Small molecules that maintain self-renewal of hESCs without growth factors

描述(申请人提供)：我们发现的合成小分子多聚体蛋白及其机制特征为胚胎干细胞的培养提供了强大的化学工具，并揭示了对胚胎干细胞自我更新的新见解：实现胚胎干细胞基本/根本自我更新状态的关键不是通过外源生长因子激活不必要的基因/途径，而是抑制各种内在的分化诱导基因。由于人类胚胎干细胞领域仍处于初级阶段(例如，人类胚胎干细胞的强大自我更新条件将是非常可取的)，无偏见和功能发现方法将非常适合，例如为假说提供工具和铺垫基础。为了解释这一概念是否也适用于人胚胎干细胞的假说，即是否可以在缺乏生长因子/细胞因子的情况下抑制胚胎干细胞的分化(在概念上类似于多聚体蛋白在胚胎干细胞中的功能/机制)，我们建议筛选100,000种不同的离散化合物，以确定在没有碱性成纤维细胞生长因子和其他生长因子的情况下，能够在化学定义的介质中维持胚胎干细胞自我更新的小分子。我们将通过各种深入的细胞/生化分析进一步确认和表征它们的作用和活性，并对选定的HIT化合物进行结构-活性-关系(SAR)研究，以优化其效力和特异性。总之，这项建议中描述的研究将加快对自我更新和分化机制的理解，使人类胚胎干细胞培养更加一致，为人类胚胎干细胞在研究和再生医学中的实际应用提供新的途径，并有助于定义和控制指导人类胚胎干细胞自我更新或分化的信号输入。与公共卫生相关：为了解决是否可以在缺乏生长因子/细胞因子的情况下抑制hESCs的分化(在概念上类似于多聚体蛋白在mESCs中的功能/机制)，我们建议筛选100,000种不同的离散化合物，以确定在没有碱性成纤维细胞生长因子和其他生长因子的情况下，能够在化学定义的介质中维持hESCs自我更新的小分子。总之，这项建议中描述的研究将为更好地了解和控制hESC的自我更新提供新的化学工具，并最终可能使使用hESCs治疗疾病的治疗方法的开发成为可能。

DESCRIPTION (provided by applicant): Our discovery of synthetic small molecule, pluripotin, and its mechanistic characterizations provided a powerful chemical tool for mESC culture and revealed a novel insight into ESC self-renewal: instead of activating dispensable genes/pathways by exogenous growth factors, the key to achieve a basic/fundamental self- renewal state of ESCs is to inhibit various intrinsic differentiation-inducing genes. Because the hESC field is still at its infancy (e.g. a robust self-renewal condition for hESCs would be highly desirable), unbiased and functional discovery approach would be highly suited, e.g. providing tools and laying ground for hypotheses. To address the hypothesis whether this notion is also true for human ESCs, i.e. whether small molecules can be identified that inhibit differentiation of hESCs in the absence of growth factors/cytokines for long-term self- renewal of hESCs (in a way conceptually similar to the function/mechanism of pluripotin in mESCs), we propose to screen 100,000 diverse and discrete compounds to identify small molecules that can maintain self- renewal of hESCs in the chemically defined media in the absence of bFGF and other growth factors. We will further confirm and characterize their effects and activities via various in-depth cellular/biochemical assays, and carry out structure-activity-relationship (SAR) studies of the selected hit compounds to optimize their potency and specificity. Collectively, the studies described in this proposal will speed up understanding of the self-renewal and differentiation mechanisms, allow much more consistency in hESC culture, provide new avenues in practical applications of hESCs in research and in regenerative medicine, and facilitate defining and controlling signaling inputs that direct self-renewal or differentiation of hESCs. PUBLIC HEALTH RELEVANCE: To address whether small molecules can be identified that inhibit differentiation of hESCs in the absence of growth factors/cytokines for long-term self-renewal of hESCs (in a way conceptually similar to the function/mechanism of pluripotin in mESCs), we propose to screen 100,000 diverse and discrete compounds to identify small molecules that can maintain self-renewal of hESCs in the chemically defined media in the absence of bFGF and other growth factors. Collectively, the studies described in this proposal will provide novel chemical tools for better understanding and controlling hESC self-renewal, and may ultimately allow development of therapeutics employing hESCs for treating diseases.