Chondroitinase ABC treatment and the phenotype of neural progenitor cells isolated from injured rat spinal cord

The aim of the present study was to investigate whether enzyme chondroitinase ABC (ChABC) treatment influences the phenotype of neural progenitor cells (NPCs) derived from injured rat spinal cord. Adult as well as fetal spinal cords contain a pool of endogenous neural progenitors cells, which play a key role in the neuroregenerative processes following spinal cord injury (SCI) and hold particular promise for therapeutic approaches in CNS injury or neurodegenerative disorders. In our study we used in vitro model to demonstrate the differentiation potential of NPCs isolated from adult rat spinal cord after SCI, treated with ChABC. The intrathecal delivery of ChABC (10 U/ml) was performed at day 1 and 2 after SCI. The present findings indicate that the impact of SCI resulted in a decrease of all NPCs phenotypes and the ChABC treatment, on the contrary, caused an opposite effect.     

Remyelination after chronic spinal cord injury is associated with proliferation of endogenous adult progenitor cells after systemic administration of guanosine

Axonal demyelination is a consistent pathological sequel to chronic brain and spinal cord injuries and disorders that slows or disrupts impulse conduction, causing further functional loss. Since oligodendroglial progenitors are present in the demyelinated areas, failure of remyelination may be due to lack of sufficient proliferation and differentiation of oligodendroglial progenitors. Guanosine stimulates proliferation and differentiation of many types of cells in vitro and exerts neuroprotective effects in the central nervous system (CNS). Five weeks after chronic traumatic spinal cord injury (SCI), when there is no ongoing recovery of function, intraperitoneal administration of guanosine daily for 2 weeks enhanced functional improvement correlated with the increase in myelination in the injured cord. Emphasis was placed on analysis of oligodendrocytes and NG2-positive (NG2+) cells, an endogenous cell population that may be involved in oligodendrocyte replacement. There was an increase in cell proliferation (measured by bromodeoxyuridine staining) that was attributable to an intensification in progenitor cells (NG2+ cells) associated with an increase in mature oligodendrocytes (determined by Rip+ staining). The numbers of astroglia increased at all test times after administration of guanosine whereas microglia only increased in the later stages (14 days). Injected guanosine and its breakdown product guanine accumulated in the spinal cords; there was more guanine than guanosine detected. We conclude that functional improvement and remyelination after systemic administration of guanosine is due to the effect of guanosine/guanine on the proliferation of adult progenitor cells and their maturation into myelin-forming cells. This raises the possibility that administration of guanosine may be useful in the treatment of spinal cord injury or demyelinating diseases such as multiple sclerosis where quiescent oligodendroglial progenitors exist in demyelinated plaques.     

Local Delivery of High-Dose Chondroitinase ABC in the Sub-Acute Stage Promotes Axonal Outgrowth and Functional Recovery after Complete Spinal Cord Transection

Chondroitin sulfate proteoglycans (CSPGs) are glial scar-associated molecules considered axonal regeneration inhibitors and can be digested by chondroitinase ABC (ChABC) to promote axonal regeneration after spinal cord injury (SCI). We previously demonstrated that intrathecal delivery of low-dose ChABC (1 U) in the acute stage of SCI promoted axonal regrowth and functional recovery. In this study, high-dose ChABC (50 U) introduced via intrathecal delivery induced subarachnoid hemorrhage and death within 48 h. However, most SCI patients are treated in the sub-acute or chronic stages, when the dense glial scar has formed and is minimally digested by intrathecal delivery of ChABC at the injury site. The present study investigated whether intraparenchymal delivery of ChABC in the sub-acute stage of complete spinal cord transection would promote axonal outgrowth and improve functional recovery. We observed no functional recovery following the low-dose ChABC (1 U or 5 U) treatments. Furthermore, animals treated with high-dose ChABC (50 U or 100 U) showed decreased CSPGs levels. The extent and area of the lesion were also dramatically decreased after ChABC treatment. The outgrowth of the regenerating axons was significantly increased, and some partially crossed the lesion site in the ChABC-treated groups. In addition, retrograde Fluoro-Gold (FG) labeling showed that the outgrowing axons could cross the lesion site and reach several brain stem nuclei involved in sensory and motor functions. The Basso, Beattie and Bresnahan (BBB) open field locomotor scores revealed that the ChABC treatment significantly improved functional recovery compared to the control group at eight weeks after treatment. Our study demonstrates that high-dose ChABC treatment in the sub-acute stage of SCI effectively improves glial scar digestion by reducing the lesion size and increasing axonal regrowth to the related functional nuclei, which promotes locomotor recovery. Thus, our results will aid in the treatment of spinal cord injury.     

