irradiated controls. A) Non-irradiated control. B) 3 weeks post-LI. C) 3
months post-LI. All are coronal photographic montages of 10x light
microscope images. All insets are 40x images of neuroblasts lining the wall of
the ventricles, positive for both BrdU and P-III tubulin (green) identifying
them as neuronal.

There is an approximate 87% decrease in the number of BrdU positive cells at the

3-month time point as compared to control, and this difference is also significant

(p value = 0.002). The 68% decrease in the number of BrdU positive cells between 3

weeks and 3 months post LI was also significant (p value = 0.03), indicating that the

observed depletion is permanent.

The depletion of mitotic cells in the SEZ is also observed in the RMS, with a

marked decrease in the visible BrdU/P-III tubulin positive migratory neuroblasts one

month following LI (Figure 4-6).

Diminished Neurosphere Yield from Lethally Irradiated Mice

As it is well accepted that the NS forming cell is the in vitro manifestation of the

NSC (63), NS from both WT and LI mice were cultured in a blind-study format to

determine if the stem cell pool in the SEZ was affected by exposure to LI.











50

p <0.05
45

S40

a 35

S30

S25
p = <0.05
20

15

10



0 1
3 weeks 3 months
Time Post-Ihradiation

Figure 4-5. Lethal irradiation significantly decreases the number of BrdU positive SEZ
neuroblasts. The total number of BrdU positive neuroblasts in three adjacent
coronal sections of either wild-type or LI mice was tabulated and placed into
the above graphical format. Three weeks following lethal irradiation (n=4),
the number of BrdU positive neuroblasts decreased to 40% of those calculated
to exist in the wild-type controls (n = 4, p value of 0.003). Three months
following lethal irradiation, this decrease is slightly greater, at 13% of control
(n = 4, p value of 0.002). Analysis by student's t-test confirmed these values
to be significant, as compared to non-irradiated controls.













































Figure 4-6. Depletion of migratory neuroblasts in the rostral migratory stream positive
for both BrdU and P-III Tubulin in lethally irradiated mice. Sagittal
montages of 20x images reveal that the visible volume of 0-III tubulin/BrdU
positive cells is severely diminished in LI mice as compared to non-irradiated
controls. A) Non-irradiated control. B) LI mouse, one month post-LI.
Animals were injected with BrdU according to the protocol described in
materials and methods. Brains were sectioned and stained for BrdU (red) and
P-III tubulin (green). Ob = olfactory bulb, rms = rostral migratory stream,
V = lateral ventricle.









NS cultured from LI brains displayed a significant decrease in yield of

approximately 77%, when compared to the non-irradiated control cultures (Figure 4-7, p

value = 0.02). This decrease closely corresponds to the decreased levels of BrdU positive

neuroblasts in vivo following lethal irradiation, further supporting the validity of this

finding. It has been reported that the stem cell population of the SEZ is between

approximately 0.02% and 1.0% of the total cells (62, 66, 82), as determined by NS yield

from dissociated SEZ tissue, and the average yield of NS from the wild-type brains in the

present study falls within this range (0.15%). The average yield of NS isolated from the

lethally irradiated brains was significantly lower, at 0.03%, indicating that exposure to

lethal doses of radiation depletes the number of NSC in the brain responsible for the

generation of NS.

Preliminary studies revealed no effect on the potential for differentiation was

observed in NS isolated from LI animals one month following irradiation as compared to

non-irradiated controls. The approximate diameter of the resultant NS was comparable to

controls (Figure 4-8), and when placed on adhesive substrates in the absence of growth

factors to induce differentiation NS from LI mice produced both astrocytes and neurons.

LI appears to only affect the yield of NS isolated from irradiated animals, with the few

spheres formed retaining a normal phenotype, although further studies are needed to

verify that irradiation in no way affects NS differentiation potential.

