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Remyelination in the central nervous system by transplatation of human fetal glial cells and co-transplantation… Xu, Yan 2000

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REMYELINATION IN THE CENTRAL NERVOUS SYSTEM BY TRANSPLANTATION OF HUMAN FETAL GLIAL CELLS AND CO-TRANSPLANTATION OF INSULIN LIKE GROWTH FACTOR-1 IN THE S H I V E R E R MOUSE MODEL B y Yan Xu M D . T h e S h a n g h a i S e c o n d M e d i c a l U n i v e r s i t y , 1 9 9 1 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E i n T h e F a c u l t y o f G r a d u a t e S t u d i e s ( D e p a r t m e n t o f S u r g e r y ) W e a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e p e q t r k e ' d s t a n d a r d T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A D e c e m b e r 1 9 9 9 © Y A N X U , 1 9 9 9 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of 'TUX The University of British Columbia Vancouver, Canada Date kcS, (111 DE-6 (2788) A B S T R A C T The shiverer mouse is a myelin-deficient animal model for glial cell transplantation studies. Human fetal glial cells and human adult oligodendrocytes were transplanted into shiverer brains. The myelination formed by transplanted oligodendrocytes was detected in the shiverer brain by immunohistochemistry using anti-myelin basic protein (MBP) antibody. Transplantation of human fetal glial cells resulted in myelination in the shiverer brain. There was no myelination observed using human adult oligodendrocytes. Furthermore, co-transplantation of the glial growth factor, Insulin like growth factor-1, with human fetal glial cells also resulted in myelination in the shiverer brain. These observations imply that human fetal glial cells may be the more suitable cell source from the central nerve system for clinical transplantation in human demyelination disease. The failure of human adult oligodendrocytes to myelination may have been due to too few oligodendrocyte progenitor cells. Co-transplantation of glial growth factors may be another possible application for glial cell transplantation in the shiverer mice. ii T A B L E OF C O N T E N T S Page ' A B S T R A C T i i LIST OF T A B L E S v LIST OF F I G U R E S v i D E D I C A T I O N . v i i A C K N O W L E D G E M E N T v i i i C H A P T E R I I N T R O D U C T I O N 1 Myelin-deficient animal model 4 Identification of the transplanted cells 5 Immunosuppression 6 Gl ia l cell to be transplanted 6 C H A P T E R II M A T E R I A L S A N D M E T H O D S 12 2.1 Animals 13 2.2 Human fetal oligodendrocyte culture 13 2.3 Human adult oligodendrocyte isolation 15 2.4 Immunocytochemical staining for 0 4 16 2.5 Transplantation procedure 16 2.5.1 Concentration of human fetal oligodendrocytes or adult oligodendrocytes before transplantation 16 2.5.2 Transplantation and Immunosuppression 17 2.6 Tissue processing 18 2.7 Immunohistochemical staining for myelin basic protein 18 C H A P T E R III C O M P A R I S O N OF S U R V I V A L A N D M Y E L I N A T I O N B Y H U M A N F E T A L O L I G O D E N D R O C Y T E S W I T H H U M A N A D U L T T O L I G O D E N D R O C Y T E S F O R T R A N S P L A N T A T I O N 20 3.1 Introduction 21 3.2 Materials and Methods 22 3.2.1 Animals 22 3.3 Results 23 3.3.1 Phenotype of human adult oligodendrocytes and fetal glial cells in culture before transplantation 23 3.3.2 Post-transplantation analysis 23 3.3.2.1 The needle tract 23 r 3.3.2.2 The morphology of the transplanted human oligodendrocytes in the host brain 23 3.3.2.3 The survival of transplanted human iii oligodendrocytes 23 3.3.2.4 The migration of transplanted human oligodendrocytes 23 3.3.2.4 The myelination of transplanted human oligodendrocytes ^ 24 3.4 Discussion 25 CHAPTER IV CO-TRANSPLANTATION OF GLIAL GROWTH FACTORS 26 4.1 Introduction 27 4.2 Material and Methods 30 4.2.1 Animal 30 4.2.2 Transplantation and Immunosuppression 30 4.3 Results 31 4.3.1 The needle tract 31 4.3.2 The morphology of transplanted human fetal glial cells 31 4.3.3 The survival and myelination of the transplanted human fetal glial cells 31 4.3.4 The migration of transplanted human fetal glial cells 32 4.4 Discussion 32 CHAPTER V DISCUSSION 34 CHAPTER VI REFERENCE 37 TABLES 56 FIGURES 57 LIST O F T A B L E S T A B L E 1. Summary of comparison of human fetal oligodendrocytes with human adult oligodendrocytes on survival, migration and remyelination in shiverer model. T A B L E 2. Summary of comparison of human fetal oligodendrocytes with human adult oligodendroyctes on survival by MBP staining in shiverer model. LIST O F FIGURES F I G U R E 1. Comparison between normal and demyelinated myelin. F I G U R E 2. Positive 04 staining cell in human fetal oligodendrocyte culture. F I G U R E 3. Needle tract in the group of human fetal oligodendrocyte transplantation. F I G U R E 4. Positive MBP staining cell with process in the group of human fetal oligodendrocyte transplantation. F I G U R E 5. Survival and remyelination of human fetal oligodendrocytes in the brain of shiverer mouse, a, positive MBP staining inside needle tract, b, positive MBP staining with short fiber gradient, c, positive MBP staining with long fiber gradient. F I G U R E 6. Negative MBP staining cell in the group of human adult oligodendrocyte transplantation. F I G U R E 7. Cell migration and gradient of positive MBP fibers in the group of human fetal oligodendrocyte transplantation. F I G U R E 8. Needle tract in the experiments of IGF-1 co-transplantation with human fetal oligodendrocytes. F I G U R E 9. Needle tract in the experiments of PBS co-transplantation with human fetal oligodendrocytes. F I G U R E 10. Positive MBP staining cell with process inside of delicate area in the experiments of IGF-1 co-transplantation with human fetal oligodendrocytes. F I G U R E 11. Survival and remyelination in the experiments of IGF-1 co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse, a, positive MBP staining inside needle tract, b, positive MBP staining with short fiber gradient, c, positive MBP staining with long fiber gradient. F I G U R E 12. Survival and remyelination in the experiments of PBS co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse, a, positive MBP staining inside needle tract, b, positive MBP staining with short fiber gradient, c, positive MBP staining with long fiber gradient. vi DEDICATION To my Parents, Dr. Xianhu Xu, Mrs. Linmei Liu To my lovely Daughter, Huihui Fan To my Husband, Dr. Dapeng Fan Their constant love and encouragement made all these possible, and will always be remembered and appreciated. vii A C K N O W L E D G M E N T S I would like to thank all my committee members, Dr. Chris Honey, Dr. William Jia and Dr. Trevor Hurwitz, for their efforts, time, commitment and support of this work. I would like to thank Dr. Hao Shen for his surgery help through all my study. viii CHAPTER 1 INTRODUCTION Myelin-deficit disorders of the central nervous system (CNS) include demyelination (loss of myelin) and myelin dysfunction. Common pathological examples in humans include multiple sclerosis (MS), spinal cord injury and adrenoleukodystrophy. These conditions are still non-curable today due to the lack of an effective therapy to restore myelin function. Electric signals of the nerve cells (neurons) are transported along their axons. Myelin is a compact hydrophobic membrane that ensheathes the axon. Without proper myelin around the axon, nerve conduction is blocked or attenuated (Figure 1). In order to restore the neuronal function in myelin-deficit diseases, the "naked" (demyelinated) nerve fibers need to be wrapped by myelin. Myelin-forming glial cells include oligodendrocytes in CNS and Schwann cells in the peripheral nerve system (PNS). Oligodendrocytes are derived from neuroepithelial progenitor cells in the subventricular zone (SVZ) of the embryonic CNS (Reynolds et al., 1988). They distribute throughout the white matter as postmitotic oligodendrocytes and wrap the growing axons during CNS development (Peters, 1960b; Miller, 1996). Oligodendrocytes are responsible for the formation and maintenance of myelin, which facilitates saltatory conduction of nerve impulses along axons (Bologa et al., 1985). Schwann cells do the same in the peripheral nervous system (Archer et al., 1994; Groves et al., 1993; Gumpel et al, 1983,1987; Hasegawa and Rosenbluth, 1991). Unlike the peripheral nervous system where Schwann cells can re-grow and form new myelin sheath, oligodendrocytes in the human central nervous system have limited ability to reproduce and remyelinate the "naked" nerve fibers in either inherited or acquired myelin disorders (Duncan, 1996). 