Tools towards the rapid derivation of glial cells from human pluripotent cells

描述（由申请人提供）：神经干细胞（NSCs）被定义为具有自我更新和分化为神经元、星形胶质细胞和少突胶质细胞的能力。然而，在中枢神经系统发育过程中，神经元和胶质细胞的产生是暂时受到调节的。在胚胎发育早期，NSCs受到限制，主要分化为神经元。从妊娠早期到妊娠晚期，NSCs具有向胶质细胞分化的能力。这表明存在一种触发胶质细胞产生的表观遗传开关。开关的时间可以在体外重现；使用原代或胚胎干细胞（ESC）衍生的NSCs。在人类ESCs中，分化后2-3个月发生向高效胶质细胞生成的转变，这一漫长的时间框架代表了胶质细胞在疾病建模和再生医学中应用的主要实际障碍。在这里，我们建议确定从神经源性到胶质源性NSCs转换的机制。为了实现这一目标，我们将建立针对胶质纤维酸性蛋白（GFAP）和水通道蛋白4 （AQP4）的胶质特异性报告人ESC系，以鉴定星形胶质细胞和胶质活性NSCs。我们的初步结果表明，包括转录因子和microRNA调节因子在内的几个因素在早期和晚期的NSCs中受到不同的调节。首先，我们将对候选蛋白质进行功能测试，以早期激活胶质细胞生成程序的能力

DESCRIPTION (provided by applicant): Neural stem cells (NSCs) are defined by their ability to self-renew and to differentiate into neurons, astrocytes and oligodendrocytes. However, during CNS development, the generation of neurons and glia is temporally regulated. In early embryonic development, NSCs are restricted and predominantly differentiate into neurons. Transitioning from early to late gestation, NSCs become competent towards glial differentiation. This suggests the presence of an epigenetic switch triggering the onset of glial production. Timing of the switch can be recapitulated in vitro; using primary or embryonic stem cell (ESC) derived NSCs. In human ESCs the switch towards efficient glial cell production occurs at 2-3 months after differentiation, a protracted time frame that represents a major practical hurdle for the application of glial cells in disease modeling and regenerative medicine. Here we propose to identify the mechanisms involved in the switch from neurogenic to gliogenic NSCs. Towards this goal we will establish a glial specific reporter human ESC lines targeting the glial fibrillary aciic protein (GFAP) and the aquaporin 4 (AQP4) to identify astrocytes and glial competent NSCs. Our preliminary results suggest that the several factors, including transcription factors as well a microRNA regulators, are differentially regulated in early versus late NSCs. Initially we will functionally test our candidate proteins for the ability to activate the gliogenic program in early NSCs. The reporter cell lines will serve as readout for a large-scale shRNA screen, aimed at identifying novel candidates that mediate the epigenetic switch in NSCs. Additionally, we found that the epidermal growth factor receptor (EGFR) is expressed in NSCs that correlate with glial competency. We will utilize EGFR to distinguish gliogenic NSCs (EGFR+ GFAP+) from glial cells (EGRF- GFAP+). Overall, the proposed studies are designed to yield novel insights into CNS fate choice and further our understanding of glial cell biology, ultimately identifying factors that may accelerate their differentiation from human pluripotent cells.