Lethal Irradiation Attenuates Engraftment of Transplanted Gfp+ Multipotent
Astrocytic Stem Cells

Three weeks after irradiation, LI animals were transplanted with gf+ MASC

(generated from neonatal forebrains) into the lateral ventricle and the cells allowed to

engraft for three weeks.














350

ii 300

250

200

S150

p = <0.05
100
W



0

Wild Type Lethal Ihadiation


Figure 4-7. Lethal irradiation significantly reduces the yield of neurospheres cultured
from the adult subependymal zone. SEZ tissue isolated in a blind-study
format was cultured to generate NS from both wild-type adult mice and adult
mice LI two months prior, with all tissues treated in identical fashion (n=3).
The resulting NS yield was determined according to the protocol described in
the methods section. The LI cultures averaged 77% fewer NS than did the
identical cultures derived from control mice (significant decrease; p value of
0.02).

A









Figure 4-8. Neurosphere differentiation potential. NS isolated from non-irradiated
control animals are capable of differentiation into astrocytes and neurons upon
attachment to adhesive substrates in the absence of EGF and bFGF. A) 10x
phase contrast image of NS derived from non-irradiated control. B) 20x
image of differentiated control NS stained for GFAP (red). C) 40x image of
differentiated control NS stained for P-III tubulin (red) and DAPI (blue).













































Figure 4-9. Attenuation of gfp multipotent astrocytic stem cell engraftment by lethal
irradiation. Control animals exhibited normal engraftment, as evidenced by
the presence of donor-derived migratory neuroblasts. A) Photographic
montage of 10x images from a sagittal section of non-irradiated animal two
weeks post transplant (red = P-III tubulin, green = gfp). B and C) 40x images
of donor-derived migratory neuroblasts in non-irradiated control. LI animals
displayed no evidence of engraftment, with no donor-derived cells seen in
either the SEZ, RMS, or OB. D) Photographic montage of 10x images from a
sagittal section of LI animal two weeks post transplant. E) 20x image of 0-III
tubulin positive migratory neuroblasts in the RMS. F) Same image as E, but
with gfp filter, revealing the absence of donor-derived migratory cells.
OB = olfactory bulb, RMS = rostral migratory stream, V = ventricle.


A




V
ob



1,111S









Analysis of 40 |tm sagittal sections revealed the presence of donor-derived cells

only in the corpus callosum, parenchyma and SEZ, but the absence of donor-derived

migratory cells in the RMS or OB (n = 5), while non-irradiated controls were observed to

contain a small but consistent number of donor-derived migratory neuroblasts in the OB

(Figure 4-9).

As the lethal dose of ionizing radiation inhibited functional engraftment of the

transplanted cells (possibly by inducing irreparable damage to the radio-sensitive support

cells of the SEZ) a lower exposure dose of 450 rads was assayed as a milder form of

injury for the potential enhancement of gfp MASC engraftment.

Effects of Sub-Lethal Irradiation on Subependymal Zone Neurogenesis

Sublethal Irradiation Results in a Transient Decrease in the Number of Mitotic
Subependymal Zone Neuroblasts.

Unlike LI, SLI (450 rads) does not complete abolish hematopoiesis in the bone

marrow of adult mice, and SLI animals to not require transplantation of bone marrow to

survive exposure. Low levels of focused gamma irradiation (1 to 3Gy) have been shown

to result in a transient increase in mitotic cell activity in the SEZ, with levels eventually

diminishing in the weeks following in a dose dependent fashion when compared to

untreated controls (6, 72).

Contrary to the observations following LI, no decrease was observed in the overall

volume of 0-III tubulin positive migratory neuroblasts in the RMS of irradiated animals 3

weeks following whole body exposure to 450rads (a dose equivalent to 4.5Gy).

BrdU quantification assays revealed that mitotic cell activity in the SEZ was significantly

decreased in the hours immediately following irradiation (Figure 4-10). The levels of









mitotic cell activity returned to near normal at 3 weeks and eventually increased to above

control levels at 6 weeks (Figure 4-11).

