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Within the two neurogenic niches of the adult mammalian brain, i.e., the subventricular zone lining the lateral ventricle and the subgranular zone of the hippocampus, there exist distinct populations of proliferating neural precursor cells that differentiate to generate new neurons. Numerous studies have suggested that epigenetic regulation by histone-modifying proteins is important in guiding precursor differentiation during development; however, the role of these proteins in regulating neural precursor activity in the adult neurogenic niches remains poorly understood. Here we examine the role of an NAD(+) -dependent histone deacetylase, SIRT1, in modulating the neurogenic potential of neural precursors in the neurogenic niches of the adult mouse brain. We show that SIRT1 is expressed by proliferating adult subventricular zone and hippocampal neural precursors, although its transcript and protein levels are dramatically reduced during neural precursor differentiation. Utilizing a lentiviral-mediated delivery strategy, we demonstrate that abrogation of SIRT1 signaling by RNAi does not affect neural precursor numbers or their proliferation. However, SIRT1 knock down results in a significant increase in neuronal production in both the subventricular zone and the hippocampus. In contrast, enhancing SIRT1 signaling either through lentiviral-mediated SIRT1 overexpression or through use of the SIRT1 chemical activator Resveratrol prevents adult neural precursors from differentiating into neurons. Importantly, knock down of SIRT1 in hippocampal precursors in vivo, either through RNAi or through genetic ablation, promotes their neurogenic potential. These findings highlight SIRT1 signaling as a negative regulator of neuronal differentiation of adult subventricular zone and hippocampal neural precursors. © 2013 Wiley Periodicals, Inc., (Copyright © 2013 Wiley Periodicals, Inc.)

The number of cells generated by a proliferating stem or precursor cell can be influenced both by proliferation and by the degree of cell death/survival of the progeny generated. In this study, the extent to which cell survival controls progenitor number was examined by comparing the growth characteristics of neurosphere cultures derived from mice lacking genes for the death-inducing Bcl-2 homologue Hara Kiri (Hrk), apoptosis-associated protein 1 (Apaf1), or the prosurvival nuclear factor-kappaB (NFkappaB) subunits p65, p50, or c-rel. We found no evidence that Hrk or Apaf1, and by inference the mitochondrial cell death pathway, are involved in regulating the number of neurosphere-derived progeny. However, we identified the p65p50 NFkappaB dimer as being required for the normal growth and expansion of neurosphere cultures. Genetic loss of both p65 and p50 NFkappaB subunits resulted in a reduced number of progeny but an increased proportion of neurons. No effect on cell survival was observed. This suggests that the number and fate of neural progenitor cells are more strongly regulated by cell cycle control than survival.

Neurotrophic factors are important for the development and maintenance of the auditory system. They have also been shown to act as survival factors for auditory neurons in animal deafness models. Studies have demonstrated recently that these neurotrophic factors not only maintain survival of auditory neurons, but that these surviving neurons retain functionality. It remains to be determined, however, if a single administration of a neurotrophic factor is sufficient to maintain auditory neuron survival after loss of hair cells, or if sustained delivery is required. This study investigated the longevity of the survival effects of BDNF on auditory neurons in deafened guinea pigs. Briefly, the left cochleae of deafened guinea pigs were infused with BDNF for 28 days via a mini-osmotic pump, and neuronal survival was analyzed at various stages after the completion of treatment. BDNF treatment prevented the degeneration of auditory neurons that normally is seen after a loss of hair cells, supporting previous studies. Our results indicate, however, that cessation of BDNF treatment leads to an accelerated decline in auditory neuron survival as compared to that observed in deafened, untreated cochleae. These findings indicate that much work remains to be done to establish a technique for the long-term survival of auditory neurons in the deaf ear., (Copyright 2003 Wiley-Liss, Inc.)