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.     

Lithium enhances the neuronal differentiation of neural progenitor cells in vitro and after transplantation into the avulsed ventral horn of adult rats through the secretion of brain-derived neurotrophic factor

This study was undertaken to elucidate the molecular mechanisms by which lithium regulates the development of spinal cord-derived neural progenitor cells (NPCs) in vitro and after transplanted in vivo . Our results show that lithium at the therapeutic concentration significantly increases the proliferation and neuronal differentiation of NPCs in vitro. Specific ELISAs, western blotting, and quantitative real-time RT-PCR assays demonstrate that lithium treatment significantly elevates the expression and production of brain-derived neurotrophic factor (BDNF) by NPCs in culture. Application of a BDNF neutralizing antibody in culture leads to a marked reduction in the neurogenesis of lithium-treated NPCs to the control level. However, it shows no effects on the proliferation of lithium-treated NPCs. These findings suggest that the BDNF pathway is possibly involved in the supportive role of lithium in inducing NPC neurogenesis but not proliferation. This study also provides evidence that lithium is able to elevate the neuronal generation and BDNF production of NPCs after transplantation into the adult rat ventral horn with motoneuron degeneration because of spinal root avulsion, which highlights the therapeutic potential of lithium in cell replacement strategies for spinal cord injury because of its ability to promote neuronal differentiation and BDNF production of grafted NPCs in the injured spinal cord.     

Antisense vimentin cDNA combined with chondroitinase ABC reduces glial scar and cystic cavity formation following spinal cord injury in rats

The formation of glial scar and cystic cavities restricts axon regeneration after spinal cord injury. Chondroitin sulphate proteoglycans (CSPGs) are regarded as the prominent inhibitory molecules in the glial scar, and their inhibitory effects may be abolished in part by chondroitinase ABC (ChABC), which can digest CSPGs. CSPGs are secreted mostly by reactive astrocytes, which form dense scar tissues. The intermediate filament protein vimentin underpins the cytoskeleton of reactive astrocytes. Previously we have shown that retroviruses carrying full-length antisense vimentin cDNA reduce reactive gliosis. Here we administered both antisense vimentin cDNA and ChABC to hemisected rat spinal cords. Using RT-PCR, Western blotting and immunohistochemistry, we found that the combined treatment reduced the formation of glial scar and cystic cavities through degrading CSPGs molecules and inhibiting intermediate filament proteins. The modified intra- and extra-cellular architecture may alter the physical and biochemical characteristics of the scar, and the combined therapy might be used to inhibit glial scar formation.     

Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury

Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of OX42 + microglial cells and glial fibrillary acidic protein + astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages.     

Transplantation of D15A-Expressing Glial-Restricted-Precursor-Derived Astrocytes Improves Anatomical and Locomotor Recovery after Spinal Cord Injury

