Repair of the injured spinal cord by transplantation of neural stem cells in a hyaluronan-based hydrogel.

Traumatic injury to the spinal cord causes cell death, demyelination, axonal degeneration, and cavitation resulting in functional motor and sensory loss. Stem cell therapy is a promising approach for spinal cord injury (SCI); however, this strategy is currently limited by the poor survival and uncontrolled differentiation of transplanted stem cells. In an attempt to achieve greater survival and integration with the host tissue, we examined the survival and efficacy of adult brain-derived neural stem/progenitor cells (NSPCs) injected within a hydrogel blend of hyaluronan and methyl cellulose (HAMC) into a subacute, clinically relevant model of rat SCI. Prior to use, HAMC was covalently modified with recombinant rat platelet-derived growth factor-A (rPDGF-A) to promote oligodendrocytic differentiation. SCI rats transplanted with NSPCs in HAMC-rPDGF-A showed improved behavioral recovery compared to rats transplanted with NSPCs in media. Rats with NSPC/HAMC-rPDGF-A transplants had a significant reduction in cavitation, improved graft survival, increased oligodendrocytic differentiation, and sparing of perilesional host oligodendrocytes and neurons. These data suggest that HAMC-rPDGF-A is a promising vehicle for cell delivery to the injured spinal cord.

Characterization of hyaluronan-methylcellulose hydrogels for cell delivery to the injured spinal cord.

No effective clinical treatment currently exists for traumatic spinal cord injury. Cell replacement therapy holds promise for attaining functional repair. Cells may be delivered directly or near the injury site; however, this strategy requires a delivery vehicle to maintain cell viability. We have identified an injectable, biocompatible, and biodegradable hydrogel scaffold composed of hyaluronan (HA) and methylcellulose (MC) that may be an effective scaffold for therapeutic cell delivery. The purpose of the present study was to determine the effects of polymer concentration on HAMC mechanical strength, gelation time, and cell viability. The yield stress of HAMC, a measure of mechanical stiffness, was tunable via manipulation of MC and HA content. Measurement of the elastic and storage moduli as functions of time revealed that HAMC gels in less than 5 min at physiological temperatures. Human umbilical tissue-derived cells encapsulated in HAMC were homogenously and stably distributed over 3 days in culture and extended processes into the scaffold. Cell viability was stable over this period in all but the most concentrated HAMC formulation. Because of its strength-tunability, rapid gelation, and ability to maintain cell viability, HAMC is a promising vehicle for cell delivery and is being tested in ongoing in vivo studies.

The effect of growth factors and soluble Nogo-66 receptor protein on transplanted neural stem/progenitor survival and axonal regeneration after complete transection of rat spinal cord.

Adult central mammalian axons show minimal regeneration after spinal cord injury due to loss of oligodendrocytes, demyelination of surviving axons, absence of growth-promoting molecules, and inhibitors of axonal outgrowth. In the present study, we attempted to address these impediments to regeneration by using a combinatory strategy to enhance cell survival and regeneration after complete spinal cord transection (SCT) in adult rats. The strategy comprised: 1) adult rat brain-derived neural stem/progenitor cells (NSPCs) preseeded on laminin-coated chitosan channels; 2) extramedullary chitosan channels to promote axonal regrowth and reduce the barrier caused by scarring; 3) local delivery of a novel rat soluble Nogo-66 receptor protein [NgR(310)ecto-Fc, referred to as NgR] to block the inhibitory effect of myelin-based inhibitors; and 4) local delivery of basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor to enhance survival and promote differentiation of transplanted cells. Compared with our previous studies where brain-derived NSPCs preseeded in extramedullary chitosan channels were implanted in the same SCT model but without growth factors and NgR, the present channel-growth factor combination produced greater numbers of surviving NSPCs after SCT. Also, the growth factors promoted preferential differentiation of NSPCs toward oligodendrocytes, while NgR significantly decreased astrocytic differentiation of NSPCs. NgR alone or in combination with NSPCs significantly enhanced the total number of myelinated fibers in the bridge and increased the area of the bridging tissue between the cord stumps. The combination of NgR, growth factors, and NSPCs had synergistic effect on bridge formation. However, only a small number of descending corticospinal tract axons grew into the central portions of the bridges as shown by anterograde tracing of the corticospinal tract with BDA. The majority of the regenerated axons in the channels originated from local host neurons adjacent to the tissue bridges. In conclusion, we showed that growth factors increased survival of transplanted NSPCs whereas NgR enhanced axonal regeneration, but the combination did not have additive effects on functional recovery or regeneration.

