Fetal Spinal Cord Research Articles

Interplay between FGF2 and BMP controls the self-renewal, dormancy and differentiation of rat neural stem cells.

Mouse and human central nervous system progenitor cells can be propagated extensively ex vivo as stem cell lines. For the rat, however, in vitro expansion has proven to be problematic owing to proliferation arrest and differentiation. Here, we analyse the establishment, in adherent culture, of undifferentiated tripotent neural stem (NS) cell lines derived from rat foetal brain and spinal cord. Rat NS cells invariably undergo growth arrest and apparent differentiation after several passages; however, conditioned medium from proliferating cultures can overcome this block, enabling continuous propagation of undifferentiated rat NS cells. We found that dormancy is induced by autocrine production of bone morphogenetic proteins (BMPs). Accordingly, the BMP antagonist noggin can replace conditioned medium to sustain continuous self-renewal. Noggin can also induce dormant cells to re-enter the cell cycle, upon which they reacquire neurogenic potential. We further show that fibroblast growth factor 2 (FGF2) is required to suppress terminal astrocytic differentiation and maintain stem cell potency during dormancy. These findings highlight an extrinsic regulatory network, comprising BMPs, BMP antagonists and FGF2 signals, that governs the proliferation, dormancy and differentiation of rat NS cells and which can be manipulated to enable long-term clonogenic self-renewal.

Non‐syndromic language delay in a child with disruption in the Protocadherin11X/Y gene pair

Protocadherin11 is located on both the X and Y chromosomes in Homo sapiens but only on the X chromosome in other hominid species. The pairing of PCDH11Y with PCDH11X arose following a duplicative 3.5 Mb translocation from the ancestral X chromosome to the Y chromosome several million years ago. The genes are highly expressed in fetal brain and spinal cord. The evolutionary consequence of this duplication has been proposed to include the sexual dimorphism of cerebral asymmetry and the hominid specific transition to the capacity for language. We report a case of a male child referred for genetic investigation of severe language delay. Microarray analysis indicated the presence of a 220 Kb intragenic deletion at Xq21.31 involving the PCDH11X gene. Fluorescence in situ hybridization using a BAC probe mapping to intron 2 of the Protocadherin11X/Y gene pair confirmed loss of the locus on both the X and Y chromosomes. The X chromosome deletion was maternally inherited, but the Y chromosome deletion was found to be a de novo occurrence in this child. This finding lends support to the hypothesis that the Protocadherin11X/Y gene plays a role in language development in humans and that rare copy number variation is a possible mechanism for communication disorders.

Effect of low-concentration ozone on calcium channels in fetal rat spinal cord neurons in vitro

Objective To investigete the effect of low-concentration ozone （O3） on voltage-gated calcium channels in fetal rat spinal cord neurons in vitro. Methods The spinal cord neurons were cultured in vitro and identified by immunohistochemistry staining. Then the cells were seeded into the culture dish and randomly divided into 3 groups： control group （group C, n = 5）, O3 15 μg/ml （group O3-15, n = 4） and O3 20 μg/ml group （group O3-20, n = 8）. The cells were exposed to O3 15 and 20 μg/ml in group O3-15 and O3-20 respectively, while the cells were exposed to air in group C. After 20 min incubation, the electrophysiological activity of calcium channels in neurons was recorded using whole-cell patch-clamp technique. Results Peak calcium current density was significantly increased in group O3-15 and O3-20 compared with group C （ P 〈 0.05）. Half-activation voltage was significantly lower in group O3-15 and O3-20 than in group C （ P 〈 0.05）. Peak calcium current density was significantly higher in group O3 -20 than in group O3-15 （ P 〈 0.05） . There was no significant difference in half-inactivation voltage among the three groups （ P 〉 0.05）. Conclusion Low concentration of O3 can make the activation of the calcium ion channel easy in fetal rat spinal cord neurons in vitro. Key words: Ozone; Calcium channels; Spinal cord; Neurons

Neuronal progenitor transplantation and respiratory outcomes following upper cervical spinal cord injury in adult rats

Despite extensive gray matter loss following spinal cord injury (SCI), little attention has been given to neuronal replacement strategies and their effects on specific functional circuits in the injured spinal cord. In the present study, we assessed breathing behavior and phrenic nerve electrophysiological activity following transplantation of microdissected dorsal or ventral pieces of rat fetal spinal cord tissue (FSC D or FSC V, respectively) into acute, cervical (C2) spinal hemisections. Transneuronal tracing demonstrated connectivity between donor neurons from both sources and the host phrenic circuitry. Phrenic nerve recordings revealed differential effects of dorsally vs. ventrally derived neural progenitors on ipsilateral phrenic nerve recovery and activity. These initial results suggest that local gray matter repair can influence motoneuron function in targeted circuits following spinal cord injury and that outcomes will be dependent on the properties and phenotypic fates of the donor cells employed.

