Kitchens D L, Snyder E Y, Gottlieb D I
Department of Neurology and Neurosurgery, Washington University School of Medicine, St. Louis, Missouri 63110.
J Neurobiol. 1994 Jul;25(7):797-807. doi: 10.1002/neu.480250705.
Individual neural progenitors, derived from the external germinal layer of neonatal murine cerebellum, were previously immortalized by the retrovirus-mediated transduction of avian myc (v-myc). C17-2 is one of those clonal multipotent progenitor cell lines (Snyder et al., 1992, Cell 68: 33-51; Ryder et al., 1990, J. Neurobiol. 21:356-375). When transplanted into newborn mouse cerebellum (CB), the cells participate in normal CB development; they engraft in a cytoarchitecturally appropriate, nontumorigenic manner and differentiate into multiple CB cell types (neuronal and glial) similar to endogenous progenitors (Snyder et al., 1992, as above). They also appear to engraft and participate in the development of multiple other structures along the neural axis and at multiple other stages (Snyder et al., 1993, Soc. Neurosci. Abstr. 19). Thus conclusions regarding these immortalized progenitors may be applicable to endogenous neural progenitors in vivo. To help identify and analyze factors that promote differentiation of endogenous progenitors, we first investigated the ability to maintain C17-2 cells in a defined, serum-free medium (N2). The cells survive in vitro in N2 but undergo mitosis at a very low rate. Addition of epidermal growth factor (EGF), however, either from mouse submaxillary gland or the human recombinant protein, appreciably stimulates thymidine incorporation and cell division approximately threefold. Basic fibroblast growth factor (bFGF) is an even more potent mitogen, promoting thymidine incorporation, cell division, and a net increase in cell number equal to that in serum. Both EGF and bFGF are active at very low nanomolar concentrations, suggesting that they interact with their respective receptors rather than a homologous receptor system. The findings demonstrate that C17-2 cells can be maintained and propagated in a fully defined medium, providing the basis for analysis of other growth and differentiation factors. That EGF and particularly bFGF are mitogenic for these cells is in accord with recent observations on primary neural tissue (Reynolds and Weiss, 1992, Science 255:1707-1710; Kilpatrick and Bartlett, 1993, Neuron 10:255-265; Ray et al., 1993, Proc. Natl. Acad. Sci. USA 90:3602-3606) suggesting that bFGF and EGF responsiveness may be fundamental properties of neural progenitors.
源自新生小鼠小脑外生发层的单个神经祖细胞，先前通过禽源 myc（v-myc）的逆转录病毒介导转导而永生化。C17-2 是那些克隆多能祖细胞系之一（斯奈德等人，1992 年，《细胞》68 卷：33 - 51 页；赖德等人，1990 年，《神经生物学杂志》21 卷：356 - 375 页）。当移植到新生小鼠小脑（CB）中时，这些细胞参与正常的 CB 发育；它们以细胞结构上合适的、非致瘤的方式植入，并分化为多种 CB 细胞类型（神经元和神经胶质细胞），类似于内源性祖细胞（斯奈德等人，1992 年，同上文）。它们似乎也能植入并参与神经轴上多个其他结构以及多个其他阶段的发育（斯奈德等人，1993 年，《神经科学学会摘要》19 卷）。因此，关于这些永生化祖细胞的结论可能适用于体内的内源性神经祖细胞。为了帮助识别和分析促进内源性祖细胞分化的因素，我们首先研究了在限定的无血清培养基（N2）中维持 C17-2 细胞的能力。这些细胞在 N2 中能在体外存活，但以非常低的速率进行有丝分裂。然而，添加来自小鼠颌下腺的表皮生长因子（EGF）或重组人源蛋白，可显著刺激胸苷掺入和细胞分裂，约增加三倍。碱性成纤维细胞生长因子（bFGF）是一种更有效的促有丝分裂原，可促进胸苷掺入、细胞分裂以及细胞数量的净增加，与血清中的情况相当。EGF 和 bFGF 在极低的纳摩尔浓度下均有活性，这表明它们与各自的受体相互作用，而非与同源受体系统相互作用。这些发现表明，C17-2 细胞可以在完全限定的培养基中维持和增殖，为分析其他生长和分化因子提供了基础。EGF 尤其是 bFGF 对这些细胞具有促有丝分裂作用，这与最近对原代神经组织的观察结果一致（雷诺兹和韦斯，1992 年，《科学》255 卷：1707 - 1710 页；基尔帕特里克和巴特利特，1993 年，《神经元》10 卷：255 - 265 页；雷等人，1993 年，《美国国家科学院院刊》90 卷：3602 - 3606 页），表明对 bFGF 和 EGF 的反应性可能是神经祖细胞的基本特性。