2 Neural transplantation into the mammalian central nervous system (CNS) has been studied since last century (Thompson, 1890). Experiments have examined the viability of transplanted neural tissue, tried to stimulate axonal regeneration and analyzed the functional recovery of injured nerve tissue (Bjorklund, 1991; Franklin et al., 1991). Recently, neuronal transplantation has been used in the treatment of degenerative disease of the CNS (Bjorklund et al., 1983,1984; Sinson et al., 1996; Svendsen et al., 1997). Most studies have focused on Parkinson's disease (PD). The major neuropathological characteristic of PD is a progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta. Degeneration of the nigrostriatal pathway leads to a decreased level of dopamine in the striatum, which causes the clinical symptoms (Hornykiewicz et al., 1989). Transplantation of new dopaminergic neurons to replace the degenerated ones is a reasonable and practical approach for the treatment of Parkinson's disease (Bjorklund, 1991; Lindvall et al., 1994). In this field, extensive studies using donors of various ages (embryo, neonate and adult) and combining a variety of growth factors and gene therapies have been conducted on different recipients (rodent, monkey and human). The following has been documented: 1. Transplanted neural cells (neurons and glial cells) can survive in the host central nervous system (Bjorklund, 1991). 2. The immunosuppressive drug cyclosporin A has been shown to enhance the survival of allografts (tissue from different animals of the same species) and xenografts (tissue from different species) (Backlund et al., 1987). 3. Fetal cell transplants survive better than adult cells (Blakemore and Franklin, 1991; Duncan 1996). 4. Co-transplantation of responsive growth factors can further promote the survival and function of transplanted 3 cells (Oppenheim et al., 1991; Gumey et al., 1992; Lewis et al., 1993; Sinson et al., 1996; Sautter et al., 1998; Svendsen et al, 1997, Yuen et al., 1996). These encouraging results have been recently extended and explored into glial cell transplantation in an attempt to replace or repair myelin-deficit disorders in the central nervous system (Duncan and Milward, 1995). Over the last decade, many studies have been conducted to attempt remyelination by introducing exogenous myelin-forming glial cells (oligodendrocytes or Schwann cells) into the demyelinated CNS of a variety of animal models (Duncan, 1996). Transplanted exogenous myelin-forming cells have been found to survive and migrate along the nerve fibers of the host CNS to further form new myelin sheaths (Franklin et al., 1993, 1997; Espinosa De Los et al., 1992; Baulac et al., 1987; Gout et al., 1988; Gumple et al , 1983; Rosenbluth et al , 1993). Remyelination was also reported to facilitate saltatory conduction of nerve impulses along axons and lead to some functional recovery (Honmou et al , 1996; Porter et al., 1986). Myelin-deficient animal model The first important issue in the study of remyelination by glial cell transplantation is how to make a glial cell deficiency animal model. Two approaches have been used and widely accepted in the literature (Blakemore and Franklin, 1991). One is the myelin-toxic/irradiation animal model (Bunge et al., 1994; Dietrich et al., 1987). Glial cells are destroyed focally in the spinal cord by a local injection of a myelin-toxic chemical such as ethidium bromide (EB). The host glial cells are then prevented from repopulating by X -irradiation. Unfortunately, many other non-myelin cells are also subject to damage. This 4 model generally is used for long-term remyelination studies in the spinal cord of normal adult animals (Blakemore and Franklin, 1991). The other ariimal model uses mutant animals with genetically deficient myelin such as the myelin-deficient rat (md) or shiverer mouse (Bird et al., 1977; Mikoshiba et al., 1982; Jacque et al., 1978; Dupouey et al., 1979; Delasalle et al., 1981). These models are characterized biochemically by the absence of myelin basic protein (MBP) in the shiverer mouse and proteolopid protein (PLP) in the md rat. Oligodendrocytes in these animal models are unable to produce normal myelin and this creates a permanent and global myelin-deficit environment. Any remyelination after glial cell transplantation into the CNS of the animals must be attributable to the transplanted myelin-forming cells. These animal models have been widely used for the study of global myelin-deficit disorders. These studies have been limited by the short life span of the mutant animal (Duncan and Milwad, 1995). The availability and surgical mortality of these mutant animals also limit their experimental use. Identification of the transplanted cells Exogenous glial cells are usually transplanted into the host myelin-deficit CNS by infusion of cell suspensions or tissue fragments. In order to evaluate the survival, migration and myelination of the transplanted cells, one must be able to distinguish the transplanted myelin-forming cells from the host glial cells and myelin. Myelin basic protein (MBP), a cytoplasmic apposition protein, is a major myelin constituent produced by oligodendrocytes in the central nervous system. Expression of MBP is considered to be a mark for oligodendrocyte differentiation and myelination in the developing CNS 5 (Zecevicet al., 1998). MBP in the myelin sheaths formed by the transplanted glial cells can be easily detected by immunocytochemistry using anti-MBP antibody. Due to the lack of myelin basic protein in the shiverer mouse, the presence of positive MBP staining at the site of glial cell transplantation is a strong evidence of remyelination achieved by transplanted glial cells (Duncan et al, 1988; Lachapelle et al., 1983). There are also more direct approaches to identify the transplanted glial cells and new myelin, such as labeling the glial cells with vital dyes or tritiated thymidine or fast blue prior to transplantation (Baron-Van Evercooren et al., 1991; Espinosa De Los et al., 1992; Gansmuller et al., 1992), using a Y-chromosome specific probe and in situ hybridization to identify male transplanted cells in the female host brain (Harvey et al., 1992), or inserting a report gene or retroviral vector into the glial cells prior to transplantation (Franklin et al., 1995; Groves et al., 1993; Tontsch et al., 1994). Immunosuppression Immunosuppression has been routinely used in the neural transplantation experiment to prevent host rejection of xenografts and allografts. There are two commonly used immunosuppressive protocols, cyclosporin A (Shevach, 1985) and anti-CD4 antibody (Rosenbluth et al., 1993; Blakemore et al., 1995; Li et al., 1994). Glial cell to be transplanted Oligodendrocytes Oligodendrocytes can be transplanted directly from tissue fragments of donor CNS or cultured in vitro prior to transplantation. Cultured oligodendrocytes can be further enriched by the addition of glial growth factors that enhance their survival and 6 proliferation. Brain tissues from newborn mouse and human fetuses have been successfully implanted