Functional Evaluation of a 3‐D Nerve Construct Utilized for Repair of a Critical Nerve Defect

Nerve grafts are often required to replace tissue damaged by disease, surgery, or extensive trauma. Limitations associated with grafting such as graft availability, donor site morbidity, and immune rejection have led investigators to develop strategies to engineer nerve tissue. The purpose of our research was to engineer a scaffold‐less 3‐D engineered nerve construct that would be functionally equivalent to native nerve. As per our labs' previously established protocol, fibroblasts isolated from F344 rat Achilles tendon and neural cells from E15 fetal spinal cords were co‐cultured and engineered into 3‐D constructs. Following 3‐D formation, neural cells oriented along the axis of strain. The functionality of a nerve is measured by its ability to conduct an electrical signal along the length of the cell axon or nerve conduction velocity. To establish an in vivo standard, conduction velocities were measured in neonatal rats ranging from 7 – 28 days. The in vivo velocities peaked at 28 days when recordings reached the expected adult conduction velocity of 16 m/s. One week after establishing a 3‐D conformation, nerve constructs were utilized in a 2 cm nerve defect repair. Following a 2 month recovery, conduction velocities and target muscle mechanical properties were evaluated. Our results suggest that our scaffold‐less 3‐D engineered nerve construct may be a viable strategy for nerve repair. Supported by R01 AR054778‐02 and gift from the Barbara & Richard Raynor Medical Foundation.

Transitional change in rat fetal cell proliferation in response to ghrelin and des-acyl ghrelin during the last stage of pregnancy

Expression of mRNA for the ghrelin receptor, GHS-R1a, was detected in various peripheral and central tissues of fetal rats, including skin, bone, heart, liver, gut, brain and spinal cord, on embryonic day (ED)15 and ED17. However, its expression in skin, bone, heart and liver, but not in gut, brain and spinal cord, became relatively weak on ED19 and disappeared after birth (ND2). Ghrelin and des-acyl ghrelin facilitated the proliferation of cultured fetal (ED17, 19), but not neonatal (ND2), skin cells. On the other hand, with regard to cells from the spinal cord and hypothalamus, the proliferative effect of ghrelin continued after birth, whereas the effect of des-acyl ghrelin on neurogenesis in these tissues was lost at the ED19 fetal and ND2 neonatal stages. Immunohistochemistry revealed that the cells in the hypothalamus induced to proliferate by ghrelin at the ND2 stage were positive for nestin and glial fibrillary acidic protein. These results suggest that in the period immediately prior to, and after birth, rat fetal cells showing proliferation in response to ghrelin and des-acyl ghrelin are at a transitional stage characterized by alteration of the expression of GHS-R1a and an undefined des-acyl ghrelin receptor, their responsiveness varying among different tissues.

Long-term survival and development of fetal ventral spinal grafts into the motoneuron-depleted rat spinal cord: Role of donor age

Fetal spinal cord (SC) tissue grafts can survive and develop into the lesioned SC, but no conclusive data are available concerning the long-term fate of transplanted material and the relation between the graft fate and the donor embryo age. Here, pre-labelled suspensions of ventral SC from E12 or E17 rat fetuses were grafted to the lumbar SC of adult rats with motoneuron depletion induced by perinatal injection of volkensin. E12 and E17 are presumably the stages when motoneuron development starts and terminates, respectively. Four or 10 months post-grafting, SCs were analyzed to check the graft survival rate and to follow the differentiation and spatial distributions of grafted cells. Neurotoxic lesion produced a 61% motoneuronal loss in the lumbar SC. In transplanted animals, all E12 fetal grafts survived until the observed time-points and developed various mature cell phenotypes. Many motoneuron-like labelled cells were found within the graft area or adjacent to it. Conversely, none of the E17 fetal grafts survived, since no graft-derived elements with neuronal morphology were found either in the site of graft placement or adjacent to it. The present findings indicate that spinal neuroblasts can survive for a long time and develop within the motoneuron-depleted SC, and that the donor embryo age is crucial for successful engraftment.