Neural stem/progenitor cells from the adult human spinal cord are multipotent and self-renewing and differentiate after transplantation.

Neural stem/progenitor cell (NSPC) transplantation is a promising therapy for spinal cord injury (SCI). However, little is known about NSPC from the adult human spinal cord as a donor source. We demonstrate for the first time that multipotent and self-renewing NSPC can be cultured, passaged and transplanted from the adult human spinal cord of organ transplant donors. Adult human spinal cord NSPC require an adherent substrate for selection and expansion in EGF (epidermal growth factor) and FGF2 (fibroblast growth factor) enriched medium. NSPC as an adherent monolayer can be passaged for at least 9 months and form neurospheres when plated in suspension culture. In EGF/FGF2 culture, NSPC proliferate and primarily express nestin and Sox2, and low levels of markers for differentiating cells. Leukemia inhibitory factor (LIF) promotes NSPC proliferation and significantly enhances GFAP expression in hypoxia. In differentiating conditions in the presence of serum, these NSPC show multipotentiality, expressing markers of neurons, astrocytes, and oligodendrocytes. Dibutyryl cyclic AMP (dbcAMP) significantly enhances neuronal differentiation. We transplanted the multipotent NSPC into SCI rats and show that the xenografts survive, are post-mitotic, and retain the capacity to differentiate into neurons and glia.Together, these findings reveal that multipotent self-renewing NSPC cultured and passaged from adult human spinal cords of organ transplant donors, respond to exogenous factors that promote selective differentiation, and survive and differentiate after transplantation into the injured spinal cord.

Effects of dibutyryl cyclic-AMP on survival and neuronal differentiation of neural stem/progenitor cells transplanted into spinal cord injured rats.

Neural stem/progenitor cells (NSPCs) have great potential as a cell replacement therapy for spinal cord injury. However, poor control over transplant cell differentiation and survival remain major obstacles. In this study, we asked whether dibutyryl cyclic-AMP (dbcAMP), which was shown to induce up to 85% in vitro differentiation of NSPCs into neurons would enhance survival of transplanted NSPCs through prolonged exposure either in vitro or in vivo through the controlled release of dbcAMP encapsulated within poly(lactic-co-glycolic acid) (PLGA) microspheres and embedded within chitosan guidance channels. NSPCs, seeded in fibrin scaffolds within the channels, differentiated in vitro to betaIII-tubulin positive neurons by immunostaining and mRNA expression, in response to dbcAMP released from PLGA microspheres. After transplantation in spinal cord injured rats, the survival and differentiation of NSPCs was evaluated. Untreated NSPCs, NSPCs transplanted with dbcAMP-releasing microspheres, and NSPCs pre-differentiated with dbcAMP for 4 days in vitro were transplanted after rat spinal cord transection and assessed 2 and 6 weeks later. Interestingly, NSPC survival was highest in the dbcAMP pre-treated group, having approximately 80% survival at both time points, which is remarkable given that stem cell transplantation often results in less than 1% survival at similar times. Importantly, dbcAMP pre-treatment also resulted in the greatest number of in vivo NSPCs differentiated into neurons (37±4%), followed by dbcAMP-microsphere treated NSPCs (27±14%) and untreated NSPCs (15±7%). The reverse trend was observed for NSPC-derived oligodendrocytes and astrocytes, with these populations being highest in untreated NSPCs. This combination strategy of stem cell-loaded chitosan channels implanted in a fully transected spinal cord resulted in extensive axonal regeneration into the injury site, with improved functional recovery after 6 weeks in animals implanted with pre-differentiated stem cells in chitosan channels.

Chitosan channels containing spinal cord-derived stem/progenitor cells for repair of subacute spinal cord injury in the rat.