into the shiverer mice and resulted in remyelination around host "naked" axons (Gumpel et al, 1983,1987). Fetal rat spinal cord fragments implanted in myelin-deficient rats have also been reported to produce remyelination (Hasegawa and Rosenbluth, 1991). Tissue fragments however, can limit the ability of the cells near the center of the fragment to penetrate and spread into the host myelin-deficit environment. The other non-oligodendrocyte components of the tissue fragment also stimulate immune rejection (Marion et al, 1990). In contrast, cell suspensions are easily prepared for injection without the above problems and allow immediate migration of the transplanted cells. The 0-2A lineage of the oligodendrocyte derived from rat optic nerve and brain has been studied extensively both in vivo and in vitro (Pfeiffer et al., 1993; Raff, 1989; Richardson et al., 1990). During their development, oligodendrocytes go through several distinct stages, which can be identified by sequential expressions of antigenic markers (Curtis etal., 1988; Gard et al., 1989; Hardy et al., 1991; Levine et al., 1988). The early oligodendrocyte progenitor stage is recognized by the surface antibody A2B5 (Fredman et al., 1984). The intermediate stage can be recognized by surface antibody 04 (Gard et al., 1989; Dubois-Dalcq, 1987; Levi et al., 1987). The mature stage can be detected by surface antibodies anti-GalC, 01 or MBP (Raff et al., 1978). The 0-2A progenitor cells can develop into either oligodendrocytes or type 2 astrocytes, depending on the microenvironment, culture condition and growth factors (Noble, 1991; Richardson et al., 1990). They can migrate during CNS development (Pringle et al., 1993; Fok-Seang et al., 7 1994). Due to their bipotential and high mobility, it has been suggested that the 0-2A progenitor is more suitable for transplantation. Remyelination obtained from various stages of the 0-2A lineage, from the early A2B5 positive progenitor (Groves et al., 1993) through to the more mature 01 positive cell (Duncan et al., 1992), have been reported. Using fluorescence-activated cell sorting or selective immunopanning, purified oligodendrocytes or progenitors have achieved more myelination after transplanted into the myelin-deficient rodents compared to mature oligodendrocytes (Duncan et al, 1992; Warrington et al, 1993). Previous studies have also shown that both allografts (Gumpel et al., 1983) and xenografts (Crang et al., 1991; Rosenbluth et al., 1993) of oligodendrocytes formed myelin in congenital myelin-deficient animals (Blakemore et al., 1988) and in the focal demyelination model (Gout et al., 1988; Vignais et al., 1993) or developing normal CNS (Lachapelle et al , 1994; Tontsch et al., 1994). In addition to primary oligodendrocytes, there are several other oligodendrocyte cell lines available for transplantation. The CG4 cell can differentiate into either mature oligodendrocytes or type-2 astrocytes. It is the most promising growth factor responsive glial cell line from the rat brain (Louis et al., 1992). Transducted with the LacZ gene, the late passages (P30-P40) of this cell line demonstrated extensive migration and axon myelination in the neonatal myelin-deficit rat spinal cord after transplantation. CG4 cells beyond the P40 stage fail to myelinate (Tontsch et al., 1994). Another growth factor responsive cell line is the EGF responsive neural stem cells from rodents (Reynolds et al., 1992). This cell line is able to proliferate as floating neurospheres in the presence of EGF in vitro, but once EGF is removed and cells are plated, their multipotential nature is 8 revealed. Transplantation of rodent neurospheres resulted in myelination in the neonatal myelin-deficit rat (Hammang et al., 1997). Oncogenes driven immortalized glial cell lines are another approach for transplantation. The most common oncogene is a temperature sensitive mutant of the SV 40 large T antigen, which maintains undifferentiated at 33°C, but proliferate at 39°C (body temperature of rodent) (Barnett et al., 1993). Once this oncogene is transplanted into the host CNS, however, its myelination is poor (Trotter et al., 1993). Schwann cell Schwann cells have been successfully used for remyelination in the spinal cord. Compared with oligodendrocytes, Schwann cells have several potential advantages for transplantation. Schwann cells can proliferate more rapidly and produce various neurotrophic factors to promote myelination (Fawcett et al., 1990; Raivich et al., 1993). Schwann cells can be readily obtained from the peripheral nerves of the same donor for autologous transplantation and thereby overcome any immune rejection. Compact remyelination has been achieved in the glia-free areas of the cat and rat spinal cords after autologous or exogenous Schwann cell transplantation (Ffonmou et al., 1996; Blakemore et al., 1985; Duncan et al., 1988). Moreover, the axons remyelinated by grafted Schwann cells have demonstrated to restore normal conduction velocity (Blight et al , 1989; Felts et al., 1992; Ffonmou et al., 1996). The Schwann cell line MSC 80 has also been found to remyelinate axons in the mature shiverer spinal cord (Boutry et al., 1992). The availability of cultured human fetal and adult Schwann cells has made it possible to transplant human fetal or adult Schwann cells into myelin-deficit animal models 9 (Rutkowski et al., 1995; Kim et al 1989, Levi et al., 1994). Although the early results have been encouraging, many aspects still remained to be elucidated. Human oligodendrocytes It would be more desirable to use human glial cells for any clinic transplantation in human patients. Compared to the extensively studied rodent oligodendrocyte, relatively little is known about transplantation of the human oligodendrocyte. Only two pioneer studies have been reported. One used human fetal brain tissue (Gumpel et al., 1987) produced myelination in the shiverer brain, and the other used a human adult glial cell suspension failed to remyelinate in the X-irradiated ethidium bromide lesioned rat (Targett et al., 1996). No research has been conducted using human fetal oligodendrocytes, although human fetal glial cells have been cultured and studied in vitro for more than a decade. Kim and Satoh (1983; 1990; 1994; 1985; 1996; 1995) have published a series of in vitro studies using human fetal glial cells. They found that human fetal glial cell cultures contain bipotential glial cells which express both oligodendrocyte and astrocyte phenotypes. These primary cultures from the human fetal brain tissue contain 0.01-0.1% cells that are galactocerebroside (GalC)+ oligodendrocytes. Human fetal brain does have a distinct developing oligodendrocyte cell lineage. The majority (93%) of these oligodendrocyte lineage cells is 04+GalC+ and 04+GalC-. Cultured human fetal oligodendrocytes can be classified into three phenotypically distinct subtypes: Ranscht-monoclonal (R-mAb)-04+cells, (R-mAb)+04- cells and the more mature (R-mAb)+04+cells. Human fetal oligodendrocytes in primary culture have a great degree of 10 proliferate capacity without the requirement of exogenous growth factors, such as plate-deprived growth factor (PDGF) or/and basic fibroblast growth factor (bFGF). Insulin like growth factor (IGF-1) promotes their differentiation. Adult human oligodendrocytes have also been isolated in vitro and an oligodendrocyte progenitor has been cultured (Kim, 1990; Armstrong et al., 1992). The purity of adult human oligodendrocytes were reported to be 90%-95% (Kim, 1990). If we are to work towards a clinic protocol using human oligodendrocytes for transplantation in myelin-deficient disease, we must first elucidate the best source of human oligodendrocytes. This thesis directly compares the two available sources, human fetal and adult cultured oligodendrocytes, in the shiverer mouse model. The progenitor cells in fetal tissue have the theoretical advantage of better survival, proliferation and migration. Using adult tissue, however, would have the potential advantage of using the patient's own cells and thereby avoiding any immune rejection. Our hypothesis is that human fetal oligodendrocytes produce more re-myelination than human adult oligodendrocytes when transplanted into the shiverer mouse brain. We also studied the ability of co-transplanting a glial growth factor, IGF-1, to enhance the remyelination of human fetal oligodendrocytes in the shiverer mouse. Our second hypothesis was that IGF-1 improved human fetal oligodendrocyte remyelination in this animal model. 