The transplantation of neural stem/progenitor cells is a promising therapeutic strategy for spinal cord injury (SCI). In this study, we tested whether combination of neurotrophic factors and transplantation of glial-restricted precursor (GRPs)-derived astrocytes (GDAs) could decrease the injury and promote functional recovery after SCI. We developed a protocol to quickly produce a sufficiently large, homogenous population of young astrocytes from GRPs, the earliest arising progenitor cell population restricted to the generation of glia. GDAs expressed the axonal regeneration promoting substrates, laminin and fibronectin, but not the inhibitory chondroitin sulfate proteoglycans (CSPGs). Importantly, GDAs or its conditioned medium promoted the neurite outgrowth of dorsal root ganglion neurons in vitro. GDAs were infected with retroviruses expressing EGFP or multi-neurotrophin D15A and transplanted into the contused adult thoracic spinal cord at 8 days post-injury. Eight weeks after transplantation, the grafted GDAs survived and integrated into the injured spinal cord. Grafted GDAs expressed GFAP, suggesting they remained astrocyte lineage in the injured spinal cord. But it did not express CSPG. Robust axonal regeneration along the grafted GDAs was observed. Furthermore, transplantation of D15A-GDAs significantly increased the spared white matter and decreased the injury size compared to other control groups. More importantly, transplantation of D15A-GDAs significantly improved the locomotion function recovery shown by BBB locomotion scores and Tredscan footprint analyses. However, this combinatorial strategy did not enhance the aberrant synaptic connectivity of pain afferents, nor did it exacerbate posttraumatic neuropathic pain. These results demonstrate that transplantation of D15A-expressing GDAs promotes anatomical and locomotion recovery after SCI, suggesting it may be an effective therapeutic approach for SCI.     

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.     

Human embryonic stem cell-derived neural precursor transplants in collagen scaffolds promote recovery in injured rat spinal cord

Background aimsSeveral studies have reported functional improvement after transplantation of in vivo-derived neural progenitor cells (NPC) into injured spinal cord. However, the potential of human embryonic stem cell-derived NPC (hESC-NPC) as a tool for cell replacement of spinal cord injury (SCI) should be considered.MethodsWe report on the generation of NPC as neural-like tubes in adherent and feeder-free hESC using a defined media supplemented with growth factors, and their transplantation in collagen scaffolds in adult rats subjected to midline lateral hemisection SCI.ResultshESC-NPC were highly expressed molecular features of NPC such as Nestin, Sox1 and Pax6. Furthermore, these cells exhibited the multipotential characteristic of differentiating into neurons and glials in vitro. Implantation of xenografted hESC-NPC into the spinal cord with collagen scaffold improved the recovery of hindlimb locomotor function and sensory responses in an adult rat model of SCI. Analysis of transplanted cells showed migration toward the spinal cord and both neural and glial differentiation in vivo.ConclusionsThese findings show that transplantation of hESC-NPC in collagen scaffolds into an injured spinal cord may provide a new approach to SCI.     

Oligodendrocytes and radial glia derived from adult rat spinal cord progenitors: morphological and immunocytochemical characterization.

Self-renewing, multipotent neural progenitor cells (NPCs) reside in the adult mammalian spinal cord ependymal region. The current study characterized, in vitro, the native differentiation potential of spinal cord NPCs isolated from adult enhanced green fluorescence protein rats. Neurospheres were differentiated, immunocytochemistry (ICC) was performed, and the positive cells were counted as a percentage of Hoescht+ nuclei in 10 random fields. Oligodendrocytes constituted most of the NPC progeny (58.0% of differentiated cells; 23.4% in undifferentiated spheres). ICC and electron microscopy (EM) showed intense myelin production by neurospheres and progeny. The number of differentiated astrocytes was 18.0%, but only 2.8% in undifferentiated spheres. The number of differentiated neurons was 7.4%, but only 0.85% in undifferentiated spheres. The number of differentiated radial glia (RG) was 73.0% and in undifferentiated spheres 80.9%. EM showed an in vitro phagocytic capability of NPCs. The number of undifferentiated NPCs was 32.8% under differentiation conditions and 78.9% in undifferentiated spheres. Compared with ependymal region spheres, the spheres derived from the peripheral white matter of the spinal cord produced glial-restricted precursors. These findings indicate that adult rat spinal cord ependymal NPCs differentiate preferentially into oligodendrocytes and RG, which may support axonal regeneration in future trials of transplant therapy for spinal cord injury.     