The 75 kD low-affinity neurotrophin receptor (p75(NTR)) is expressed in developing and axotomised spinal motor neurons. There is now convincing evidence that p75(NTR) can, under some circumstances, become cytotoxic and promote neuronal cell death. We report here that a single application of antisense p75(NTR) oligodeoxynucleotides to the proximal nerve stumps of neonatal rats significantly reduces the loss of axotomised motor neurons compared to controls treated with nonsense oligodeoxynucleotides or phosphate-buffered saline. Our investigations also show that daily systemic intraperitoneal injections of antisense p75(NTR) oligodeoxynucleotides for 14 days significantly reduce the loss of axotomised motor neurons compared to controls. Furthermore, we found that systemic delivery over a similar period continues to be effective following axotomy when intraperitoneal injections were 1) administered after a delay of 24 hr, 2) limited to the first 7 days, or 3) administered every third day. In addition, p75(NTR) protein levels were reduced in spinal motor neurons following treatment with antisense p75(NTR) oligodeoxynucleotides. There were also no obvious side effects associated with antisense p75(NTR) oligodeoxynucleotide treatments as determined by behavioural observations and postnatal weight gain. Our findings indicate that antisense-based strategies could be a novel approach for the prevention of motor neuron degeneration associated with injuries or disease., (Copyright 2001 Wiley-Liss, Inc.)

Upregulated expression of the low-affinity neurotrophin receptor (p75) in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE) has recently been demonstrated. To investigate whether p75 plays a role in disease pathogenesis, we adopted a gene therapy approach, utilizing antisense oligonucleotides to downregulate p75 expression during EAE. Phosphorothioate antisense oligonucleotides (AS), nonsense oligonucleotides (NS) or phosphate buffered saline (PBS) were injected daily for 18 days after immunization of SJL/J (H-2s)-mice with myelin proteolipid protein (PLP) peptide 139-151. In the AS group, there was a statistically significant reduction in both the mean maximal disease score (1.85 in the AS, 2.94 in the NS and 2.75 in the PBS-groups, respectively, P < 0.025) and in the cumulative disease incidence ( approximately 60% in the AS group and approximately 90% in the control groups). Histological and immunohistochemical analysis showed reduced inflammation and demyelination, as well as reduced p75 expression at the blood-brain barrier (BBB) in the AS-treated mice in comparison with both control groups. There was no difference, however, in p75 expression on neural cells within the CNS between the three groups of mice. We conclude that p75 could play a proactive role in the pathogenesis of EAE and may exert its effect at the level of the BBB., (Copyright 2000 Wiley-Liss, Inc.)

Stem cells from the adult forebrain of mice were stimulated to form clones in vitro using fibroblast growth factor-2 (FGF-2). At concentrations above 10 ng/ml of FGF-2, very few clones gave rise to neurons; however, if FGF-2 was removed after 5 days, 20-30% of clones subsequently gave rise to neurons. The number of neuron-containing clones and the number of neurons per clone was significantly enhanced, if insulin-like growth factor (IGF)-1 or heparin were added subsequent to FGF-2 removal. The spontaneous production of neurons after FGF-2 removal was shown to be due to endogenous IGF-1, since antibodies to IGF-1 and an IGF-1 binding protein totally inhibited neuronal production. Similarly, these reagents also abrogated the neuron-promoting effects of heparin. Thus, it appears that endogenous IGF-1 may be a major regulator of stem cell differentiation into neurons. Furthermore, it was found that high levels of IGF-1 or insulin promoted the maturation and affected the neurotransmitter phenotype of the neurons generated., (Copyright 2000 Wiley-Liss, Inc.)

The peripheral nervous system consists of multiple neural lineages derived from the neural crest (NC). Pax-3 is expressed in the NC and when mutated in the splotch mouse (Sp) results in the loss of derivatives from this precursor cell population. We have investigated the role of Pax-3 in regulating the generation of neurons from NC-derived precursor cells in vitro. Pax-3 mRNA in NC cultures is initially expressed in all NC but is subsequently only retained in neurons, suggesting a role in their generation. To determine whether Pax-3 is involved in neuron development, we first examined the generation of sensory-like neurons in NC cultures from Sp mice. Fivefold less sensory-like neurons were generated in NC cultures from Sp homozygous mice as compared to wild-type littermates. The role of Pax-3 in sensory neuron generation was then directly examined in dorsal root ganglia cultures by down-regulating the expression of Pax-3 protein with antisense oligonucleotides. It was found that antisense oligonucleotides inhibited 80-90% of newly generated sensory neurons; however, there was no significant effect on the survival of sensory neurons or the precursor population. These results suggest that Pax-3 has a role in regulating the differentiation of peripheral neurons.

