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（NSC）的定义是其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，神经元和神经胶质细胞的产生受到时间调节。在早期胚胎发育中，NSC受到限制并主要分化为神经元。从妊娠早期到晚期的过渡，NSC变得有能力向神经胶质分化。这表明存在一个表观遗传开关，触发神经胶质细胞的产生。转换的时间可以在体外重现;使用原代或胚胎干细胞（ESC）衍生的NSC。在人类胚胎干细胞中，向高效神经胶质细胞生产的转变发生在分化后2-3个月，这是一个漫长的时间框架，代表了神经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，我们建议确定的机制参与开关从神经源性神经干细胞胶质细胞。为了实现这一目标，我们将建立一个胶质细胞特异性报告人ESC系，以胶质细胞酸性蛋白（GFAP）和水通道蛋白4（AQP 4）为靶点，以鉴定星形胶质细胞和胶质细胞活性神经干细胞。我们的初步结果表明，包括转录因子以及microRNA调节因子在内的几种因子在早期与晚期NSC中受到差异调节。最初，我们将在功能上测试我们的候选蛋白在早期激活胶质细胞生成程序的能力。 神经干细胞报告细胞系将作为大规模shRNA筛选的读数，旨在鉴定介导NSC中表观遗传开关的新候选物。此外，我们发现表皮生长因子受体（EGFR）在神经干细胞中表达，与神经胶质能力相关。我们将利用EGFR来区分胶质源性NSC（EGFR+ GFAP+）和胶质细胞（EGRF-GFAP+）。总的来说，拟议的研究旨在对CNS命运选择产生新的见解，并进一步了解神经胶质细胞生物学，最终确定因素 这可能会加速它们从人类多能细胞的分化。

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.