Villa A, Snyder E Y, Vescovi A, Martínez-Serrano A
Department of Molecular Biology, Center of Molecular Biology Severo Ochoa, Autonomous University of Madrid-CSIC, Campus Cantoblanco, Madrid, 28049, Spain.
Exp Neurol. 2000 Jan;161(1):67-84. doi: 10.1006/exnr.1999.7237.
The ready availability of unlimited quantities of neural stem cells derived from the human brain holds great interest for basic and applied neuroscience, including therapeutic cell replacement and gene transfer following transplantation. We report here the combination of epigenetic and genetic procedures for perpetuating human neural stem cell lines. Thus we tested various culture conditions and genes for those that optimally allow for the continuous, rapid expansion and passaging of human neural stem cells. Among them, v-myc (the p110 gag-myc fusion protein derived from the avian retroviral genome) seems to be the most effective gene; we have also identified a strict requirement for the presence of mitogens (FGF-2 and EGF) in the growth medium, in effect constituting a conditional perpetuality or immortalization. A monoclonal, nestin-positive, human neural stem cell line (HNSC.100) perpetuated in this way divides every 40 h and stops dividing upon mitogen removal, undergoing spontaneous morphological differentiation and upregulating markers of the three fundamental lineages in the CNS (neurons, astrocytes, and oligodendrocytes). HNSC.100 cells therefore retain basic features of epigenetically expanded human neural stem cells. Clonal analysis confirmed the stability, multipotency, and self-renewability of the cell line. Finally, HNSC.100 can be transfected and transduced using a variety of procedures and genes encoding proteins for marking purposes and of therapeutic interest (e.g., human tyrosine hydroxylase I).
从人脑中获取大量神经干细胞的可行性，对基础神经科学和应用神经科学具有重大意义，包括治疗性细胞替代和移植后的基因转移。我们在此报告了用于维持人类神经干细胞系的表观遗传和基因程序的组合。因此，我们测试了各种培养条件和基因，以找到最适合人类神经干细胞持续、快速扩增和传代的条件。其中，v-myc（源自禽逆转录病毒基因组的p110 gag-myc融合蛋白）似乎是最有效的基因；我们还确定了生长培养基中必须存在促有丝分裂原（FGF-2和EGF），实际上构成了一种条件性永存或永生化。以这种方式维持的单克隆、巢蛋白阳性人类神经干细胞系（HNSC.100）每40小时分裂一次，去除促有丝分裂原后停止分裂，经历自发形态分化并上调中枢神经系统中三个基本谱系（神经元、星形胶质细胞和少突胶质细胞）的标志物。因此，HNSC.100细胞保留了表观遗传扩增的人类神经干细胞的基本特征。克隆分析证实了该细胞系的稳定性、多能性和自我更新能力。最后，HNSC.100可以使用各种程序和编码用于标记目的和具有治疗意义的蛋白质的基因（例如人酪氨酸羟化酶I）进行转染和转导。