The cytokine leukemia inhibitory factor (LIF) favors the survival and growth of axons in vitro and in vivo. Fibronectin has been shown to enhance nerve regeneration when added in combination with various growth factors including LIF. The goal of this study was to evaluate the effect of LIF plus fibronectin on the regeneration of transected nerve and functional recovery of reinnervated skeletal muscle, in one experimental model of peripheral nerve repair, at two recovery times. The rat sciatic nerve was cut at mid-thigh level and a silicone cuff containing either saline (control), LIF, or LIF plus fibronectin (L+F) was used to bridge the proximal and distal nerve stumps leaving a 1 cm gap between them. Rats were then explored at 6 or 12 weeks following the initial surgery. Regenerating nerves were assessed by measuring the diameter of myelinated axons, conduction velocity, and number of myelinated fibers. Muscle reinnervation was assessed by measuring muscle mass, force of contraction, and histologically for changes in muscle fiber type (type I and type II). In this report we demonstrate that at 6 weeks there were significant increases in 1) nerve conduction velocity, 2) myelinated axon diameter, and 3) number of myelinated axons over that of control (saline-treated) animals. Both LIF groups demonstrated a shift in type II muscle fiber area compared to saline-treated controls, with the L+F group having a significant increase in muscle mass. At 12 weeks there was an improved recovery over and above that demonstrated at 6 weeks. Muscle mass was 65% and 42% greater than control for LIF and L+F, respectively. Force of contraction, conduction velocity, myelinated fiber number, and diameter were also significantly greater for both LIF- and L+F-treated rats than saline-treated rats. These results demonstrate that LIF significantly improves the regeneration of damaged peripheral nerves and the preservation of muscle viability, resulting in greatly enhanced recovery of skeletal muscle function.

The low-affinity p75 receptor for nerve growth factor (p75NGFR) has been implicated in mediating neuronal cell death in vitro. A recent in vitro study from our laboratory showed that the death of sensory neurons can be prevented by reducing the levels of p75NGFR with antisense oligonucleotides. To determine if p75NGFR also functions as a death signal in vivo, we have attempted to reduce its expression in peripheral sensory neurons by applying antisense oligonucleotides to the proximal end of the transected sciatic or median and ulnar nerves. We report here that antisense oligonucleotides, when applied to the proximal stump of a transected peripheral nerve, are retrogradely transported and effectively reduce p75NGFR protein levels in sensory neurons located in the dorsal root ganglia. Furthermore, treatment of the proximal nerve stump with antisense p75NGFR oligonucleotides significantly reduced the loss of these axotomized sensory neurons. These findings further support the view that p75NGFR is a death signaling molecule and that it signals death in axotomized neurons in the neonatal sensory nervous system.

The effects of leukemia inhibitory factor (LIF) on muscle atrophy and the reinnervation of muscle were investigated. The rat medial gastrocnemius (MG) nerve was either cut (denervation groups), crushed (reinnervation group) or left intact (normal group). Muscles were injected with LIF in phosphate buffered saline (PBS) containing pluronic gel, the contralateral control muscles were injected with the vehicle alone. The muscles from the LIF-injected denervation groups were analysed for muscle fibre area; this was found to be significantly larger than controls. The greatest change was observed in the reinnervation group, where the muscle fibre area following LIF treatment was 53% (Type 1) and 84% (Type 2) greater. In addition, nerve fibre diameters were analysed in the reinnervation-treated group and these were also significantly larger. However, LIF injected into normally innervated muscle resulted in a decrease in muscle fibre area. These results show that LIF ameliorates denervation-induced muscle atrophy and improves regeneration of muscle and nerve.

Being genetically homogeneous, clonal cell lines are potentially important for investigating many aspects of cellular differentiation. We describe here the creation of clonal cell lines by immortalization of neuronal precursor cells from the adult mouse olfactory epithelium. Unlike neurons elsewhere in the vertebrate nervous system, the olfactory sensory neuron can be replaced throughout the lifespan of the animal. However, little is known about the molecular aspects of olfactory neurogenesis. Continuous cell lines were generated by retroviral transduction of the n-myc proto-oncogene into the mitotically active basal cells of the olfactory epithelium which give rise to the sensory neuron. Twenty-one clonal cell lines were produced which could be divided into three distinct morphological classes: one with flat, epithelial-like cells only; another with round, flat, and bipolar cells; and a third with large flat and large bipolar cells. These morphological classes had different patterns of intermediate filament expression, as shown by immunocytochemistry and immunoblot analysis. All cells in all cell lines expressed the intermediate filament protein vimentin. Most bipolar cells, but not other cell types, expressed neurofilament protein and in one morphological class the bipolar cells co-expressed neurofilament and glial fibrillary acidic protein. Several cell lines expressed mRNA for OMP, a marker of mature olfactory sensory neurons, and GOLF, a guanine nucleotide binding protein involved in olfactory sensory transduction. It is concluded that these cell lines were immortalized from sensory neuron precursors late in the lineage pathway. Other cell lines appear to have been immortalized at earlier stages in the lineage pathway. These cell lines therefore provide useful tools for the investigation of neuronal differentiation and sensory transduction in the olfactory epithelium.