11 CHAPTER n MATERIALS AND METHODS 2.1 Animals The genetically mutant myelin-deficit shiverer mouse model was used in this study. A total of forty shiverer mice (C57BL/6J-MBP, stock No. 002492) were used. They were originally obtained from Jackson Company in Canada and maintained through brother-sister mating for generations. Twenty-one days after birth, they were weaned and identified by their shivering behavior. Animals were kept in a 12-hour light/dark cycle and fed with standard rodent diet. At the age of six weeks (20-26 gm), they were divided into two experiments (twenty in each experiment). Experiment I, comparison of fetal and adult human oligodendrocytes transplantation; Experiment II, IGF-1 co-transplantation of fetal human oligodendrocyte. All experiments were performed according to the guidelines of the Canadian Council for Animal Care and approved by the animal care unit of the University of British Columbia. 2.2 Human fetal glial cell culture The entire primary cultures were prepared according to methods previously published (Satoh et al., 1994, 1996;Yong et al., 1988; Kim, 1985). Human fetal brains from therapeutic abortion were obtained from the Department of Neuropathology at Vancouver General Hospital. The brain tissue was diced in the 100-mm glass Petri dish (Costar, Nepean, Ontario) and incubated with 0.25% trypsin (2.5% trypsin, Sigma, Oakville, Ontario) and 40 ug/ml DNase (Sigma, Oakville, Ontario) in 0.01M phosphate-buffered saline (PBS, Sigma, Oakville, Ontario) for twenty minutes at 37°C. Tissue fragments were then digested into single cell suspension by gently pipetting using 10-ml disposable pipette (Costar, Nepean, Ontario). Fetal bovine serum (20%, Gibco BRL, 13 Burlington, Ontario) was added to the suspension to stop trypsinization. The suspension was then centrifuged at 400g for twenty minutes to collect the dissociated cells. Following two washings in 0.01M PBS (Sigma, Oakville, Ontario), the mixed brain cells were finally suspended in a feeding medium of Dulbecco's Modified Eagle Medium (DMEM, Sigma, Oakville, Ontario) containing 10% fetal bovine serum, lOOU/ml penicillin-streptomycin (Gibco BRL, Burlington, Ontario) and 20 |il/ml gentamicin (Gibco BRL, Burlington, Ontario). The cell suspension was then plated at a density of 3X106 cells/ml in 75 cm 2 culture flasks (Costar, Nepean, Ontario). They were incubated at 37°C incubator with 5% C0 2-95% air atmosphere and grew for ten days. This culture technique allows only glial cells to survive since most neurons die in non Poly-Lysin coated flask over ten days. The glial cell suspension, consisting mostly of oligodendrocytes and astrocytes, was used for intracerebral transplantation. After ten days, the cells were trypsinized (0.25% trypsin, 2.5% trypsin, Sigma, Oakville, Ontario) and washed twice with 0.01M PBS (Sigma, Oakville, Ontario) for transplantation. For immunocytochemistry, the cells were plated on Poly-L-Lysin (10 |ig/ml, Sigma, Oakville, Ontario) coated 20-mm-diameter glass coverslips (Fisher, Nepean, Ontario). 2.3 Human adult oligodendrocyte isolation The whole primary culture was performed according to methods previously published (Kim et al., 1983; Kim, 1990). Fresh white matter fragments of human adult brain were obtained from epilepsy patients during the surgery at Vancouver General Hospital. The tissues did not include the epileptic focus and was taken from deep white 14 matter near the ventricle. Tissue fragments were incubated in 0.25% trypsin (2.5% trypsin, Sigma, Oakville, Ontario) and 40 p:g/ml DNase (Sigma, Oakville, Ontario) in 0.01 M PBS (Sigma, Oakville, Ontario) at 37°C for forty-five minutes. Then the fragments were dissociated into cell suspension by gentle pipetting (Costar, Nepean, Ontario). The suspension was passed through a nylon mesh (Tetko, Kansas, MO) with a pore size of 100 |im in order to get rid of tissue chunks. The filtrate was mixed with 30% Percoll (Pharmacia, Uppsala, Sweden) and 3% 10X Hanks' balance salt solution (Sigma, Oakville, Ontario) in 0.01M PBS (Sigma, Oakville, Ontario) and centrifuged at 4000g at 4°C for thirty minutes. The middle layer that contained an enriched population of oligodendrocytes and was collected and diluted three folds with 0.01M PBS (Sigma, Oakville, Ontario), then centrifuged at 400g for fifteen minutes to remove the Percoll. The cell pellet was washed with 0.01M PBS (Sigma, Oakville, Ontario) twice and then suspended in a feeding medium of D M E M (Sigma, Oakville, Ontario) containing 5% fetal bovine serum (Gibco BRL, Burlington, Ontario), 20 (ig/ml gentamicin (Gibco BRL, Burlington, Ontario), 100 U/ml penicillin-streptomycin (Gibco BRL, Burlington, Ontario). The oligodendrocytes were then plated onto 100-mm-diameter plastic Petri dishes (Costar, Nepean, Ontario). Most oligodendrocytes were not able to cling to the plastic dishes after twenty-four hour whereas astrocytes, endothelial cells and microglia attached to the bottom of the dish. The floating cells (oligodendrocytes) were collected and seeded on Poly-L-Lysin (10 (Xg/ml, Sigma, Oakville, Ontario) coated 20-mm-diameter glass coverslips (Fisher, Nepean, Ontario) for immunocytochemistry studies. The rest of 15 the oligodendrocyte suspension was washed with 0.0IM PBS (Sigma, Oakville, Ontario) for transplantation. 2.4 Immunocytochemical staining for 04 The cell surface marker for the intermediate oligodendrocyte progenitor, 04, was detected with an anti-04 antibody (Bansal et al, 1989). Cells on coverslips were fixed in 95% acetic alcohol for one minute at room temperature and then washed with 0.0IM PBS. The coverslips were then incubated with a mouse monoclonal anti-04 (20 |Xg/ml, Boehringer Mannheim, Quebec City, Quebec) at 4°C overnight. After washing with 0.0IM PBS, coverslips were incubated with a biotinylated anti mouse IgG (1:250, Sigma, Oakville, Ontario) for one hour at room temperature and then incubated in A B C Elite Kit (Vector, Burlingame, Ontario) for another one hour at room temperature. The reaction product was revealed with a DAB-Nickel solution for ten minutes at room temperature. Finally, the coverslips were counter-stained with Methyl Green (Sigma, Oakville, Ontario), dehydrated and coverslipped with permount (Fischer, Nepean, Ontario) for observation under light microscope. 2.5 Transplantation procedure 2.5.1 Concentration of human fetal glial cells or adult oligodendrocytes before transplantation For human fetal glial cells, after removing the old culture medium, the culture was washed twice with 0.01M PBS. 5 ml of 2.5% trypsin and 40 ug/ml DNase in 0.01M PBS was added to culture flask, and incubated at 37°C for five minutes. The dissociated oligodendrocytes were collected and gently pipetted, then centrifuged at 300g for ten 16 minutes. The cells were washed twice by 0.0IM PBS and resuspended in 0.0IM PBS to a concentration of 5X105 cells/[xl for transplantation. For human adult oligodendrocytes, floating oligodendrocytes were collected and washed twice with 0.0IM PBS, then the cells were resuspended in 0.01M PBS to a concentration of 5X105 cells/ui for transplantation. The concentration of both human fetal glial cells and adult oligodendrocytes in 0.01M PBS was 5 X10 5 cells per microliter for transplantation. 