Cadmium effects on human fetal spinal cord

The biological effects of Cadmium (Cd) on the central nervous system is poorly understood; it is thought that during brain development, when the blood-brain barrier is not well established, Cd may enter the brain and exerts its toxic effects. It has also been suggested that several diseases such as Parkinson disease, amyotrophic lateral sclerosis (ALS) as well as malformations such as spina bifida and forelimb ectrodactyly may be related to Cd exposure. In this study we evaluated the role of Cd in affecting motor neurons and glial cells of the ventral horns of human fetal spinal cord. Sections of human fetal spinal cord (9-12 weeks) were exposed for 24 h to 10 and 100 μM CdCl2. Morphology was evaluated by Haematoxilyn-Eosin staining; distribution and number of motor neurons as well as of glial cells were studied by immunohistochemistry and by Western blot; apoptosis were investigated by TUNEL assay and by Western blot. The cell density in the ventral horns, after CdCl2 treatments, appeared dramatically decreased and the number of apoptotic cells became higher in comparison to control specimens as demonstrated by the high expression level of three different apoptotic markers (NGFR p75, caspase 8 and PARP). The number and the distribution of motor neurons, expressing β tubulin III and positive to choline acetyltransferase, appeared significantly decreased after the treatment with different concentrations of CdCl2. Activation and increase of glial cells were confirmed by the high expression level of Glial Fibrillary Acidic Protein (GFAP). In this study we demonstrate the role of Cd, one of the most diffuse environmental pollutant linked to industrial development, in inducing apoptosis of motor neurons in the ventral horns of human fetal spinal cord. Motor neurons death is accompanied by a surrounding intense glyosis leading to a significant overthrow of the morphological architecture of spinal cord during its development.

Characterization of a human fetal spinal cord stem cell line, NSI-566RSC, and its induction to functional motoneurons

Specific neuronal subtypes, especially motoneurons (MNs), derived from human stem cells provide a significant therapeutic potential for spinal cord diseases, such as amyotrophic lateral sclerosis (ALS) and spinal cord injury. So far, in vitro, MNs have only been successfully induced from embryonic stem cells (hESC) and human fetal cortical progenitors. Although neural progenitors from spinal cord would be a likely source for generating MNs, there has been no study reporting successful in vitro differentiation of MNs from spinal cord progenitors. This study first characterized a polyclonal spinal cord stem cell line isolated from an 8 week-old fetus. Then a paradigm was introduced to successfully induce MNs from this cell line, which was demonstrated by immunostaining using the MN markers HB9, Islet1 and choline acetyl transferase (ChAT). The combination of HB9 and ChAT immunostainings indicated that approximately 20% of the cells were MNs after this induction protocol. The presence of other cell types in the differentiated culture was also analysed. Finally, the electrophysiological properties of these differentiated MNs were characterized to confirm their functional integrity. The majority of these MNs fired repetitive action potentials (APs), which is an indicator of functional maturation. The recordings of spontaneous excitatory postsynaptic currents (EPSCs) confirmed the formation of synapses onto these MNs. This study reports the first successful differentiation of MNs from human spinal cord stem cells in vitro, providing a novel approach for obtaining functional MNs when designing the therapeutic strategy for spinal cord diseases or injuries.

Transplantation-mediated strategies to promote axonal regeneration following spinal cord injury.

Devastating central nervous system injuries and diseases continue to occur in spite of the tremendous efforts of various prevention programs. The enormity and annual escalation of healthcare costs due to them require that therapeutic strategies be responsibly developed. The dysfunctions that occur after injury and disease are primarily due to neurotransmission damage. The last two decades of both experimental and clinical research have demonstrated that neural and non-neural tissue and cell transplantation is a viable option for ameliorating dysfunctions to markedly improve quality of life. Moreover, significant progress has been made with tissue and cell transplantation in studies of pathophysiology, plasticity, sprouting, regeneration, and functional recovery. This article will review information about the ability and potential, particularly for traumatic spinal cord injury, that neural and non-neural tissue and cell transplantation has to replace lost neurons and glia, to reconstruct damaged neural circuitry, and to restore neurotransmitters, hormones, neurotrophic factors, and neurotransmission. Donor tissues and cells to be discussed include peripheral nerve, fetal spinal cord and brain, central and peripheral nervous systems' glia, stem cells, those that have been genetically engineered, and non-neural ones. Combinatorial approaches and clinical research are also reviewed.