Neurogenesis in the adult olfactory epithelium is highly regulated in vivo. Little is known of the molecular signals which control this process, although contact with the olfactory bulb or with astrocytes has been implicated. Explants of mouse olfactory epithelium were grown in the presence or absence of several peptide growth factors. Basic fibroblast growth factor (FGF2) stimulated differentiation of sensory neurons in adult and embryonic olfactory epithelium. Other growth factors tested were ineffective. FGF2-stimulated neurons were born in vitro and expressed neurofilament, neural cell adhesion molecule, and beta-tubulin. The cells also expressed olfactory marker protein, a marker for mature olfactory sensory neurons in vivo. These bipolar neurons did not express glial fibrillary acidic protein or low-affinity nerve growth factor receptor. These results indicate that neither astrocytes nor olfactory bulb are necessary for differentiation of olfactory sensory neurons in vitro.

Interferon-gamma (IFN-gamma) can induce class I major histocompatibility complex (MHC) antigen (H-2) expression on virtually all neuroepithelial cells isolated from embryonic day 9 (E9) mice. However, a subpopulation of cells become refractory to H-2 induction (H-21-) by E10 and the percentage of H-2 noninducible cells increases during development. Cell sorting, by flow cytometry or magnetic bead immunoselection, has shown that H-21- cells give rise exclusively to neuronal cells, and by E12, the majority of the neuronal progenitors are found within this population. It has also been found that 98% of the H-21- also express the neuron-associated marker, A2B5. Cells of the glial cell lineage retain the ability to express class I antigens throughout development. From these studies, it is clear that the neuroepithelium contains cells committed to the neuronal cell lineage as early as E10 in the mouse.

Leukemia inhibitory factor (LIF) has several characteristics of a neurotrophic factor for sensory neurons. Here we have investigated whether LIF also supports the survival of axotomised sensory neurons in vivo. Newborn rat pups received a unilateral sciatic nerve transection and the injury site was treated with gelfoam soaked in phosphate buffered saline (PBS), nerve growth factor (NGF), or LIF. Neuronal nucleoli in the L5 dorsal root ganglia were counted, appropriate corrections applied, and the resultant neuronal loss expressed as a percentage of the contralateral intact side. In animals where LIF was administered neuronal loss was significantly reduced: 2 days after LIF treatment neuronal loss was 19.5% compared to 43% in PBS-treated animals; 3 days after LIF treatment neuronal loss was 20.4% compared to 40.2% in PBS-treated animals; however, 7 days after LIF treatment there was no significant reduction in the number of neurons lost. The degree of rescue of sensory neurons in vivo by LIF was found to be similar to NGF, which was not surprising as both factors supported the survival of a similar population of sensory neurons in vitro. Rescue was not observed when LIF-containing gelfoam was placed away from the axotomised nerve, suggesting that LIF's action may be associated with its retrograde transport or direct signalling at the site of nerve injury.

The influence of accessory cells on the generation of neurons and neuronal survival has been studied in vitro using an immortalised, cloned cell line, Ast-1, which has many of the functional and phenotypic characteristics of cells of the astrocytic lineage. It was found that monolayers of Ast-1 cells were equivalent to monolayers of primary astrocytes in their ability to promote the generation of neurofilament positive neurons from neuroepithelial cells obtained from embryonic day 10 (E10) mice; and both were superior to NIH 3T3 cells. Ast-1 cell monolayers were also found to provide a suitable substrate for the prolonged survival (at least 3 days in vitro) of neurofilament positive neurons obtained from E17 mice, whereas neurons plated onto NIH 3T3 cells were all dead after 2 days. Medium conditioned by Ast-1 cells displayed similar biological activities to that of the monolayers: it increased the number of neurons generated from the E10 neuroepithelial cells, whether they were plated directly onto glass coverslips or onto monolayers of NIH 3T3 cells; and it increased the survival of E17 neurons plated directly onto glass coverslips. In addition, the Ast-1 conditioned medium was shown to promote the survival of the neuroepithelial cells. These results confirm that one of the mechanisms by which astrocytes or their precursors may regulate neuronal development is by secreting soluble growth factors, as has been previously documented in the case of fibroblast growth factor (FGF) (Hatten et al., 1988; Drago et al., 1991a). However, it appears in this system that FGF is not responsible for the demonstrated biological activities, and the Ast-1 action appears to be mediated by putatively novel factor(s).