2.5.2 Transplantation and Immunosuppression Shiverer mice were anesthetized with an intraperitoneal injection of sodium pentobarbitol (30mg/ml, Sigma, Oakville, Ontario). After shaving the hairs of the scalp, the head of mouse was fixed in the stereotactic frame. The scalp was cleaned with 70% alcohol and cut along the middle line until the Bregma was identified. A hole was drilled through the skull with a burr at the coordinates for the striatum. The coordinates were 0.8 mm anterior to Bregma, 1.6 mm to the right of midline, and 3.0-4.0 mm deep to the cortical surface. A 26 gauge needle connected to a 10 ul Hamilton syringe on a constant syringe infusion mini-pump 22 (Harvard Apparatus, St. Laurent, Quebec) was inserted into the burr hole to a depth of 4 mm from the cortical surface. 1.5 ul human fetal glial cells or human adult oligodendrocyte suspension was then delivered into the right striatum at a rate of 0.3 pil/minute. The needle then was raised 1 mm and an additional 1.5 ul was injected at the same rate. In order to prevent host immune rejection of the graft, 17 Cyclosporine A (5 mg/kg, 5 mg/ml, Sandoz, Dorval, Quebec) was administered daily by intraperitoneal injection for 21 consecutive days after the surgery. 2.6 Tissue processing Twenty-two animals survived three weeks after cell transplantation (at least five in each group). Animals were deeply anesthetized by intraperitoneal injection of high dose sodium pentobarbital (90 mg/ml, Sigma, Oakville, Ontario) and transcardially perfused with cold 0.9% saline (Sigma, Oakville, Ontario) followed by cold 4% paraformaldehyde (Sigma, Oakville, Ontario). The brains were dissected out and post-fixed overnight in 4% paraformaldehyde (Sigma, Oakville, Ontario) and then cryoprotected in 20% sucrose (Sigma, Oakville, Ontario) in 0.05M PBS (Sigma, Oakville, Ontario) for another twelve hours. The striatum was serially sectioned at 30 |im with a vibratome (TPI 1000, Montreal, Quebec). The sections containing cell transplantation injection sites were collected in 0.05M PBS (Sigma, Oakville, Ontario) for immunohistochemistry studies. 2.7 Immunohistochemical staining for myelin basic protein (MBP) Brain tissue sections were prepared by the method described by Shu et al. (Shu et al, 1988). Sections were first rinsed twice in 0.05M PBS (Sigma, Oakville, Ontario) for twenty minutes, incubated in 0.6% H2O2 (Fischer, Nepean, Ontario) in 0.05M PBS (Sigma, Oakville, Ontario) for ten minutes. After rinsing three times in 0.05M PBS (Sigma, Oakville, Ontario), sections were incubated in horse anti-mouse MBP-Dig (1:250, Serotec, Raleigh, NC) in 0.05M PBS (Sigma, Oakville, Ontario) with 2% normal horse serum (Vector, Burlingame, Ontario), 3% of 10% Triton X-100 (Sigma, Oakville, Ontario) 18 and 1% of 2% sodium-azide (Sigma, Oakville, Ontario) for two nights at room temperature. Sections were then rinsed three times in 0.05M PBS (Sigma, Oakville, Ontario) and incubated in biotinylated sheep anti-horse IgG (1:200, Sigma, Oakville, Ontario) in 0.05M PBS (Sigma, Oakville, Ontario) with 2% normal horse serum (Vector, Burlingame, Ontario), 3% of 10% Triton X-100 (Sigma, Oakville, Ontario) and 1% of 2% sodium-azide (Sigma, Oakville, Ontario) for one hour at room temperature. Sections were then rinsed three times and incubated in A B C Elite Kit (1:500, Vector, Burlingame, Ontario) for one hour at room temperature. Sections were then rinsed twice in 0.05M PBS (Sigma, Oakville, Ontario) and once in 0.2M acetate buffer (Sigma, Oakville, Ontario). Finally, sections were incubated in glucose oxidase-DAB-Nickel solution for five minutes at room temperature. The immunohistochemical reaction was terminated with two rinses with 0.05M PBS (ten minutes each). Sections were mounted on Chrom Alum coated slides (Fischer, Nepean, Ontario) for dehydration and then coverslipped with paramount (Fischer, Nepean, Ontario). 19 CHAPTER IH COMPARISON OF SURVIVAL AND MYELINATION BY HUMAN FETAL OLIGODENDROCYTES WITH HUMAN ADULT OLIGODENDROCYTES FOR TRANSPLANTATION 20 3.1 Introduction Although various kinds of oligodendrocyte lineage can form myelin following transplantation, many studies have shown that the myelination ability of these cells depends on their developmental stage. Studies in rodents have shown that immature oligodendrocytes have a much higher capability for myelination and migration than adult oligodendrocytes after being transplanted into host CNS (Duncan 1996). For example, although transplantation of both early A2B5 positive 0-2A progenitor (Groves et al., 1993) and the more mature 01 positive cell (Duncan et al., 1992) resulted in transplant derived myelination, the more extensive remyelination was achieved with more immature A2B5 positive progenitor cells (Warrington et al., 1993). Oligodendrocytes from fetal canine brain produced more myelin when transplanted into the spinal cord than those from neonatal canine brain (Archer et al., 1997). Immature oligodendrocytes are also more motile than mature oligodendrocytes. The pre 0-2A cell and 0-2A cell of the oligodendrocyte lineage has shown extensive migration ability both in vitro and in vivo (Small et al., 1987; Levison et al., 1993; Pringle et al., 1993). Following transplantation, progenitor cells migrate into the areas of demyelination, then undergo one or more cell divisions and finally differentiate into oligodendrocytes, which wrap demyelinated axons with new myelin sheaths. Extending this work to human oligodendrocytes would be suggested for future research. Several reports, however, have demonstrated pre-oligodendrocytes in adult human brain tissue (Armstrong et al., 1992; Gogate et al., 1994). We set out to directly 21 compare the survival and myelination capability of human oligodendrocytes in the shiverer transplant model. 3.2 Materials and Methods 3.2.1 Animals A total of twenty neonate shiverer mice (C57BL/6J-MBP, stock No. 002492) were used. They were divided into two experimental groups with ten mice in each group. Group I received human fetal oligodendrocyte transplantation while Group II received human adult oligodendrocyte transplantation (Summary in Table 1). 3.3 Results 3.3.1 Phenotype of human adult oligodendrocytes and fetal glial cells in culture before transplantation In the fetal human glial cell culture, we detected 04 positive staining oligodendrocyte progenitor cells with no, uni-, bi-, or multipolar- process (Figure 2), although the number of 04 positive cells was low. In the human adult oligodendrocyte cell culture, only MBP positive but no 04 positive cell was detected. 3.3.2 Post-transplantation analysis 3.3.2.1 The needle tract The entry point of the infusion needle in the cortex was usually identified by a slight depression on the brain surface. In transverse brain tissue sections, the M B P positive surviving cells were easily seen by their dark brown reaction product (Figure 3). In MBP negative staining sections, we found the needle tract by identifying its glial scar. 22 3.3.2.2 The morphology of the transplanted human oligodendrocytes in the host brain The morphology of the transplanted oligodendrocytes with positive M B P staining was difficult to assess due to the distortion of the transplanted cells during the tissue processing (which included detergent treatment and freezing). Cellular processes, however, were observed arising from MBP positive staining cell bodies and running perpendicular to the fibers (Figure 4). 3.3.2.3 The survival of transplanted human oligodendrocytes The survival of transplanted oligodendrocytes was evidenced by the presence of MBP positive staining. In the human fetal group, MBP positive staining cells were found in the transplantation area of all 5 surviving mice. The intensity of staining was quite different from one mouse to another (Figure 5 a, b, c). In the adult group, there was no MBP positive staining visualized (Figure 6) in any of 7 surviving mice. The results are summarized below in Table 2. The results were analyzed using Fisher's exact test. Fetal human oligodendrocytes had better survival (p<0.05) than adult human oligodendrocytes in this experimental paradigm. 