We have previously reported the isolation of an EGF-responsive precursor from the embryonic and adult mouse striatum. This precursor exhibits self renewal and the ability to produce a sphere of undifferentiated cells which can be induced to differentiate into neurons and glia. RT-PCR analysis of these spheres of undifferentiated cells revealed the expression of mRNA for the trkB neurotrophin receptor, both with and without the catalytic domain, and little or no expression of trkA or trkC. We examined the actions of BDNF on the fate of EGF-generated neural precursors. Ten days after a one-time exposure to BDNF, single EGF-generated spheres showed a twofold increase in neuron number and a marked enhancement in neurite outgrowth. Examination of neuronal nuclei with immunochemical probes for c-fos and bromodeoxyuridine revealed that the actions of BDNF were directly upon neuronal cells and did not involve division of neuronal precursors. The twofold increase in neuronal number due to BDNF, observed after 10 d in vitro, was significantly reduced after 21 d in vitro and was not apparent at 27 d in vitro. Quantitative analyses revealed that while repeated application of BDNF did not prevent the loss of neuron number over time, it did result in a significant increase in neurite numbers. Moreover, delayed addition of BDNF mimicked the increase in neuronal numbers seen when BDNF was present throughout. These BDNF actions did not appear to involve the enhancement of a novel neuronal phenotype, with all effects being due to increase in the numbers and neurite outgrowth of neurons that colocalize GABA and substance P. These findings suggest that BDNF markedly enhances the antigenic and morphologic differentiation of EGF-generated neuronal precursors. BDNF alone does not appear to act as a survival factor for neuronal precursors nor is it sufficient for preventing their death over time.

The mitogenic actions of epidermal growth factor (EGF) were examined in low-density, dissociated cultures of embryonic day 14 mouse striatal primordia, under serum-free defined conditions. EGF induced the proliferation of single progenitor cells that began to divide between 5 and 7 d in vitro, and after 13 d in vitro had formed a cluster of undifferentiated cells that expressed nestin, an intermediate filament present in neuroepithelial stem cells. In the continued presence of EGF, cells migrated from the proliferating core and differentiated into neurons and astrocytes. The actions of EGF were mimicked by the homolog transforming growth factor alpha (TGF alpha), but not by NGF, basic fibroblast growth factor, platelet-derived growth factor, or TGF beta. In EGF-generated cultures, cells with neuronal morphology contained immunoreactivity for GABA, substance P, and methionine-enkephalin, three neurotransmitters of the adult striatum. Amplification of embryonic day 14 striatal mRNA by using reverse transcription/PCR revealed mRNAs for EGF, TGF alpha, and the EGF receptor. These findings suggest that EGF and/or TGF alpha may act on a multipotent progenitor cell in the striatum to generate both neurons and astrocytes.

Neurogenesis in the mammalian central nervous system is believed to end in the period just after birth; in the mouse striatum no new neurons are produced after the first few days after birth. In this study, cells isolated from the striatum of the adult mouse brain were induced to proliferate in vitro by epidermal growth factor. The proliferating cells initially expressed nestin, an intermediate filament found in neuroepithelial stem cells, and subsequently developed the morphology and antigenic properties of neurons and astrocytes. Newly generated cells with neuronal morphology were immunoreactive for gamma-aminobutyric acid and substance P, two neurotransmitters of the adult striatum in vivo. Thus, cells of the adult mouse striatum have the capacity to divide and differentiate into neurons and astrocytes.

Neural stem cells in the lateral ventricles of the adult mouse CNS participate in repopulation of forebrain structures in vivo and are amenable to in vitro expansion by epidermal growth factor (EGF). There have been no reports of stem cells in more caudal brain regions or in the spinal cord of adult mammals. In this study we found that although ineffective alone, EGF and basic fibroblast growth factor (bFGF) cooperated to induce the proliferation, self-renewal, and expansion of neural stem cells isolated from the adult mouse thoracic spinal cord. The proliferating stem cells, in both primary culture and secondary expanded clones, formed spheres of undifferentiated cells that were induced to differentiate into neurons, astrocytes, and oligodendrocytes. Neural stem cells, whose proliferation was dependent on EGF+bFGF, were also isolated from the lumbar/sacral segment of the spinal cord as well as the third and fourth ventricles (but not adjacent brain parenchyma). Although all of the stem cells examined were similarly multipotent and expandable, quantitative analyses demonstrated that the lateral ventricles (EGF-dependent) and lumbar/sacral spinal cord (EGF+bFGF-dependent) yielded the greatest number of these cells. Thus, the spinal cord and the entire ventricular neuroaxis of the adult mammalian CNS contain multipotent stem cells, present at variable frequency and with unique in vitro activation requirements.