Central Canal Ependymal Cells Proliferate Extensively in Response to Traumatic Spinal Cord Injury but Not Demyelinating Lesions

The adult mammalian spinal cord has limited regenerative capacity in settings such as spinal cord injury (SCI) and multiple sclerosis (MS). Recent studies have revealed that ependymal cells lining the central canal possess latent neural stem cell potential, undergoing proliferation and multi-lineage differentiation following experimental SCI. To determine whether reactive ependymal cells are a realistic endogenous cell population to target in order to promote spinal cord repair, we assessed the spatiotemporal dynamics of ependymal cell proliferation for up to 35 days in three models of spinal pathologies: contusion SCI using the Infinite Horizon impactor, focal demyelination by intraspinal injection of lysophosphatidylcholine (LPC), and autoimmune-mediated multi-focal demyelination using the active experimental autoimmune encephalomyelitis (EAE) model of MS. Contusion SCI at the T9–10 thoracic level stimulated a robust, long-lasting and long-distance wave of ependymal proliferation that peaked at 3 days in the lesion segment, 14 days in the rostral segment, and was still detectable at the cervical level, where it peaked at 21 days. This proliferative wave was suppressed distal to the contusion. Unlike SCI, neither chemical- nor autoimmune-mediated demyelination triggered ependymal cell proliferation at any time point, despite the occurrence of demyelination (LPC and EAE), remyelination (LPC) and significant locomotor defects (EAE). Thus, traumatic SCI induces widespread and enduring activation of reactive ependymal cells, identifying them as a robust cell population to target for therapeutic manipulation after contusion; conversely, neither demyelination, remyelination nor autoimmunity appears sufficient to trigger proliferation of quiescent ependymal cells in models of MS-like demyelinating diseases.     

Chondroitinase ABC promotes axonal re-growth and behavior recovery in spinal cord injury

In spinal cord injury, the injury could trigger some inhibitory signal cascades to promote chondroitin sulfate proteoglycans (CSPGs), the structures of scar tissues, formation. CSPGs could limit axonal regeneration mainly through the glycosaminoglycan (GAG) chain in the lesion site were suggested. We hypothesized that the digestion of CSPGs by chondroitinase ABC (ChABC) might decrease the inhibitory effects of limiting axonal re-growth after spinal cord injury. We compared the digesting products of CSPGs such as 2B6 by ChABC with the untreated control group and found no immunostaining of 2B6 in control group. The smaller size scars of ChABC-treatment were observed via CS-56, a type of CSPGs, 8 weeks after transection by immunohistochemistry. The inhibitory effects of CSPGs withdraw GAGs following ChABC-treatment would reduce, and immunopositive GAP-43 newly outgrown fibers were identified. In the animal trials, ChABC-treatment could improve motor function through BBB locomotor's test and reduce limiting ability of scar tissues to promote axonal regeneration via changing the structure of CSPGs by immunohistochemistry with GAP-43.     

IT Delivery of ChABC Modulates NG2 and Promotes GAP-43 Axonal Regrowth After Spinal Cord Injury

Chondroitin sulphate proteoglycans (CSPGs) with the major component NG2 have an inhibitory effect on regeneration of damaged axons after spinal cord injury. In this study, we investigate whether the digestion of CSPGs by chondroitinase ABC (ChABC) may decrease the NG2 expression and promote axon regrowth through the lesion site. Rats underwent spinal cord compression injury and were treated with ChABC or vehicle through an intrathecal catheter delivery at 2, 3, and 4 days after injury. In addition, animals were behaviorally scored using BBB test in weekly intervals after SCI. Based on immunocytochemical analyses, we have quantified distribution of NG2 glycoprotein and GAP-43 in spinal cord tissue in both experimental groups. Multiple injections of ChABC caused decrease of NG2 expression at lesion site at 5 and 7 days, but not at 14 and 28 days in comparison with vehicle-treated rats and significantly enhanced GAP-43 expression during the entire survival. The densitometry analysis showed significantly higher GAP-43 immunoreactivity (1.8–2.2-fold) in the regrowing axons and cell bodies within the central lesion cavity when compared with vehicle group. Longitudinally oriented and disorganized GAP-43-labeled axons were able to infiltrate and penetrate damaged tissue. The outgrowth of GAP-43 axons after CHABC delivery was significantly longer (≤0.457 mm) when compared with the length of axons in vehicle-treated rats (≤0.046 mm). Present findings suggest that degradation of NG2 with acute IT ChABC treatment may promote ongoing (long-lasting) axonal regenerative processes at late survival (14 and 28 days), but with no significant impact on the improvement of motor function.     

