Sato Kenichi, Kanno Junko, Tominaga Teiji, Matsubara Yoichi, Kure Shigeo
Department of Medical Genetics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Sendai 980-8574, Japan.
Brain Res. 2006 Feb 3;1071(1):24-33. doi: 10.1016/j.brainres.2005.11.039. Epub 2006 Jan 10.
We studied the de novo and salvage pathways of DNA synthesis in sphere-forming neural stem cells obtained from mouse embryos by a neurosphere method. The former pathway needs folic acid (FA) for nucleotide biosynthesis, while the latter requires deoxyribonucleosides (dNS). We examined the proliferative activity of sphere-forming cells in E14.5 embryos by counting the number of spheres formed in media that lacked FA and/or dNS. Proliferation failure and apoptosis occurred in a deficient medium lacking of both FA and dNS. Spheres formed in the deficient medium supplemented with dNS, without FA, did not produce neuron, but rather only seem to generate astrocytes and oligodendrocytes when plated under differentiation condition in culture. On the other hand, a subpopulation of cultured cells formed spheres in the deficient medium supplemented with FA alone in an appropriate concentration, and did possess the self-renewing and multipotential characteristics of neural stem cells. Spheres formed in the media containing low dose Azathioprine and methotrexate, inhibitors of de novo DNA synthesis, were selectively prevented from producing neurons even in the presence of FA. These results suggested that activating de novo DNA synthesis was needed for neural stem cells to proliferate with multipotentiality.
我们通过神经球法研究了从小鼠胚胎获得的成球神经干细胞中DNA合成的从头合成途径和补救途径。前者途径需要叶酸（FA）用于核苷酸生物合成，而后者需要脱氧核糖核苷（dNS）。我们通过计算在缺乏FA和/或dNS的培养基中形成的球的数量，检测了E14.5胚胎中成球细胞的增殖活性。在缺乏FA和dNS的缺陷培养基中发生增殖失败和凋亡。在补充有dNS但无FA的缺陷培养基中形成的球，在培养中置于分化条件下接种时，不产生神经元，而是似乎仅产生星形胶质细胞和少突胶质细胞。另一方面，培养细胞的一个亚群在仅补充适当浓度FA的缺陷培养基中形成球，并且确实具有神经干细胞的自我更新和多能特性。在含有低剂量硫唑嘌呤和甲氨蝶呤（从头DNA合成抑制剂）的培养基中形成的球，即使在有FA存在的情况下也被选择性地阻止产生神经元。这些结果表明，神经干细胞以多能性增殖需要激活从头DNA合成。