3.3.2.4 The migration of transplanted human oligodendrocytes In the fetal group, a characteristic gradient of MBP positive fibers was observed in a pattern radiating from the injection site. The quantity and the length of MBP positive fibers appeared to be very different from one mouse to another (Figure 5 a, b, c). Figure 5a showed that positive MBP staining was only inside needle tract. Figure 5b showed 23 that positive MBP staining along the needle tract with short fiber gradient. Figure 5c showed that positive MBP staining along the needle tract with long fiber gradient. Figure 7 shows that a typical cell among the fibers that had migrated away from the injection site. 3.3.2.4 The myelination of transplanted human oligodendrocytes MBP positive fibers were observed in all 5 shiverer brains in the fetal group. The quantity and the length of MBP positive fibers were different from one mouse to another (Figure 5 a, b, c). This demonstrated that human fetal oligodendrocytes could survive transplantation and function (by myelinating host neurons) in this transplant paradigm. However, there was no any MBP positive fiber detected in the adult group. 3.4 Discussion Following transplantation into myelin-deficient shiverer mice, we have compared the abilities of human fetal glial cells and human adult oligodendrocytes on the survival, migration and myelination. It was clear that the fetal source had significantly better survival than the adult source. The fetal source also demonstrated cell migration and myelination. This was consistent with others work using non-human oligodendrocytes (Franklin and Blakemore, 1991, 1997; Duncan, 1996; Duncan and Milward, 1995). Oligodendrocyte progenitors were demonstrated with 04 antibody staining (Sommer and Schachller 1981; Pfeiffer et al., 1993). Our results were consistent with the previous reports that i) 04 positive cells are a minor component (l%-5%) of fetal human glial cell cultures and ii) 04 positive cells in fetal human glial cell culture have a mixture of immature and mature cell types (Satoh et 24 al., 1994). We assume this small portion of oligodendrocyte progenitors differentiate into oligodendrocytes that express MBP in the shiverer mice. The failure of survival and function in adult group may be due to too few oligodendrocyte progenitors. This result was also consistent with previous report that human adult glial cell suspension failed to remyelinate in the X-irradiated ethidium bromide lesioned rat (Targett et al., 1996). In the human fetal glial cell culture, astrocyte was a major population. It may promote oligodendrocyte proliferation or myelination by means of producing glia growth factors. Transplantation may also stimulate host astrocyte to produce glia growth factor. These two astrocyte populations may function together to support oligodendrocyte progenitor proliferation and differentiation. However, the adult human oligodendrocyte was demonstrated that had no response to any growth factor (Young et al., 1988). This may be another possibility to explain this significantly different ability on survival and myelination between human adult oligodendrocyte and human fetal glia after transplantation into shiverer mice. The very intensive MBP positive staining may not only contribute to oligodendrocyte progenitor differentiation, but also contribute to its proliferation stimulated by glial growth factor from astrocyte. For future study, we can detect the proliferation of transplanted human fetal glial cells using anti-Brdu staining in order to estimate coordination between oligodendrocyte progenitor proliferation and myelination. 25 CHAPTER IV CO-TRANSPLANTATION OF GLIAL GROWTH FACTORS 4.1 Introduction Whether we can achieve our final goal of clinical remyelination will eventually depend on whether the transplanted myelin-forming glial cells can survive, proliferate, migrate and remyelinate the demyelinated axons in the new hostile host environment. As discussed in Chapter I, the growth and differentiation of mammalian glial cells are regulated by extracellular signals, including growth factors and cytokines. These molecules can locally influence the growth and behavior of their target cells by binding to specific cell surface receptors and triggering a cascade of intracellular events. We proposed to evaluate if infusing glial growth factors simultaneously with glial cell transplantation could improve the survival and performance of the transplanted cells. Co-transplantation of neuronal growth factors has been successfully conducted in neuronal transplantation experiments. Nerve growth factors and neurotrophic factors have been shown to improve the proliferation, growth, and function of neurons in vitro (Hohn et al, 1990; Longo et al, 1993; Bjerre et al, 1975; Apfel et al, 1991, 1992; DiCicco-Bloometal, 1993; Gold et al , 1991; Hory-Lee et al, 1993; DiStefano et al, 1992). The neurotrophic factors, BDNF (brain derived nerve factor), NT-3 (neurotrophin-3) and CNTF (ciliary neurotrophic factor) can rescue neurons and stimulate their re-growth after neuronal injuries (Oppenheim et al, 1995, 1992; Yan et al, 1992; Sendtner et al, 1992; Tetzlaff et al, 1994; Giel et al, 1996). Co-transplantation of neurotrophic factors with neurons into the CNS via osmotic minipump has enhanced transplanted neuronal survival and functional (Oppenheim et al , 1991; Gurney et al , 1992; Lewis et al, 1993; Sinson et al, 1996; Sautter et al, 1998). Infusion of BDNF into the vicinity of axotomized 27 corticospinal and rubrospinal neurons has also promoted the survival and functional recovery of these injured neurons (Giehl, 1996; Kobayashi and Fan 1997). Co-transplantation of growth factor has also been successfully applied in the treatment of peripheral nervous system disorders and Parkinson's disease in patients (Rask et al., 1995; Petty et al., 1994; Bradley et al , 1995; ACTS, 1996). For glial cells, in vitro studies have tested the effects of growth factors on survival, proliferation and migration of oligodendrocytes (Gard et al., 1995; Mckay et al., 1997). Several glial growth factors and their receptors have been identified in the CNS (Gospodarowicz et al., 1984; Gammeltoft et al., 1987). Glial growth factors can act on various stages of the oligodendrocyte lineage and at various times during the myelination process by influencing their proliferation, migration and differentiation (Gard et al., 1995; Mckay et al., 1997). Platelet-deprived growth factor (PDGF), whose receptors are restricted to cells of glial origin, has been reported to play an important role in the control of normal glial cell growth (Heldin et al., 1981c; Ross et al., 1986). This astrocyte type-1-derived mitogen has been shown to strongly control the proliferation and differentiation of 0-2A progenitor cells in the developing optic nerve (Richardson et al., 1988; Noble et al., 1988; Raff et al., 1988). Moreover, it has been found that cell migration is highly associated with a bipolar morphology and PDGF can induce cell mobility by changing the multipolar progenitors into a bipolar morphology (Small et al., 1987; Nobel et al., 1988). Basic fibroblast growth factor (bFGF) has been reported to stimulate the proliferation and short-term survival of oligodendrocyte progenitors in vitro (Gard, 1993) 28 and to be responsible for the proliferation of oligodendrocyte progenitor cells in the developing optic nerve in vivo (Richardson, 1988). Oligodendrocyte progenitor development is delayed in vitro by PDGF because 04+GalC" progenitors are transiently reverted to A2B5+04" preprogenitor-like cells (Gard, 