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually α-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment with ChABC was successful in returning neuronal glycosylation to normal conditions at all timepoints for MAA, PNA and SNA-I staining, and by day 8 in the case of WFA. This study demonstrated neuronal cell surface glycosylation changes in an inhibitory environment and indicated a return to normal glycosylation after treatment with ChABC, which may be promising for identifying potential therapies for neuronal regeneration strategies.     

Chondroitin sulfate glycosaminoglycans control proliferation, radial glia cell differentiation and neurogenesis in neural stem/progenitor cells

Although the local environment is known to regulate neural stem cell (NSC) maintenance in the central nervous system, little is known about the molecular identity of the signals involved. Chondroitin sulfate proteoglycans (CSPGs) are enriched in the growth environment of NSCs both during development and in the adult NSC niche. In order to gather insight into potential biological roles of CSPGs for NSCs, the enzyme chondroitinase ABC (ChABC) was used to selectively degrade the CSPG glycosaminoglycans. When NSCs from mouse E13 telencephalon were cultivated as neurospheres, treatment with ChABC resulted in diminished cell proliferation and impaired neuronal differentiation, with a converse increase in astrocytes. The intrauterine injection of ChABC into the telencephalic ventricle at midneurogenesis caused a reduction in cell proliferation in the ventricular zone and a diminution of self-renewing radial glia, as revealed by the neurosphere-formation assay, and a reduction in neurogenesis. These observations suggest that CSPGs regulate neural stem/progenitor cell proliferation and intervene in fate decisions between the neuronal and glial lineage.     

硫酸软骨素酶ABC治疗减少大鼠脊髓损伤引起的酪氨酸蛋白激酶A4的表达

本研究旨在探讨大鼠脊髓损伤(spinal cord impairment,SCI)后硫酸软骨素酶ABC (chondroitinase ABC,ChABC)对酪氨酸蛋白激酶A4 (ephrin A4,EphA4)表达变化的影响.选取成年雌性SD大鼠,随机分为假手术组、生理盐水(NS)组和ChABC组.NS组和ChABC...     

Mixed primary culture and clonal analysis provide evidence that NG2 proteoglycan-expressing cells after spinal cord injury are glial progenitors

NG2(+) cells in the adult rat spinal cord proliferate after spinal cord injury (SCI) and are postulated to differentiate into mature glia to replace some of those lost to injury. To further study these putative endogenous precursors, tissue at 3 days after SCI or from uninjured adults was dissociated, myelin partially removed and replicate cultures grown in serum-containing or serum-free medium with or without growth factors for up to 7 days in vitro (DIV). Cell yield after SCI was 5-6 times higher than from the normal adult. Most cells were OX42(+) microglia/macrophages but there were also more than twice the normal number of NG2(+) cells. Most of these coexpressed A2B5 or nestin, as would be expected for glial progenitors. Few cells initially expressed mature astrocyte (GFAP) or oligodendrocyte (CC1) markers, but more did at 7 DIV, suggesting differentiation of glial precursors in vitro. To test the hypothesis that NG2(+) cells after SCI express progenitor-like properties, we prepared free-floating sphere and single cell cultures from purified suspension of NG2(+) cells from injured spinal cord. We found that sphere cultures could be passaged in free-floating subcultures, and upon attachment the spheres clonally derived from an acutely purified single cell differentiated into oligodendrocytes and rarely astrocytes. Taken together, these data support the hypothesis that SCI stimulates proliferation of NG2(+) cells that are glial progenitor cells. Better understanding the intrinsic properties of the NG2(+) cells stimulated by SCI may permit future therapeutic manipulations to improve recovery after SCI.     

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.