The expression of clusterin has been shown to be elevated in several models of experimentally induced programmed cell death and in association with a number of neurodegenerative conditions. In order to test whether this protein is expressed in neurons during development, the expression of clusterin was examined in the developing nervous system, using immunohistochemistry and mRNA analysis. Clusterin expression was observed in the earliest neurons of the cortical plate on embryonic day (E) 12. Thereafter, the intensity of clusterin staining continued to increase in an age-dependent manner, with the greatest intensity of staining being found in the postnatal mature brain. Virtually all neurons were clusterin-positive and there was no evidence for the appearance of clusterin-positive cells specifically during epochs of programmed neuronal death in the embryo. This study suggests that clusterin has a role in neuronal maturation and it is unlikely to be associated exclusively with neuronal cell death.

Recent evidence from our laboratory has shown that leukemia inhibitory factor (LIF) can act early in peripheral nervous system development. We have investigated a potential role of LIF in the developing spinal cord. In explants and dissociated cultures of spinal cord primordium, LIF stimulated a profuse neurite outgrowth. To determine if these effects were related to neuronal differentiation, cells were plated at low cell density and stained for neurofilament. LIF stimulated an increase in the number of newly differentiated neurons, without inducing proliferation of the precursors. Given that LIF has previously reported effects as a cholinergic switching factor for sympathetic neurons, we investigated whether LIF had similar effects in these spinal cord cultures. LIF increased the number of cholinergic neurons in proportion to its overall effect on the stimulation of all neurofilament positive neurons in the culture. These data show that LIF stimulates the generation of spinal cord neurons from their precursors and further implicates a role for LIF in nervous system development.

In order to study mammalian neural crest differentiation in vitro, a series of clonal neural crest (NC) cell lines have been generated by infection of migrating mouse neural crest cells with two recombinant retroviruses containing either the c-myc or N-myc proto-oncogenes. Many cell lines were generated which could be subdivided into three groups based on their appearance in culture. Eleven of these cell lines representative of each of the morphological groups were characterized for the expression of six antigenic markers expressed by neural cells. In addition, mRNA was prepared from these cell lines and analyzed for the expression of a number of neural specific genes. These analyses show that the cell lines are representative of the following cell types: (1) neural crest-like cell lines that do not differentiate in 10% serum; (2) progenitor cell lines, some of which can partially differentiate in culture; and (3) mature neuronal cell lines or bipotential cell lines. Southern blot analysis of DNA from these lines indicated that they have multiple integration sites for the provirus and suggest that phenotypically different cell types have arisen from a single cell. None of the cell lines showed any proliferative or morphological response to nerve growth factor (NGF), whereas over two-thirds of the lines showed both marked proliferative and morphological responses to fibroblast growth factor (FGF). These data indicate that we have generated a range of cell lines representative of a spectrum of mouse neural crest derivatives.

We have developed an in vitro culture system to study the regulation of proliferation and differentiation of neural precursor cells contained within the neuroepithelium of embryonic day 10 mice. A number of soluble growth factors have been tested for their ability to regulate these early events and, of these factors, we have found that the fibroblast growth factors [FGFs] can directly stimulate the proliferation and survival of the neuroepithelial cells. At least 50% of the neuroepithelial cells divide in the presence of FGF whereas in the absence of FGF all of the cells die within 6 days of culture. At higher concentrations of FGF, the cells change from being nonadherent round cells in tight clusters into a more flattened cell type which adheres to the substratum. This morphological change is accompanied by the expression of both neurofilament and GFAP, which are definitive markers of the two major cell types in the central nervous system: neurons and glia. In addition a neuroepithelial cell line, which does not rely on FGF for survival or proliferation, expresses both of these markers in response to FGF. These results indicate that FGF is stimulating the differentiation of the neuroepithelial cells into mature neurons and glia.

