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

描述（由申请人提供）：我们对合成小分子pluripotin及其机制表征的发现为mESC培养提供了强大的化学工具，并揭示了对ESC自我更新的新见解：不是通过外源性生长因子激活ESCs基因/途径，而是实现ESC基本/根本自我更新状态的关键是抑制各种内在分化诱导基因。由于hESC领域仍处于起步阶段（例如，hESC的稳健自我更新条件将是非常期望的），因此无偏见和功能性的发现方法将是非常适合的，例如，提供工具并为假设奠定基础。为了解决这一观点是否也适用于人ESC的假设，即是否可以鉴定在不存在用于hESC长期自我更新的生长因子/细胞因子的情况下抑制hESC分化的小分子（在概念上类似于mESC中pluripotin的功能/机制），我们建议筛选100，000种不同的和离散的化合物来鉴定可以在化学成分确定的培养基中在不存在bFGF和其它生长因子的情况下维持hESC自我更新的小分子。我们将通过各种深入的细胞/生化测定进一步确认和表征其效果和活性，并对选定的热门化合物进行结构-活性-关系（SAR）研究，以优化其效力和特异性。总的来说，本提案中描述的研究将加快对自我更新和分化机制的理解，使hESC培养具有更大的一致性，为hESC在研究和再生医学中的实际应用提供新的途径，并促进定义和控制指导hESC自我更新或分化的信号输入。公共卫生相关性：为了解决是否可以鉴定在不存在生长因子/细胞因子的情况下抑制hESC分化的小分子，以使hESC长期自我更新（在概念上类似于mESC中pluripotin的功能/机制），我们建议筛选100，000种不同的和离散的化合物，以确定小分子，可以保持自我，在不存在bFGF和其它生长因子的化学成分确定的培养基中更新hESC。总的来说，本提案中描述的研究将为更好地理解和控制hESC自我更新提供新的化学工具，并可能最终允许开发使用hESC治疗疾病的治疗方法。

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.