OBJECTIVE: We evaluated the survival and differentiation capacity of neural stem/progenitor cells (NSPCs) derived from the adult rat spinal cord and seeded on intramedullary chitosan channels that were implanted in a subacute rat spinal cord injury model. METHODS: We implanted into the injured spinal cord a chitosan channel filled with NSPCs harvested from the spinal cord of adult transgenic rats expressing green fluorescent protein 3 weeks after extradural 35g clip compression injury at T8. The NSPC-chitosan channel group was compared with 2 control groups not receiving channels: 1 receiving a direct intramedullary injection of NSPCs into the lesion cavity and 1 receiving trauma alone. The survival and differentiation of NSPCs were evaluated with immunohistochemical and histopathological techniques, and functional improvement was assessed for 6 weeks with the Basso, Beattie, and Bresnahan locomotor score. RESULTS: The NSPC-chitosan channel group showed enhanced survival of NSPCs compared with NSPCs transplanted directly into the lesion cavity, although there was no significant difference in functional recovery between the treatment and control groups. In addition, the intramedullary implantation of the chitosan channel did not worsen the functional deficit after the 35g clip injury. CONCLUSIONS: Chitosan channels enhanced the survival of transplanted NSPCs in the subacutely injured spinal cord. Functional deficits were not exacerbated by the intramedullary transplantation of chitosan channels into the site of injury.

Endogenous radial glial cells support regenerating axons after spinal cord transection.

During the development of central nervous system, radial glial cells support target-specific neuronal migration. We recently reported that after implantation of chitosan channels with complete spinal cord transection, the tissue bridging the spinal cord stumps contained axons and radial glial cells. The purpose of this study was to clarify the role of the radial glial cells in the tissue bridges. Chitosan channels were implanted in rats with thoracic spinal cord transection. After 14 weeks, all animals had tissue bridges in the channels that contained many radial glial cells in longitudinal arrangement, some of which were in contact with axons in the bridges. We suggest that radial glial cells can guide regenerating axons across the bridge in the channel after spinal cord transection.

Functional immobilization of interferon-gamma induces neuronal differentiation of neural stem cells.

Stem cell transplantation provides significant promise to regenerative strategies after injury in the central nervous system. Neural stem/progenitor cells (NSPCs) have been studied in terms of their regenerative capacity and their ability to differentiate into neurons when exposed to various soluble factors. In this study, interferon-gamma (IFN-gamma) was compared with brain-derived neurotrophic factor (BDNF) and erythropoietin and was shown to be the best single growth factor for inducing neuronal differentiation from adult rat brain-derived NSPCs. Next, IFN-gamma was surface immobilized to a methacrylamide chitosan (MAC) scaffold that was specifically designed to match the modulus of brain tissue and neuronal differentiation of NSPCs was examined in vitro by immunohistochemistry. Bioactive IFN-gamma was successfully immobilized and quantified by ELISA. Both soluble and immobilized IFN-gamma on MAC surfaces showed dose dependent neuronal differentiation with soluble saturation occurring at 100 ng/mL and the most effective immobilized IFN-gamma dose at 37.5 ng/cm(2), where significantly more neurons resulted compared with controls including soluble IFN-gamma.

Neural stem/progenitor cells differentiate in vitro to neurons by the combined action of dibutyryl cAMP and interferon-gamma.

Transplantation of neural stem/progenitor cells (NSPCs) is a promising strategy for repair of the diseased/injured central nervous system (CNS); however, controlling their differentiation remains a significant hurdle. This study is aimed at controlling differentiation and specifically at screening exogenous factors to direct NSPC differentiation into neurons in vitro. In this study, adult rat SVZ-derived NSPCs were treated with several factors and screened individually and in combination for changes in cellular morphology, neuronal marker expression, quantitative real-time qRT-PCR, and electrophysiological properties. These in vitro screens showed that of all the different treatments, dibutyryl cyclic AMP (dbcAMP) and interferon-gamma (IFN-gamma) enhanced neuronal differentiation most significantly compared to the 1% fetal bovine serum (FBS) controls. Importantly, the combined treatment of NSPCs with dbcAMP and IFN-gamma promoted greater neuronal differentiation as reflected by an increase in beta-III tubulin expression and morphological differentiation. Interestingly, the neurons that were generated from the NSPCs in vitro in the presence of dbcAMP and IFN-gamma, alone or in combination, responded to exogenous glutamate (Glu), but not gamma-aminobutyric acid (GABA), indicating that these neurons express glutamate receptors. These NSPC-derived neurons may be promising for neural regenerative strategies in the CNS.