Thy-1 is a glycoprotein primarily expressed in the nervous system of a large number of vertebrate species. A concanavalin-A binding glycoprotein with a molecular weight of 25,000 daltons has been isolated from cat brain, which corresponds to the Thy-1 molecule isolated in other species. Monoclonal antibodies were generated against this cat Thy-1 molecule, and immunohistochemical staining on adult cat brain frozen sections demonstrated a localisation of the molecule to regions rich in synapses. In the cerebellum, the immunoperoxidase staining with the antibodies was intense in the molecular layer, the fibres of the granular layer, and the Purkinje cell bodies. This distribution of Thy-1 is consistent with that seen with the rodent and chicken Thy-1 molecule. In the retina, one antibody (WEHY-MS-1) detected the cat Thy-1 molecule in the ganglion cell layer and on fibres of the inner plexiform and inner nuclear layers. Another antibody (WEHY-MS-2) produced a similar retinal staining pattern except that the ganglion cell layer was devoid of Thy-1. Although the Thy-1 molecule in the rodent and chicken retina have been reported in the ganglion cell and inner plexiform layers, there have been no consistent reports of Thy-1 in the inner nuclear layer. This suggests that this putative cat Thy-1 molecule may have a retinal distribution pattern different from that seen in other species.

The major function of the oligodendrocyte is to myelinate axons in the central nervous system (CNS). Two of the components of myelin, galactocerebroside (galc) and myelin basic protein (MBP), have been used as markers of oligodendrocyte maturation in the developing CNS, and it has been found that galc+ cells arise initially, which then mature into MBP+ oligodendrocytes several days later. We have been interested in the control of expression of MBP and have followed its appearance in cultures of brain cells isolated from 4 day-old mice. In low serum (0.5% foetal bovine serum), approximately 330 MBP+ cells arise per 2 x 10(5) brain cells after 3 days incubation. We have examined the ability of several growth factors to influence the expression of MBP in these cultures, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and the fibroblast growth factors (acidic and basic FGF). EGF was found to suppress strongly the developmental expression of MBP in these cultures, but the suppression was reversible, since the number of MBP+ cells approached control numbers 3 days after removal of EGF from the cultures. It was also found that MBP could be down-regulated in mature MBP+ oligodendrocytes. The action of EGF in these cultures could be mimicked by transforming growth factor-alpha (TGF alpha). The effects of EGF appear to be associated primarily with MBP production in oligodendrocytes since expression of galc is unaffected by EGF.

The expression of Thy-1 on developing sensory neurons cultured in vitro is strictly regulated and follows a temporal course identical to that observed in situ. We have investigated the role of trans-acting factors in this innate regulation by constructing heterokaryons between mature Thy-1.1+ expressing neurons and immature Thy-1.2- neurons. It has been observed by immunofluorescence that within 16 hr of fusion, Thy-1.1 expression is suppressed in such heterokaryons. However, this suppression is reversible, and after 3-4 days in vitro, at the time at which Thy-1.2 is normally expression, there is re-expression of the Thy-1.1 molecule producing Thy-1.1+/Thy-1.2+ co-expressing heterokaryons. As nuclear fusion does not occur, it appears that a developmentally regulated diffusible suppressor molecule is responsible for these observations.

To study the role of c-myc and N-myc in the immortalization of neural precursors, we infected neuroepithelial cells isolated from 10-day-old mouse embryos with a new retrovirus vector, pzen, harboring either the c-myc or the N-myc oncogene. The immortalized cell lines contain high levels of the virally expressed myc protein. The amount of myc proteins correlated with the capacity of the cells to differentiate spontaneously in vitro into neurons and glia; cell lines expressing high levels of myc protein can differentiate spontaneously while cell lines expressing low levels of the myc protein resemble epithelial cells. Addition of acidic or basic fibroblast growth factor enhanced differentiation of most cell lines. Some of the cell lines produced neurotrophic growth factors capable of supporting the growth of other cell lines at low density. There was no significant difference between cell lines immortalized with c-myc or with N-myc. Most of the immortalized cells lines generated from bipotential precursors are capable of differentiating into neurons and glia.