Diminished Neurosphere Yield from Sublethally Irradiated Mice

Blind-analysis of NS yield (same protocol as the LI study, n = 4) from SLI brains at

3 weeks revealed a 30% decrease in the number of NS as compared to non-irradiated

controls (Figure 4-12, p = 0.032), with control NS yield again falling into the

aforementioned acceptable range of 0.2 to 1.0% (0.12%). As in the LI studies, no

observable effect was observed in preliminary studies of NS potential in SLI mice,

although further studies are needed to verify this initial observation.

Sublethal Irradiation Enhances Engraftment of Transplanted Gfp+ Multipotent
Astrocytic Stem Cells

Three weeks following irradiation, SLI animals were transplanted with gf+

MASC into the lateral ventricle and the cells were allowed to engraft for three weeks.

Analysis of 40 |tm sagittal sections revealed that in a portion of the irradiated animals a 4

fold higher number of gfp migratory cells were present in the OB as compared to non-

irradiated controls (n = 14, Figure 4-13). Taken as a whole, the average number of g+

migratory cells in the OB of SLI animals was twice the average number seen in non-

irradiated controls (significant value, p = 0.014) indicating that SLI significantly

enhances the engraftment potential of transplanted gfp+ MASC, albeit in a variable

fashion (Figure 4-14).




























Figure 4-10. Mitotic subependymal zone neuroblasts are transiently depleted following
sub-lethal irradiation. 6 hours following irradiation, the SEZ is almost
completely devoid of BrdU (red) cells as compared to non-irradiated controls.
3 weeks following irradiation, BrdU positive cells in the SEZ are near to the
levels observed in controls. A) Non-irradiated control. B) 6 hr post-SLI. C)
3 weeks post-SLI. All are coronal photographic montages of 10x light
microscope images. All insets are 40x images of neuroblasts lining the wall of
the ventricles, positive for both BrdU and P-III tubulin (green) identifying
them as neuronal.











120.
pp=<00.05

S100


80


0 0 60


c 40


neuroblasts than in non-irradiated controls (p = 0.001, n = 3). This depletion<0.05
20 p <0.05



6 hours 24 horn's 48 hours 3 weeks 6 weeks

Time after Irradiation

Figure 4-11. Sublethal irradiation results in transient, recoverable depletion of mitotic
subependymal zone neuroblasts. The total number of BrdU positive
neuroblasts in three adj acent coronal sections of either control or SLI mice
was tabulated and placed into the above graphical format for quantification of
the number of BrdU positive SEZ neuroblasts calculated as percent of control.
Six hours following SLI there are approximately 83% fewer BrdU positive
neuroblasts than in non-irradiated controls (p = 0.001, n = 3). This depletion
persists 24 (78%,p = 0.001, n =3) and 48 hours (90%,p = 0.001, n = 3)
following SLI. Three weeks following irradiation the number of mitotic SEZ
neuroblasts recovers to near control levels, and eventually increases to 13%
above control levels at 6 weeks (p = 0.003, n = 3).











160


S140
II

S120o
a p = <0.05
I-
100


so80

S60





20


0

Control Sub-Lethal Irradiation

Figure 4-12. Sublethal irradiation significantly reduces the generation of neurospheres
cultured from adult subependymal zone. SEZ tissue was isolated in a blind
format from both non-irradiated control adult mice and adult mice SLI three
weeks prior and cultured to generate NS, with all tissues treated in identical
fashion. The resulting NS yield was determined according to the protocol
described in the methods section. The SLI cultures averaged 30% fewer NS
than did the identical cultures derived from control mice (significant decrease;
p = 0.032, n = 4).




