The effect of immobilized platelet derived growth factor AA on neural stem/progenitor cell differentiation on cell-adhesive hydrogels.

Neural stem/progenitor cells (NSPCs) hold great promise in regenerative medicine; however, controlling their differentiation to a desired phenotype within a defined matrix is challenging. To guide the differentiation of NSPCs, we first created a cell-adhesive matrix of agarose modified with glycine-arginine-glycine-aspartic acid-serine (GRGDS) and then demonstrated the multipotentiality of NSPCs to differentiate to the three primary cell types of the central nervous system on this matrix: neurons, oligodendrocytes and astrocytes. We then examined whether immobilized platelet derived growth factor AA (PDGF-AA) would promote differentiation similarly to the same soluble factor and found similar percentages of NSPCs differentiated to oligodendrocytes as determined by immunohistochemistry (IHC) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Interestingly, the gene expression of the differentiated oligodendrocytes was similar for 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) but different for myelin oligodendrocyte glycoprotein (MOG) in the presence of soluble PDGF-AA vs. immobilized PDGF-AA. These results demonstrate for the first time, that it is possible to control the differentiation of NSPCs, and specifically to oligodendrocytes, in cell-adhesive matrices with immobilized PDGF-AA.

Bioengineering neural stem/progenitor cell-coated tubes for spinal cord injury repair.

The aim of this study was to understand the survival and differentiation of neural stem/progenitor cells (NSPCs) cultured on chitosan matrices in vivo in a complete transection model of spinal cord injury. NSPCs were isolated from the subependyma of lateral ventricles of adult GFP transgenic rat forebrains. The GFP-positive neurospheres were seeded onto the inner lumen of chitosan tubes to generate multicellular sheets ex vivo. These bioengineered neurosphere tubes were implanted into a completely transected spinal cord and assessed after 5 weeks for survival and differentiation. The in vivo study showed excellent survival of NSPCs, as well as differentiation into astrocytes and oligodendrocytes. Importantly, host neurons were identified in the tissue bridge that formed within the chitosan tubes and bridged the transected cord stumps. The excellent in vivo survival of the NSPCs coupled with their differentiation and maintenance of host neurons in the regenerated tissue bridge demonstrates the promise of the chitosan tubes for stem cell delivery and tissue regeneration.

Extramedullary chitosan channels promote survival of transplanted neural stem and progenitor cells and create a tissue bridge after complete spinal cord transection.

Transplantation of neural stem and progenitor cells (NSPCs) is a promising strategy for repair after spinal cord injury. However, the epicenter of the severely damaged spinal cord is a hostile environment that results in poor survival of the transplanted NSPCs. We examined implantation of extramedullary chitosan channels seeded with NSPCs derived from transgenic green fluorescent protein (GFP) rats after spinal cord transection (SCT). At 14 weeks, we assessed the survival, maturation, and functional results using NSPCs harvested from the brain (brain group) or spinal cord (SC group) and seeded into chitosan channels implanted between the cord stumps after complete SCT. Control SCT animals had empty chitosan channels or no channels implanted. Channels seeded with brain or spinal cord-derived NSPCs showed a tissue bridge, although the bridges were thicker in the brain group. Both cell types showed long-term survival, but the number of surviving cells in the brain group was approximately five times as great as in the SC group. In both the brain and SC groups at 14 weeks after transplantation, many host axons were present in the center of the bridge in association with the transplanted cells. At 14 weeks astrocytic and oligodendrocytic differentiation in the channels was 24.8% and 17.3%, respectively, in the brain group, and 31.8% and 9.7%, respectively, in the SC group. The channels caused minimal tissue reaction in the adjacent spinal cord. There was no improvement in locomotor function. Thus, implantation of chitosan channels seeded with NSPCs after SCT created a tissue bridge containing many surviving transplanted cells and host axons, although there was no functional improvement.

Modular biodegradable biomaterials from surfactant and polyelectrolyte mixtures.