Regeneration of spinal cord with cell and gene therapy

Transplantation of fetal spinal cord cells (FSCC) can promote regeneration of injured spinal cord, while Schwann cells (SC) and some growth factors have a similar effect. However, the synergistic effects and optimal combination of these modalities have not yet been evaluated. In the current study, the efficiency of cell therapy of FSCC and/or SC, with/without growth factors (nerve growth factor [NGF] and brain-derived neurotrophic factor [BDNF]) was examined, with the aim of establishing an optimized protocol for spinal cord injury. One hundred and twenty adult rats were randomly divided into six groups with 20 rats in each group. One week after the thoracic spinal cord injury model had been created, the rats were treated with different therapeutic modalities: Dulbecco's modified Eagles medium (DMEM) in Group I, FSCC in Group II, FSCC plus SC in Group III, FSCC plus SC over-expressing NGF in Group IV, FSCC plus SC over-expressing BDNF in Group V, and FSCC plus SC over-expressing both NGF and BDNF in Group VI. Subsequently, the rats were subjected to behavioral tests once a week after injury, while histology, immunohistochemistry and electron microscopy were performed at one and three month post-operation. Both SC and FSCC promoted regeneration of spinal cord injury when used separately, while a combination of the two types of cell resulted in better recovery than either alone. Both growth factors (NGF and BDNF) enhanced the outcomes of cell therapy, while synergistic effects meant that a combination of each individual component (group VI) achieved the best results according to locomotion scale, histology and immunoreactivity in the injured cords. SC, NGF and BDNF can enhance the outcome of FSCC therapy, while the combination of FSC with SC, NGF and BDNF is possibly the optimal protocol for clinical treatment of acute spinal cord injury.

The identification of proteoglycan, collagen and neuron in precursor cells from human fetal spinal cord

After spinal cord injury (SCI), a loss of myelinating oligodendrocytes and neurons occurs. The functional recovery of injured spinal cords is the principal objective of SCI repair. Cell transplantation may prove beneficial to help replace lost myelin and spinal cord circuitry. In this study, we demonstrated that neural precursor cells (hNPCs) from human fetal spinal cord express three types of proteoglycan proteins—chondroitin sulfate, keratan sulfate, and cartilage proteoglycan (an extracellular matrix detected in normal spinal cord), and non-proteoglycan matrix collagen. Both proteoglycan and collagen evidenced profound immunoreactivity in double-stained cell clusters. In addition, whether or not hNPCs were capable of differentiating into a variety of cells, including GABAergic and cholinergic neurons, were assessed. The differentiated cells of eight passages grown on a monolayer expressed the human nuclear protein (HNu), the progenitor marker nestin, GAD, ChAT, TJU, and MAP-2. These results indicate that hNPCs may prove to be candidate cells for therapeutic SCI strategies.

Effects of Schwann cells on proliferation and differentiation of neural stem cells from rat spinal cord

Objective To evaluate the effect of Schwann cells on in vitro proliferation and differentiation of rat spinal cord derived stem cells.Mellaods We cultured and identified neural stem cells(NSCs)from fetal rat spinal cord and Sehwann cells(SCs)from seiatie nerve of SD rats of 24 hour age.NSCs were cultured under4 eondidom according to group assignment:control group(NSCs+basic culture),group A(NSCs+BDNF+basic culture),group B(NSCs+SCs+basic culture),group C(NSCs+SCs+BDNF+basic culture).Morphological changes of NSCs were observed.The number of cells was counted and their diameters measured.By using inmaunoeytochemical method,expression of specific neuron-like cell markers were detected.The percentage of NSCs differentiated into neuron-like cells was calculated and the average length of processes was measured.Results NSCs from fetal rat spinal cord expressed Nestin and differentiated into neurons and astrocytes The quantity and average diameter of NSCs in groups A,B and C were larger than those of the control group at the 72nd hour of culture(P＜0.05).The percentage of NSCs differentiated into MAP-2 positiVe neuron-like cells and the average length of the processes were also larger in groups A,B and C than in the control group at the 3rd,7th and 14th day of culture(P＜0.05).Conclusion Plat Sehwann cells can promote proliferation of NSCs from fetal rat spinal cord and irtduee its direelional differentiation. Key words: Schwann cells; Spinal cord; Neural stem cells; Differentiation