Figure 4-13. Gfp+ multipotent astrocytic stem cells transplanted into the lateral
ventricles of control and sub-lethally irradiated mice produce donor-derived
migratory cells in the host olfactory bulb. Age matched mice (SLI and non-
irradiated controls) were sacrificed three weeks following intra-ventricle
transplantation of passage 3 gf+ MASC. 40 [im sagittal sections of the
transplanted hemisphere were collected in a serial fashion, stained for P-III
tubulin (red) and analyzed by light and confocal microscopy for the presence
ofgfp+ donor-derived cells (green). A and D): 10x photographic montages of
control and SLI mice (respectively). Circles represent the presence of donor-
derived migratory neuroblasts, with representative cells captured at 40x
(insets). B and C) 63x confocal images ofgfp+ migratory cells in the
olfactory bulb of non-irradiated control mice. E and F) 63x confocal images
ofgfp+ migratory cells in the olfactory bulb of SLI mice. Scale bars = 40[m.














p = <0.05


.5 25


02



2|0----
t 15
0


It
E 5 -----


0 1


Control


Sub Lethal Irradiation


Figure 4-14. Sublethal irradiation enhances engraftment of donor-derived neuroblasts.
Quantification of 14 transplanted animals per condition revealed the presence
of approximately twice as many donor-derived migratory neuroblasts in the
olfactory bulb proper of SLI animals as compared to non-irradiated controls (p
= 0.014). Transplanted hemispheres were sectioned three-weeks following
transplantation of passage 3 gfp+ MASC into the lateral ventricle. All
sections containing the olfactory bulb were collected in a serial fashion and
stained for the presence of 3-III tubulin.














CHAPTER 5
DISCUSSION AND CONCLUSIONS

Neurogenesis in the subependymal zone (SEZ) of the adult mouse persists for the

life of the animal, alluding to the existence of a persistent neural stem cell (NSC) pool in

this region. While NSCs cannot yet be prospectively purified like the hematopoietic stem

cell (HSC), they can be cultured under specific conditions to form clonal neurospheres

(NS) and multipotent astrocytic stem cells (MASC); potential NSC manifestations with

multipotent characteristics attributed to stem cells.

The extensive investigation of the HSC has produced a gold-standard definition of

a stem cell: a single cell that is capable of producing all cell types of a particular organ

for the life of the animal via asymmetrical division in which both an exact duplicate of

the stem cell and a lineage-committed progenitor daughter cell are generated.

Additionally, the stem cell can reconstitute its native niche following transplantation, and

this ability is retained following secondary transplantation, a phenomenon known as

serial reconstitution. Functional transplantation of a stem cell and its subsequent

reconstitution of the niche is a vital requirement, as the isolated cell can now be

considered a useful tool for tissue repair. Candidate stem cells from other organs would

need to meet the same criteria if they are to be classified as true stem cells.

Both NS and MASC transgenic for gfp exhibited low levels of engraftment upon

transplantation into the lateral ventricles of adult mice, with few donor derived migratory

neuroblasts in the RMS observed in the weeks immediately following transplantation.

Neither the NS nor the MASC displayed the ability to be re-isolated following









transplantation into proven regions of neurogenesis, a limitation resulting in the inability

of either cell types to survive subsequent serial transplantation. As neurological

development continues shortly after birth and potentially provides an environment rich in

extra cellular cues favored by NSCs, transplantation into the brains of neonatal mice was

performed in an effort to enhance the engraftment of transplanted NS and MASC. While

the levels of engraftment were visibly higher than those seen in adult transplants, neither

cell type survived secondary isolation attempts, even with the use of selective protocols

designed to enrich for gfp+ cells.

Long-term analysis of transplanted animals revealed the existence of donor-derived

granule neurons and astrocytic cells in the olfactory bulb, parenchyma and SEZ, but a

noticeable absence of migratory neuroblasts in the rostral migratory stream (RMS) of the

transplanted animals. This potentially alludes to transient engraftment as opposed to

long-term engraftment.

