Epigenetic Controls in hESC Dopaminergic Fate

描述(申请人提供)：干细胞，包括胚胎干细胞和体细胞干细胞，在人类疾病的组织和功能恢复方面具有巨大的潜力。然而，我们缺乏对干细胞分化为特定表型的确切过程的了解，甚至缺乏对特定疾病具有最大治疗潜力的干细胞的最佳来源的了解，这反映了我们对基本干细胞生物学知识的空白，仍然是有效临床翻译的障碍。对“茎”基因的研究表明，单靠基因表达并不足以确保或定义可塑性或谱系特征。我们假设：(A)通过表观遗传过程表征人类干细胞将为未分化的人类胚胎干细胞(HESCs)的多能性及其分化级联提供潜在的机制。 (B)由染色质重塑动力学建立的贯穿发育的表观遗传标记可用于定义人类干细胞发育连续体的效力、可塑性和谱系承诺；以及(C)此类标记可用于判断细胞的治疗潜力。这项研究的目标是研究hESC(NIH注册代码：WA01，WA07和WA09)在分化为人类神经干细胞(HNSC)然后是多巴胺(DA)表型时的表观遗传控制。在我们的实验室建立了分化多能hESCs的策略，在确定的培养条件下，向多能hNSCs分化，然后向DA神经元分化，并产生了一些也可以定向到DA表型的hNSC系，我将通过检测染色质状态的进展来表征分化过程，并确定表观遗传标志。将hESC分化过程中的表观遗传标记与CNS来源的hNSCs及其分化的DA神经元的表观遗传标记进行比较。已确定的表观遗传标志将用于定义和比较人类干细胞的可塑性和潜力。这些特征将通过使用体内生物测定(老龄大脑中DA功能障碍的小鼠模型)来测试和预测给定干细胞状态的治疗潜力来进一步确认。在追求这项提案的目标的过程中，我将在Snyder实验室得到指导，这对我未来作为一名独立的人类干细胞研究员的职业发展将具有重要意义。

DESCRIPTION (provided by applicant): Stem cells, both embryonic and somatic, hold great potential for tissue and function restoration in human diseases. However, our lack of understanding regarding the precise process by which stem cells differentiate towards a particular phenotype or even the optimal source of stem cells with the greatest therapeutic potential for a particular disease reflects gaps in our knowledge of the fundamental stem cell biology and remains an obstacle to effective clinical translation. The search for "stemness" genes has suggested that gene expression alone is not sufficient to insure or define either plasticity or lineage specification. We hypothesize (a) that characterization of human stem cells by epigenetic processes will provide an underlying mechanism for the pluripotency of undifferentiated human embryonic stem cells (hESCs) and the differentiation cascades of their progeny; (b) that epigenetic marks, as established across development by the dynamics of chromatin remodeling, can be used to define potency, plasticity, and lineage-commitment along the continuum of human stem cell development; and (c) that such marks can be used to judge the therapeutic potential of a cell. The goal of this research proposal is to study the epigenetic controls of the hESC (NIH registry code: WA01, WA07, and WA09) as it differentiates towards a human neural stem cell (hNSC) and then a dopaminergic (DA) phenotype. Having established in our lab strategies for differentiating pluripotent hESCs towards becoming multipotent hNSCs and then towards DA neurons under defined culture conditions, and having generated a number of hNSC lines which can also be directed towards a DA phenotype, I will characterize the differentiation process by examining the progression of chromatin states and identify epigenetic landmarks. The profile of epigenetic marks in hESC differentiation will be compared to that of the CNS-derived hNSCs and their differentiated DA neurons. The identified epigenetic landmarks will be used to define and compare the plasticity and potential of human stem cells. These characteristics will be further affirmed by using an in vivo bioassay (a mouse model of DA dysfunction in the aged brain) to test and predict the therapeutic potential of a given stem cell state. The mentorship I will receive in the Snyder lab while pursuing the goals of this proposal will be significant for developing my future career as an independent human stem cell investigator.