Polymeric assemblies are used in many biomaterials applications, ranging from drug-bearing nanoparticles to macroscopic scaffolds. Control over their biodegradation rates is usually achieved through synthetic modification of their molecular structure. As a simpler alternative, we exploit the associative phase separation in mixtures of bioderived surfactants and polyelectrolytes. The gel fiber scaffolds are formed via phase inversion, using a homologous series of fatty acid salts-sodium caprate (NaC10), laurate (NaC12), and myristate (NaC14), and a water-soluble chitosan derivative, N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride (HTCC). Their dissolution times are modulated through the selection of the fatty acid molecule and vary in a predictable manner from minutes (for NaC10-HTCC), to hours (for NaC12-HTCC), to days (for NaC14-HTCC). These variations are linked to differences in surfactant-polyelectrolyte binding strength and scale with the equilibrium binding constants of their mixtures. These fibers were found to be both cytocompatible and cell-adhesive using neural stem/progenitor cells, suggesting their potential for utility in biomedical applications.

Progenitor cells from the porcine neural retina express photoreceptor markers after transplantation to the subretinal space of allorecipients.

Work in rodents has shown that cultured retinal progenitor cells (RPCs) integrate into the degenerating retina, thus suggesting a potential strategy for treatment of similar degenerative conditions in humans. To demonstrate the relevance of the rodent work to large animals, we derived progenitor cells from the neural retina of the domestic pig and transplanted them to the laser-injured retina of allorecipients. Prior to grafting, immunocytochemical analysis showed that cultured porcine RPCs widely expressed neural cell adhesion molecule, as well as markers consistent with immature neural cells, including nestin, Sox2, and vimentin. Subpopulations expressed the neurodevelopmental markers CD-15, doublecortin, beta-III tubulin, and glial fibrillary acidic protein. Retina-specific markers expressed included the bipolar marker protein kinase Calpha and the photoreceptor-associated markers recoverin and rhodopsin. In addition, reverse transcription-polymerase chain reaction showed expression of the transcription factors Dach1, Hes1, Lhx2, Pax6, Six3, and Six6. Progenitor cells prelabeled with vital dyes survived as allografts in the subretinal space for up to 5 weeks (11 of 12 recipients) without exogenous immune suppression. Grafted cells expressed transducin, recoverin, and rhodopsin in the pig subretinal space, suggestive of differentiation into photoreceptors or, in a few cases, migrated into the neural retina and extended processes, the latter typically showing radial orientation. These results demonstrate that many of the findings seen with rodent RPCs can be duplicated in a large mammal. The pig offers a number of advantages over mice and rats, particularly in terms of functional testing and evaluation of the potential for clinical translation to human subjects. Disclosure of potential conflicts of interest is found at the end of this article.

A comparison of neural differentiation and retinal transplantation with bone marrow-derived cells and retinal progenitor cells.

Retinal progenitor cells (RPCs) are immature precursors that can differentiate into retinal neurons, including photoreceptors. Recently, it has been reported that bone marrow-derived cells may also be capable of differentiation into cells of central nervous system lineage, including retinal neurons. We compared these two cell types to evaluate their potential as a source of cells for retinal transplantation. Marrow stromal cells (MSCs) and macrophages were isolated from enhanced green fluorescence protein mice. MSCs were cultured with brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor to induce neuronal differentiation. RPCs were cultured under the same conditions or with 10% fetal bovine serum. Neuronal marker expression was examined and compared between MSCs and RPCs. MSCs, macrophages, and RPCs were also cultured with explanted retinas from rhodopsin knockout mice to study their potential for retinal integration. MSCs expressed neuronal and retina-specific markers by reverse transcription-polymerase chain reaction and immunocytochemistry. Both types of cells migrated into retinal explants and expressed neurofilament 200, glial fibrillary acidic protein, protein kinase C-alpha, and recoverin. RPCs expressed rhodopsin, a photoreceptor marker we never detected in MSCs. A majority of bone marrow derived-macrophages differentiated into cells that resembled microglia, rather than neural cells, in the explanted retina. This study shows that RPCs are likely to be a preferred cell type for retinal transplantation studies, compared with MSCs. However, MSCs may remain an attractive candidate for autologous transplantation.

Sorbitol causes preferential selection of Muller glial precursors from late retinal progenitor cells in vitro.