NSC engraftment could be defined in this case as long-term contribution to

neuroblast pool in the adult animal following transplantation, with functionality exhibited

by the existence of terminally differentiated donor-derived cells in the neural circuitry of

the olfactory bulb. HSC engraftment into the bone marrow of myeloablated mice is

evidenced by the robust generation of daughter cells representing both the myeloid and

lymphoid lineages. The obvious evidence of functional engraftment by the transplanted

HSC is that the myeloablated animal survives the previous lethal dose of radiation; the

depleted bone marrow becomes repopulated by the transplanted HSC and its resultant

progeny. Long-term (i.e. 3 months or longer) engraftment and hematopoietic

contribution is a critical requirement in the definition of the HSC, as short-term, transient









engraftment can be supplied by hematopoietic progenitor cells. The ability to survive

serial-transplantation while retaining the capacity to provide long-term bone marrow

reconstitution fulfills the final requirement for classification as a true stem cell.

Currently, the liver hepatocyte is the only adult stem cell other than the HSC that has

displayed the potential for serial, functional engraftment. In a liver repopulation assay,

transplanted hepatocytes functionally contributed to the regenerating liver in a robust,

serial fashion with contribution observed following the sixth transplantation (58).

The results obtained reported here indicate that the in vitro derived NSCs were not

capable of re-isolation following transplantation into proven regions of neurogenesis.

Whether this is due to the cell population transplanted or the niche into which the cells

were placed is not known. The failure observed in the adult model could be attributed to

the decreased level of neurogenesis in the adult animal, resulting in fewer engrafted cells

and subsequently fewer isolatable cells. However, because transplants into neonatal mice

yielded the same results the cause likely lies in the cells transplanted rather than in the

niche itself. Both NS and MASC cultures are heterogeneous in nature, with cells existing

in varying stages of maturation. It is possible that the cell types observed to be

multipotent and proliferative in culture are progenitor cells derived from a relatively

small number of NSCs, implying that not every NS or MASC in culture is a NSC.

Transplantation into the SEZ results in mono-potent engraftment with only neurons

and neuroblasts observed in the RMS and olfactory bulb, an observation concurrent with

progenitor cell activity. Stable engraftment by the NSC should result in an increase of

donor-derived NSC via asymmetric division, allowing for subsequent re-isolation.

While initial engraftment was observed in the weeks immediately following









transplantation as evidenced by the presence of donor derived cells in the SEZ, RMS and

olfactory bulb, the engraftment ultimately was found to be transitory. As it was not

possible in the experiments performed to re-isolate a donor-derived NS or MASC, the

data presented here would indicate that the in vitro manifestations of the NSC are not true

stem cells, but rather a form of neural progenitor cell lacking the characteristics attributed

to true stem cells and may not be a viable cell source for potential stem cell therapy of

neurological injury and disease.

It is entirely possible that the observed neurogenesis in the adult brain is not the

result of an endogenous, isolatable NSC pool, but rather the product of migratory adult

stem cells that undergo a phenotypic shift upon integration into neurogenic regions of the

brain and subsequently give rise to more lineage committed neural progenitor cells.

Observations supporting the contribution of the HSC to adult neurogenesis are as

numerous as they are conflicting. Donor-derived microglia and astrocytes (17) and

neurons (8, 49) have been reported to exist in the brains of animals exposed to lethal

irradiation and reconstituted with HSC transgenic for reporter proteins (17), while a

recent investigation has argued strongly for the existence of non-fusion product, donor-

derived neurons in the hippocampus of humans following sex-mismatched bone marrow

transplant (12). It has also been suggested that 0.5% of astrocytes in the adult brain are

generated from a bone marrow derived cell (17), and it is generally accepted that the NSC

of the SEZ (the type B cell) is a form of astrocyte (4, 5, 15). With this in mind, the

proposal that adult neurogenesis is driven by an adult stem cell residing in the bone

marrow may not be implausible.









