Horiuchi Makoto, Itoh Takayuki, Pleasure David E, Tomooka Yasuhiro
Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda City, Chiba 278-8510, Japan.
Brain Res. 2005 Dec 20;1066(1-2):24-36. doi: 10.1016/j.brainres.2005.09.061. Epub 2005 Dec 5.
We previously established cell lines from brains of p53-deficient embryos, and have now characterized one of these lines, FBD-103a, in detail. Recloning FBD-103a yielded 3 types of subclones: 5 generated the neuronal lineage (Type 1), 3 generated the glial lineage (Type 2), and 10 gave rise to both lineages as the parental line (Type 3), indicating that FBD-103a is a multipotent neural progenitor cell line indistinguishable from true neural stem cells. RT-PCR analyses of transcription factor expression indicated that the transition of multipotent Type 3 clones to either neuronally or glially differentiated progeny was marked by down-regulation of Ascl1/Mash1 and Olig1 and up-regulation of Nrsf/Rest. As expected for neural stem cells, FBD-103a and Type 3 clones formed neurospheres when cultured on a non-adhesive substrate in a serum-free medium containing fibroblast growth factor-2 (FGF2). Interestingly, the transition between Type 3 and Type 1 neuronal- or Type 2 glial-specified cells proved to be reversible; Type 1 and Type 2 subclones could also form neurospheres, from which both neuron-generating and glia-generating progenies could be derived. Moreover, when maintained on an adherent substratum that prevented neurosphere formation, but with FGF2 and without serum, Type 2 clones could generate Type 3 multipotent cells. Thus, at least in the absence of p53, specialized cell-cell interactions within neurospheres are not essential for persistence or recapture of the capacity for self-renewal and multipotency by cells differentiating along glial pathways, a result of possible significance to the pathogenesis of malignant brain tumors.
我们之前从p53基因缺失的胚胎大脑中建立了细胞系，现在已经对其中一个细胞系FBD - 103a进行了详细的特性分析。对FBD - 103a进行再次克隆产生了3种类型的亚克隆：5个产生神经细胞谱系（1型），3个产生神经胶质细胞谱系（2型），10个产生与亲代细胞系相同的两种谱系（3型），这表明FBD - 103a是一种多能神经祖细胞系，与真正的神经干细胞难以区分。转录因子表达的逆转录聚合酶链反应（RT - PCR）分析表明，多能3型克隆向神经或神经胶质分化后代的转变以Ascl1/Mash1和Olig1的下调以及Nrsf/Rest的上调为标志。正如神经干细胞所预期的那样，当在含有成纤维细胞生长因子2（FGF2）的无血清培养基中于非黏附性底物上培养时，FBD - 103a和3型克隆形成了神经球。有趣的是，3型与1型神经或2型神经胶质指定细胞之间的转变被证明是可逆的；1型和2型亚克隆也能形成神经球，从中可以产生产生神经细胞和产生神经胶质细胞的后代。此外，当维持在阻止神经球形成的黏附性基质上，但添加FGF2且无血清时，2型克隆可以产生3型多能细胞。因此，至少在没有p53的情况下，神经球内特定的细胞 - 细胞相互作用对于沿着神经胶质途径分化的细胞维持或重新获得自我更新和多能性的能力并非必不可少，这一结果可能对恶性脑肿瘤的发病机制具有重要意义。