1993). bFGF has been shown to promote mitogenic activity of the 04+GalC" progenitors to a mature level in vitro (Gard, 1993). The promotion effect of bFGF and PDGF on the proliferation and migration of oligodendrocyte progenitor cells has been observed in vivo (Mckinnon, 1993). Insulin-like growth factors (IGFs) exist in the normal brain at different developmental ages, including the early postnatal period when myelination is at a high level (D'Ercole et al, 1984; Brown et al, 1986; Lund et al, 1986; Baskin et al, 1988). Astrocytes have been reported to produce IGF-1 (Ballotti et al, 1987; Bondy et al, 1991; Komoly et al, 1992). IGF-1 has been reported to play a crucial role in the development and myelination of oligodendrocytes by promoting their proliferation, differentiation and regulating myelin gene expression both in vitro and in vivo (McMorris et al, 1993, 1986; Carson et al, 1993). The myelin content per oligodendrocyte is significantly increased in transgenic mice that overexpress IGF-1 (McMorris et al, 1993). Moreover, IGF-1 also supports the survival of younger oligodendrocytes and their progenitors in the developing rat optic nerve culture (Barres et al , 1992). No growth factors have been reported to be effective for the proliferation of adult human oligodendrocytes and astrocytes in culture (Young et al, 1988). For fetal human glial cells in culture, it has been reported that they have a great proliferative capacity without the requirement of exogenous growth factors (Kim et al 1983, 1992, and 1994). 29 Human fetal oligodendrocyte did not response to PDGF or bFGF, but exogenous IGF-1 did accelerate their differentiation in vitro (Satoh et al , 1994). Although both in vitro and in vivo studies have demonstrated the benefit of glial growth factors in the promotion of survival and migration of oligodendrocytes, no experiment has been tried to apply this into glial cell transplantation. In light of the successful results from neuronal transplantation, the combination of myelin forming cells and glial growth factors transplantation represents a new promising therapeutic strategy in the treatment of demyelination disorders. In this chapter, we assess the feasibility of co-transplantation of human fetal oligodendrocytes with IGF-1 in the shiverer mouse model. We also compare the survival, migration and myelination of the fetal cells with those that were transplanted without IGF-1 infusion. 4.2 Material and Methods 4.2.1 Animal A total of twenty neonate shiverer mice (C57BL/6J-MBP, stock No. 002492) were used divided into two experimental groups with ten mice in each group. Group I received human fetal oligodendrocytes and IGF-1 infusion while Group II received human fetal oligodendrocytes and PBS infusion. In addition, the human fetal glial transplant group from Chapter III was also compared to the experimental group. The fetal oligodendrocyte culture and transplantation techniques were the same as Chapter III. 30 4.2.2 Transplantation and Immunosuppression For IGF-1 or PBS co-transplantation, a cannula (0.36 mm in diameter; stainless steel one, Alzet, Newark, DE) was inserted beside the transplanted cells in the striatum at the following coordinates: 0.8 mm anterior to Bregma, 2.1 mm to the right of midline, and 3.5 mm deep to the cortical surface. The cannula was anchored in position with Loctite 454 (Loctite, Mississauga, Ontario). An osmotic minpump (Alzet no. 2002, Newark, DE) (0.5 ul/hour) filled with either vehicle alone (0.01M sterile PBS supplemented with 100 U penicillin-streptomycin (Gibco BRL, Burlington, Ontario) or IGF-1 (Human, Recombinant, Sigma, Oakville, Ontario) at a concentration of 100 ng/ml was then connected to the infusion cannulae with a one cm long catheter tube (Alzet, Newark, DE). Each pump delivered 1.2 ng IGF-1 per day in a total volume of 168 ul for 14 days. All animals received Cyclosporine A 5 mg/kg intraperitoneally for 14 days. Animals were sectioned 21 days after the transplantation and their brains prepared for MBP staining as in Chapter III. 4.3 Results 4.3.1 The needle tract The needle tract and minipump infusion tracts combined together in both Group I and Group II and were observed by dark brown MBP positive staining along the edge of the area (Figure 8). The width of needle tract was very different from one mouse to another. Some tissue inside the tract in the Group I (fetal cells+IGF-1) was delicate (Figure 9). This was not observed in the Group II (fetal cells+PBS). 31 4.3.2 The morphology of transplanted human fetal glial cells Cell bodies with a process were observed with MBP positive staining in the Group I (Figure 10). These cells were located inside the needle tract and their processes interconnected. No cell bodies were observed to have migrated outside the needle tract. There was no obvious cell bodies observed in the Group II. 4.3.3 The survival and myelination of transplanted human fetal glial cells MBP positive staining was observed in all mice in both groups. However, the intensity of staining was very different from one mouse to another (Figure 11 a, b, c; Figure 12 a, b, c). Figure 11, 12 a showed that positive MBP staining inside needle tract. Figure 11, 12 b showed that positive MBP staining along the needle tract with short fiber gradient. Figure 11,12 c showed that positive MBP staining along the needle tract with long fiber gradient. 4.3.4 The migration of transplanted human fetal glial cells The MBP positive fibers were observed to radiated from the injection site (Figure 11 c; 12 c). The quantity and the length of MBP positive fibers varied more within the groups than them before (Figure 11, 12). Within IGF-1 co-transplantation group, Figure 11 a, b, c showed different length of MBP positive fiber, whereas Figure 12 a, b, c showed different length of MBP positive fiber in PBS co-transplantation group. 4.4 Discussion Human fetal glial cells and glial growth factor, IGF-1, were co-transplanted into the brains of myelin-deficient shiverer mice. 32 There was no difference in survival, migration and myelination between groups co-transplanted with IGF-1 or PBS control. IGF-1 co-transplantation did not act on human fetal glial cell survival and myelination in shiverer mice. There are several potential reasons for the lack of difference between the groups. First, the time course may have been too short to allow a significant difference to occur. Second, the ideal IGF-1 concentration and distance from implantation are unknown. We used the concentration that produced effects in vitro but the situation may be different in vivo. Third, transplantation may stimulate host astrocyte to produce growth factor to support fetal glial cell survival in PBS co-transplantation group. Comparing with the experiment of human fetal glial cell transplantation, it was obvious that the needle tract was wider, and the transplant was delicate. The immunohistochemical method for detecting transplant survival may not have been sensitive enough. We could not conclude the effect of IGF-1 co-transplantation on the survival and myelination of transplanted human fetal glial cells. Future trials could use mRNA quantification and protein quantification... This study did demonstrate that the shiverer mouse model could be used for co-transplant experiments and that chronic infusion of glial growth factors is possible in this animal. 