PURPOSE: The replacement of glucose by sorbitol in growth medium causes selection of astroglial cells from heterogeneous primary cultures derived from the brains of newborn mice. The present study was undertaken to investigate the effects of sorbitol on in vitro selection of Müller glial precursors from expanded late retinal progenitor cells (RPCs). METHODS: RPCs used in these studies were isolated from the neural retina of postnatal day one green fluorescent protein (GFP) transgenic mice. The resulting GFP positive neurospheres were dissociated into a single cell suspension and grown on poly-D-lysine/laminin coated tissue culture flasks or slides to generate adherent RPCs. These adherent cells were treated with glucose free medium containing 25 mM sorbitol for 7 days and the expression of retinal-specific cell markers was determined by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot analysis RESULTS: In vitro studies showed that sorbitol treatment of late RPCs altered cellular morphology. Immunocytochemical studies showed an increase in the proportion of cells expressing glial cell markers, most of which co-expressed CRALBP, GFAP, and vimentin. An increase in the proportion of cells expressing PKCalpha, a bipolar cell marker, was also observed. RT-PCR analysis showed down-regulation of nestin transcripts with a concomitant increase in CRALBP, GFAP, vimentin and PKCalpha. These findings were confirmed by immunoblot analysis, where down-regulation of nestin expression with simultaneous up-regulation of CRALBP, GFAP and PKCalpha was observed. CONCLUSIONS: Sorbitol treatment of multipotent late RPCs, in the absence of glucose, results in the preferential selection of Müller glial precursors and their subsequent differentiation into cells that morphologically resemble Müller cells and co-express multiple glial markers.

Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells.

Retinal progenitor cells (RPCs) are multipotent central nervous system precursors that give rise to all of the cell types of the retina during development. Several groups have reported that mammalian RPCs can be isolated and expanded in culture and can differentiate into retinal neurons upon grafting to the mature, diseased eye. However, cell delivery and survival remain formidable obstacles to application of RPCs in a clinical setting. Because biodegradable polymer/progenitor constructs have been shown to be capable of tissue generation in other compartments, we evaluated the survival, migration, and differentiation of RPCs delivered on PLLA/PLGA polymer substrates to the mouse subretinal space and compared these results to conventional injections of RPCs. Polymer composite grafts resulted in a near 10-fold increase in the number of surviving cells after 4 weeks, with a 16-fold increase in cell delivery. Grafted RPCs migrated into the host retina and expressed the mature markers neurofilament-200, glial fibrillary acidic protein, protein kinase C-alpha, recoverin, and rhodopsin. We conclude that biodegradable polymer/progenitor cell composite grafts provide an effective means of increasing progenitor cell survival and overall yield when transplanting to sites within the central nervous system such as the retina.

Effects of ciliary neurotrophic factor on differentiation of late retinal progenitor cells.

Ciliary neurotrophic factor (CNTF) has been shown to be a potent regulator of retinal cell differentiation. The present study was undertaken to investigate the effects of CNTF on in vitro differentiation of expanded late retinal progenitor cells. Retinal progenitor cells used in these studies were isolated from the neural retina of postnatal day-1 green fluorescent protein (GFP) transgenic mice. The resulting GFP-positive neurospheres were dissociated into a single-cell suspension and grown on poly-D-lysine/laminin-coated tissue culture flasks or slides to generate adherent retinal progenitor cells. These adherent cells were treated with 20 ng/ml of CNTF for up to 14 days, and expression of specific retinal cell markers was determined by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and immunoblot analysis. In vitro studies showed that CNTF treatment of late retinal progenitor cells resulted in changes in cellular morphology. Immunocytochemical studies showed an increase in the proportion of cells expressing markers of bipolar cells but not rod differentiation. In addition, an increase in the proportion of cells expressing glial cell markers was observed. RT-PCR analysis showed downregulation in Hes1, Nestin, Notch1, and Pax6 transcripts along with a concomitant increase in protein kinase C (PKC) alpha and glial fibrillary acidic protein (GFAP) transcripts. These findings were confirmed by immunoblot analysis, where downregulation in Nestin expression and simultaneous upregulation in PKC alpha and GFAP were observed. The data indicate that CNTF treatment of multipotential late retinal progenitors increases the proportion of cells that express markers of bipolar neurons and glia.