Neural stem cell delivery to the spinal cord in an ovine model of fetal surgery for spina bifida

We introduce the notion of prenatal neural stem cell (NSC) delivery to the spinal cord as an adjuvant to fetal repair of spina bifida. Fetal lambs with experimental myelomeningocele (MMC; n = 25) were divided in 3 groups: group I, no repair; group II, standard surgical MMC coverage; and group III, MMC coverage plus delivery of a murine NSCs clone into the spinal cord defect. Donor cells constitutively expressed lacZ encoding for Escherichia coli beta-galactosidase, yet they were further labeled by exposure to either BrdU and/or to the fluorescent membrane dye PKH-26. Blinded initial clinical evaluations and multiple spinal cord analyses were undertaken soon after birth. Both survival and the incidence of major paraparesis were significantly worse in group I compared with groups II and III. In group III, NSC density was highest within the most damaged areas of the spinal cord, with selective engraftment within those regions. Donor NSCs retained an undifferentiated state in vivo, producing neurotrophic factors within the defect. No animals in group III had a worsened condition following this intervention. Neural stem cells retain an undifferentiated state and produce neurotrophic factors in the short term after delivery to the fetal spinal cord, in the setting of experimental MMC. Further scrutiny of NSC delivery to the spinal cord as a therapeutic strategy against spina bifida is warranted.

Directed Evolution of Motor Neurons from Genetically Engineered Neural Precursors

Stem cell-based therapies hold therapeutic promise for degenerative motor neuron diseases, such as amyotrophic lateral sclerosis, and for spinal cord injury. Fetal neural progenitors present less risk of tumor formation than embryonic stem cells but inefficiently differentiate into motor neurons, in line with their low expression of motor neuron-specific transcription factors and poor response to soluble external factors. To overcome this limitation, we genetically engineered fetal rat spinal cord neurospheres to express the transcription factors HB9, Nkx6.1, and Neurogenin2. Enforced expression of the three factors rendered neural precursors responsive to Sonic hedgehog and retinoic acid and directed their differentiation into cholinergic motor neurons that projected axons and formed contacts with cocultured myotubes. When transplanted in the injured adult rat spinal cord, a model of acute motor neuron degeneration, the engineered precursors transiently proliferated, colonized the ventral horn, expressed motor neuron-specific differentiation markers, and projected cholinergic axons in the ventral root. We conclude that genetic engineering can drive the differentiation of fetal neural precursors into motor neurons that efficiently engraft in the spinal cord. The strategy thus holds promise for cell replacement in motor neuron and related diseases. Disclosure of potential conflicts of interest is found at the end of this article.

Neural agrin controls maturation of the excitation-contraction coupling mechanism in human myotubes developing in vitro

The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: 1) aneurally cultured, 2) cocultured with fetal rat spinal cord explants, 3) aneurally cultured in medium conditioned by cocultures, and 4) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca(2+) imaging indicated that coculturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism. The effect of spinal cord explants was mimicked by treatment with medium conditioned by cocultures or by addition of 1 nM of recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors (RyRs) and dihydropyridine-sensitive L-type Ca(2+) channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both RyRs and L-type Ca(2+) channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.

Transplanted Neural Progenitor Cells Survive and Differentiate but Achieve Limited Functional Recovery in the Lesioned Adult Rat Spinal Cord