33 CHAPTER V DISCUSSION In present study, we transplanted human fetal oligodendrocytes and human adult oligodendrocytes into myelin-deficient shiverer mice. We also co-transplanted the glial growth factor, IGF-1, with human fetal glial cells into the brains of shiverer mice. We demonstrated that human fetal glial cells have a significantly better survival and ability to myelinate in this myelin-deficient environment. The shiverer mouse was demonstrated to be a good model for glial growth factor (IGF-1) co-transplantation. Although various kinds of oligodendrocyte lineage can form myelin following transplantation, many studies have shown that the myelination ability of the transplanted myelin-forming cells depends on their developmental stage. Studies in rodents have shown that immature oligodendrocytes have a much higher capability for myelination and migration after being transplanted into host CNS than adult oligodendrocytes (Duncan 1997; Groves et al., 1993; Duncan et al., 1992; Warrington et al., 1993; Archer et al., 1997; Small et al., 1987; Levison et al., 1993; Pringle et al., 1993). Our observations with human oligodendrocytes were consistent with these previous findings in rodents. Although human fetal oligodendrocytes represent only a small portion of the population in glial cell culture, they are able to play a significant role in remyelinating the host myelin- deficient environment. It is likely that signals from the host's demyelinated environment trigger differentiation of oligodendrocyte precursors into mature oligodendrocytes as demonstrated in our study. In vitro studies have shown that glial progenitor cells can develop into either oligodendrocytes or astrocytes depending on the culture medium (Raff 35 et al, 1983). Similar signals from the in vivo glial-deficient area can also affect the destination of transplanted progenitor cells (Franklin et al, 1995; Espinosa De Los Monteros et al, 1993). Furthermore, oligodendrocytes and astrocytes differentiate in different areas of the lesion following transplantation indicating an environment effect (Baltimore et al, 1994). Many studies have suggested that local environmental factors determine survival, differentiation pathway and performance of transplanted cells. Oligodendrocyte precursors cultured from neonatal rat pups survive well in the spinal cord of x-irradiated syngeneic adult rats, but have poor survival after being transplanted into the spinal cord of normal adult rats (O'Leary et al, 1997). Similarly, transplanted CG4 cells can survive, migrate and remyelinate axons in the X-irradiated and demyelinated spinal cord, but not in the normal spinal cord (Franklin et al, 1996a). Multiple extracellular signals are likely required for transplanted myelin-forming cells to survive, migrate and myelinate. (Baltimore et al, 1994; Barres et al, 1993). 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Growth factors for human glial cells in culture. GLIA 1988, 1: 113-123. Yan Q, Elliott J, Snider WD. Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death. Nature 1992, 360: 753-755. Yuen EC and Mobley WC. Therapeutic potential of neurotrophic factors for neurological disorders. Annals Neurol 1996 Sept, 40(3): 346-354. Zecevic N, Andjelkovic A, Matthieu JM, Tosic M . Myelin basic protein immunoreactivity in the human embryonic CNS. Brain Res Dev Brain Res 1998, Jan 14(105): 1,97-108. 56 Group I Group II cell source human fetal oligodendrocyte human adult oligodendrocyte cell suspension 3ul, 5X105/ul 3ul 5X105/ul injection site striatum striaum immunosuppression cyclosporine A, 5 mg/kg x 21d cyclosporine A, 5mg/kg x 2Id post-injection time 21 days 21 days tissue processing frozen section, 30 um frozen section, 30 urn section staining MBP MBP T A B L E 1. Summary of comparison of human fetal oligodendrocytes with human adult oligodendrocytes on survival, migration and remyelination in shiverer model. Survival No survival Fetal Group 5 0 Adult Group 0 7 T A B L E 2. Summary of comparison of human fetal oligodendrocytes with human adult oligodendroyctes on survival by MBP staining in shiverer model :57 Nerve impluse-Neuron Axon ' Nerve implUse •Normal myelin Oligodendrocyte Neuron Axon 'if^K^J Abnormal myelin Oligodendrocyte Modified from 'Research on multiple Sclerosis', by courtesy of Dr. C W H Adams and of C C Thomas/Springfield Normal mouse CNS Shiverer mouse CNS Reproduced from 'Neurology Mutation Affecting Myelination' , by courtesy of Drs. A .L . Ganser and D.A. Kirschner and of Elsevier/North Holland F I G U R E 1. Comparison between normal and demyelinated myelin. 58' F I G U R E 2 . Positive 04 staining cell in human fetal oligodendroycte culture. 20X 59 F I G U R E 3. Needle tract in the group of human fetal oligodendrocyte transplantation. 20X 60 F I G U R E 4. Positive MBP staining cell with process in the group of human fetal oligodendrocyte transplantation. 61 62 40X F I G U R E 5. Survival and remyelination of human fetal oligodendrocytes in the brain of shiverer mouse, a, positive MBP staining inside needle tract. 63 F I G U R E 5. Survival and remyelination of human fetal oligodendrocytes in the brain of shiverer mouse, b, positive MBP staining with short fiber gradient. 64 F I G U R E 5. Survival and remyelination of human fetal oligodendrocytes in the brain of shiverer mouse, b, positive MBP staining with short fiber gradient. 65 F I G U R E 5. Survival and remyelination of human fetal oligodendrocytes in the brain of shiverer mouse. c , positive MBP staining with long fiber gradient. 66 F I G U R E 5. Survival and remyelination of human fetal oligodendrocytes shiverer mouse, c, positive MBP staining with long fiber gradient. 67 F I G U R E 6. Negative MBP staining cell in the group of human adult oligodendrocyte transplantation. 20X 68 F I G U R E 7. Cell migration and gradient of positive MBP fibers in the group of human fetal oligodendrocyte transplantation. •69 F I G U R E 8. Needle tract in the experiments of IGF-1 co-transplantation with human fetal oligodendrocytes. 71 F I G U R E 9. Needle tract in the experiments of PBS co-transplantation with human fetal oligodendrocytes. 40X 72 F I G U R E 10. Positive MBP staining cell with process inside of delicate area in the experiments of IGF-1 co-transplantation with human fetal oligodendrocytes. 73 F I G U R E 11. Survival and remyelination in the experiments of IGF-1 co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse, a, positive MBP staining inside needle tract. 74 F I G U R E 11. Survival and remyelination in the experiments of IGF-1 co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse, a, positive MBP staining inside needle tract. 75 F I G U R E 11. Survival and remyelination in the experiments of IGF-1 co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse. b, positive MBP staining with short fiber gradient. 76 F I G U R E 11. Survival and remyelination in the experiments of IGF-1 co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse, c, positive MBP staining with long fiber gradient. 77 F I G U R E 11. Survival and remyelination in the experiments of IGF-1 co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse. c, positive MBP staining with long fiber gradient^ 78 F I G U R E 12. Survival and remyelination in the experiments of PBS co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse, a, positive MBP staining inside needle tract. 79 F I G U R E 12. Survival and remyelination in the experiments of PBS co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse, a, positive MBP staining inside needle tract. 80 F I G U R E 12. Survival and remyelination in the experiments of PBS co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse. b, positive MBP staining with short fiber gradient. ~~ 81 60X F I G U R E 12. Survival and remyelination in the experiments of PBS co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse. b, positive MBP staining with short fiber gradient. 82 F I G U R E 12. Survival and remyelination in the experiments of PBS co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse, c, positive MBP staining with long fiber gradient. 83 F I G U R E 12. Survival and remyelination in the experiments of PBS co-transplantation with human fetal oligodendrocytes in the brain of shiverer mouse, c, positive MBP staining with long fiber gradient X4 

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