While the neonatal transplant model provides an ideal system for relatively robust

engraftment of transplanted NS and MASC, it is impractical for the investigation of adult

neurogenesis and adult NSC potential as the adult brain is more quiescent than the

developing neonatal brain. A technique is necessary to render the neurogenic niche (in

this case, the SEZ) more receptive to transplanted cells. The current tactic in the field of

hematopoiesis to maximize engraftment of transplanted HSC is to first deplete the host

bone marrow of HSC by exposure to lethal doses of radiation (myeloablation). This

renders the hematopoietic stem cell niche more receptive to transplanted cells, allowing

for stable long-term, multi-lineage engraftment. It was hypothesized that depletion of the

native NSC pool by lethal irradiation would render the SEZ more receptive to

transplanted cells and subsequently enhance the engraftment efficiency of the

transplanted NSC.

Because a single, high dose of ionizing radiation is sufficient to permanently

deplete the bone marrow of viable HSC in adult mice, a similar result should be observed

in the SEZ, with the pool of NSC being similarly depleted. The number of mitotic cells

in the SEZ was decreased by approximately 60% three weeks following irradiation and

this decrease was slightly lower three months later, at 87%, indicating a long-term, if not

permanent depletion. The levels of migrating neuroblasts in the RMS reflected this

decrease, with the chains decreasing in volume at both three week and three month time-

points.

If the NSC pool itself had been diminished by the radiation exposure, the number

of NS cultured from those brains should reflect this decrease in the number of BrdU

positive neuroblasts. In fact, it was observed that there were approximately 77% fewer









NS isolated from the brains of LI adult mice that had been irradiated two months earlier.

Preliminary observations indicated that only the yield of NS appeared to be affected, with

the resulting spheres being similar in size and multipotency to control NS. In addition,

the resulting cultures from control tissue supported earlier findings that the number of

NSC in the SEZ is between 0.02 and 1.0% (62, 66, 82), with the average NS yield in

cultures being approximately 0.15% of the SEZ tissue cultured. The percent yield of NS

cultured from the SEZ of LI mice was diminished, at 0.03%. These observations lead to

the conclusion that the SEZ is significantly depleted of NSC following LI, and that this

depletion is long-term, if not permanent. The reason for this permanent depletion of

neurogenesis is not entirely clear, although recent studies have indicated that the neuro-

inflammatory response to injury in the hippocampus inhibits neurogenesis by disruption

of normal stem cell function (52).

While the most prevalent in vitro manifestation of the NSC is the NS, the MASC

has recently emerged as an alternative to the NS. The NSC in vivo is generally accepted

to be a slowly dividing astrocytic cell described as the type B cell (4) residing in the

ciliated ependymal layer of the SEZ. Work by Laywell et al. demonstrates that by

surgically dissecting the SEZ of neonatal animals and culturing the subsequently

generated dissociated cells in neural media on adhesive substrates, a monolayer of

astrocytes is produced that contains a sub-population of MASC capable of generating

multipotent NS (43). Transplantation of gf+ MASC into the lateral ventricles of both

neonatal and adult C57BL/6 mice results in observable engraftment, with donor-derived

cells present in not only the SEZ but also in the RMS and OB as migratory neuroblasts









(89). Taken as a whole, these observations allude to the MASC being an acceptable in

vitro manifestation of the NSC.

Transplantation of gf+ MASC into the lateral ventricles of LI mice three weeks

following irradiation did not result in the expected increase of donor-derived migratory

cells in OB. Non-irradiated controls exhibited normal engraftment levels while

transplanted LI animals were observed to only contain donor-derived cells in the corpus

callosum and SEZ. While it is unknown why the transplanted cells did not engraft

normally into the neurogenic regions of LI animals, recent work has proposed that

deleterious effects to the microvasculature occur in the dentate gyms following 10Gy of

focused irradiation rendering the region unreceptive to transplanted cells (51) and a

similar phenomenon may occur here.