Endogenous repair after injury in the adult CNS is limited by a number of factors including cellular loss, inflammation, cavitation and glial scarring. Spinal cord neural progenitor cells (SCNPCs) may provide a valuable cellular source for promoting repair following spinal cord injury. SCNPCs are multipotent, can be expanded in vitro, have the capacity to differentiate into CNS cell lineages and are capable of long-term survival following transplantation. To determine the extent to which SCNPCs may contribute to spinal cord repair SCNPCs isolated from rat fetal spinal cord were expanded ex vivo and transplanted into the adult rat spinal cord after a dorsal column crush lesion. The survival and distribution of transplanted cells were examined at 24 h, 1, 2 and 6 weeks after injury. Transplanted cells were identified at all time points, located mainly at the lesion perimeter, indicating good post-transplant cell survival. Furthermore, SCNPCs maintained their ability to differentiate in vivo, with approximately 40% differentiating into cells with a glial morphology, whilst 8% displayed a neural morphology. Transplanted animals were also assessed on a number of behavioral tasks measuring sensorimotor and proprioceptive function to determine the extent to which SCNPC transplants might attenuate lesion-induced functional deficits. SCNPCs failed to promote significant functional recovery, with a small improvement observed in only one of the four tasks employed, primarily related to improvements in sensory function. Tracing of the corticospinal tract and ascending dorsal column pathway revealed no regeneration of the axons beyond the lesion site. These data indicate that, although transplanted SCNPCs show good survival in the spinal cord injury environment, combination with other treatment strategies is likely to be required for these cells to fully exert their therapeutic potential.

Angiogenin loss‐of‐function mutations in amyotrophic lateral sclerosis

Heterozygous missense mutations in the coding region of angiogenin (ANG), an angiogenic ribonuclease, have been reported in amyotrophic lateral sclerosis (ALS) patients. However, the role of ANG in motor neuron physiology and the functional consequences of these mutations are unknown. We searched for new mutations and sought to define the functional consequences of these mutations. We sequenced the coding region of ANG in an independent cohort of North American ALS patients. Identified ANG mutations were then characterized using functional assays of angiogenesis, ribonucleolysis, and nuclear translocation. We also examined expression of ANG in normal human fetal and adult spinal cords. We identified four mutations in the coding region of ANG from 298 ALS patients. Three of these mutations are present in the mature protein. Among the four mutations, P(-4)S, S28N, and P112L are novel, and K17I has been reported previously. Functional assays show that these ANG mutations result in complete loss of function. The mutant ANG proteins are unable to induce angiogenesis because of a deficiency in ribonuclease activity, nuclear translocation, or both. As a correlate, we demonstrate strong ANG expression in both endothelial cells and motor neurons of normal human spinal cords from the developing fetus and adult. We provide the first evidence that ANG mutations, identified in ALS patients, are associated with functional loss of ANG activity. Moreover, strong ANG expression, in normal human fetal and adult spinal cord neurons and endothelial cells, confirms the plausibility of ANG dysfunction being relevant to the pathogenesis of ALS.

G.P.2.19 Comparison of foetal muscle and spinal cord in SMA: Morphology, cell death and Bcl2/Bcl-X expression

G.P.2.19 Comparison of foetal muscle and spinal cord in SMA: Morphology, cell death and Bcl2/Bcl-X expression

Phenotypic characterization of neural stem cells from human fetal spinal cord: Synergistic effect of LIF and BMP4 to generate astrocytes

If cell based therapy for spinal cord injury is to become a reality, greater insights into the biology of human derived spinal cord stem cells are a prerequisite. Significant species differences and regional specification of stem cells necessitates determining the effects of growth factors on human spinal cord stem cells. Fetal spinal cords were dissociated and expanded as neurospheres in medium with bone morphogenetic protein 4 (BMP4), leukemia inhibitory factor (LIF) or BMP4 and LIF. First-generation neurospheres comprised a heterogeneous population of neural cell types and after plating emergent cells included neurons, oligodendrocytes and GFAP(+) cells which coexpressed stem cells markers and those of the neuronal lineage and were thus identified as GFAP(+) neural precursor cells (NPC). When plated, neurospheres maintained in BMP4 demonstrated a reduced proportion of emergent oligodendrocytes from 13 to 4%, whereas LIF had no statistically significant effect on cell type distribution. Combining BMP4 and LIF reduced the proportion of oligodendrocytes to 3% and that of neurons from 37 to 16% while increasing the proportion of GFAP(+) NPC from 45 to 79%. After 10 passages in control media aggregates gave rise to multiple neural phenotypes and only continued passage of neurospheres in the presence of BMP4 and LIF resulted in unipotent aggregates giving rise to only astrocytes. These results provide a means of obtaining pure populations of human spinal-cord derived astrocytes, which could be utilized for further studies of cell replacement strategies or in vitro evaluation of therapeutics.