As LI was not observed to enhance engraftment of transplanted MASC, a milder

form of injury to the SEZ in the form of SLI (450rads) would assessed for enhanced

engraftment of the transplanted cells. Recent studies in stem cell biology have revealed

that injury to the candidate site of transplantation is critical for engraftment and/or

contribution of the transplanted stem cell, as the recipient niche is normally relatively

quiescent in a healthy adult animal and induction of injury often renders the niche more

receptive to transplanted cells (83). Indeed, SLI animals transplanted with gfp+ MASC

were observed to contain significantly more donor-derived migratory cells in the OB as

compared to non-irradiated controls. It is important to note that although this increase

was statistically significant it was highly variable with fully half of the transplanted SLI

animals exhibiting levels of engraftment similar to controls. The reason for this









variability is not known, although it could easily be attributed to the inherent

inconsistency observed to occur in adult transplants.

BrdU incorporation experiments to determine the mitotic cell levels at the time of

transplantation revealed that while the numbers of mitotic SEZ neuroblasts were

significantly depleted immediately following exposure, the levels returned to near normal

by three weeks and were found to be significantly higher at six weeks. This observation

was further supported by blind NS culture assays of SLI animals in which the observed

yield ofNS cultured from brains exposed to SLI 3 weeks earlier was significantly

decreased by 30%. As was observed in the LI study, the NS yield from non-irradiated

controls was again between 0.02 and 1% (0.12%). This mild injury to the brain may

potentially enhance neurogenic activity and allow for increased engraftment by

transplanted NSCs.

Candidate NSC have recently been isolated from the brain utilizing antibodies that

bind to unique cell surface antigens, such as CD15 and CD133 (10, 80). Isolated cells are

then subjected to culture conditions that will produce NS, indicating that the cell

population isolated contains NSCs. In the field of hematopoiesis, attempted in vitro

manipulation and subsequent expansion of cultured HSC has been shown to result in

decreased pluripotency and engraftment by the HSC, with only hematopoietic progenitor

cells lacking the capacity for self-renewal being successfully expanded in vitro (14) in

theory due to the incomplete reconstruction of the bone marrow microenvironment in

vitro. Transplantation of non-cultured, primary HSC isolated from donor bone marrow

still yields the most robust, long-term engraftment into the niche, and it is entirely

possible that the in vitro manipulation of NSC results in a similar loss of multipotency









and engraftment ability. Ideally, the NSC would be isolated directly from primary SEZ

tissue prior to manipulation or transplantation, but in order for the NSC to be identified

and isolated, a model system for robust engraftment is necessary for the analysis of the

functional ability of the NSC.

By utilizing the injury model described here, candidate cell populations isolated

according to surface antigen expression could be transplanted to the injury-activated SEZ,

followed by later analysis of the RMS for increased, long-term production of donor

derived neuroblasts. The resulting observations would then allow for a definitive

conclusion to be made as to if the isolated cell population expressed the characteristics of

a true stem cell.
















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BIOGRAPHICAL SKETCH

Gregory Paul Marshall II was born on August 6th, 1976 in Gainesville, FL. After

graduating from Bradford High School in Starke, FL in 1994, he attended St. Andrews

Presbyterian College in Laurinburg, NC where he earned a Bachelor of Science degree

in biology and met Kathleen Kelly, his future wife. After a two year employment with

Dr. Susan Frost as head laboratory technician, Greg entered into the University of

Florida's College of Medicine Interdisciplinary Program in Biomedical Sciences in the

year 2000. After joining the laboratory of Dr. Edward Scott, Greg began his research

into the characterization of neural stem cells. He has presented posters of his research at

the Keystone Symposia in 2003 and at the Annual Society for Neuroscience meeting in

2004, and has recently submitted a first author article for publication in the journal Stem

Cells.