Investigating regulators controlling differentiation potential of ES cells

总结 多能干细胞，如胚胎干（ES）细胞和诱导多能干（iPS）细胞，可 在体外无限期增殖而不改变其特征（自我更新），同时保持其 有可能在成年生物体中产生几乎所有细胞类型（多能性）。由于这种特殊的 ES和iPS细胞已经被广泛研究，并用作理解这些特征的工具。 早期胚胎发育的分子基础，也是药物发现和 建立各种疾病模型。为了充分利用它们在治疗应用中的潜力， 来完全理解这两个独特的特征是如何被调制的。以前的研究在很大程度上 专注于自我更新的理解，使我们能够更好地阐明监管机制， 由关键转录因子（TF）、信号传导途径和其他相关基因组特征介导。 另一方面，了解从自我更新到细胞命运指定的退出机制， 和参与多能干细胞适当分化的因素尚未被系统地 考察拟议研究的长期目标是调查监管机构控制 多能干细胞的分化潜能。在我们之前的NIGMS奖项支持的研究中， 我们已经揭示了多种转录因子、表观遗传调节因子和基因组特征，这些特征影响了 ES细胞的分化潜能。在这些因子中，我们发现Yap 1，一个转录共调控因子， Hippo通路下游的调节因子，是自我更新的必需品，但也是自我更新所必需的。 ES细胞的分化。我们进一步揭示了Yap 1在保护ES细胞免受 在分化过程中过度的细胞死亡。我们还观察到，细胞密度，紧密联系在一起， Hippo信号传导活性，不仅显著影响自我更新的全局基因表达程序， 胚胎干细胞，而且他们的分化潜力。然而，ES细胞的潜在机制 在细胞密度和存活与死亡决定的背景下的分化是难以捉摸的。解决 这个关键的知识差距，我们的目标的建议将是1）确定，在单细胞水平， 当ES细胞分化时，生存与死亡的决定是如何做出的，2）定义密度的结果- ES细胞自我更新过程中依赖的基因表达特征和增强子的使用， 3）鉴定控制密度依赖性基因表达程序的效应子， 阐明其调控机制。从提案中获得的信息将提供新的 深入了解生物医学研究中由细胞密度不一致引起的再现性问题 不同的实验技术。此外，该提案的结果将提供一个 操作干细胞以控制细胞朝向所需谱系的命运并有助于 干细胞疗法的进展

SUMMARY Pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can proliferate indefinitely in vitro without changes in their characteristics (self-renewal) while keeping their potential to give rise to almost all cell types in adult organisms (pluripotency). Due to such exceptional characteristics, ES and iPS cells have been extensively studied and used as tools for understanding the molecular basis of early embryo development and also serve as useful instruments in drug discovery and establishing various disease models. To fully utilize their potential in therapeutic applications, it is crucial to completely understand how these two unique characteristics are modulated. Prior studies have largely focused on understanding of self-renewal, allowing us to better illuminate the regulatory mechanisms mediated by key transcription factors (TFs), signaling pathways, and other associated genomic features. On the other hand, understanding of exit mechanisms from self-renewal towards cell fate specification, and factors involved in proper differentiation of pluripotent stem cells have not yet been systematically examined. The long-term objective of the proposed research is to investigate regulators controlling differentiation potential of pluripotent stem cells. In our previous research supported by NIGMS awards, we have revealed multiple TFs, epigenetic regulators, and genomic features that influence the differentiation potential of ES cells. Among those factors, we showed that Yap1, a transcriptional co- regulator, downstream of the Hippo pathway, is dispensable for self-renewal but required for differentiation of ES cells. We furthermore revealed the roles of Yap1 in safeguarding ES cells from excessive cell death during differentiation. We additionally observed that cell density, tightly linked to the Hippo signaling activity, significantly affects global gene expression programs of not only self-renewing ES cells, but also their differentiation potential. However, underlying mechanisms of ES cell differentiation in the context of cell density and survival vs. death decision have been elusive. To address this critical gap in knowledge, our objectives of the proposal will be 1) to determine, at the single cell level, how the survival vs. death decision is made when ES cells differentiate, 2) to define outcomes of density- dependent gene expression signatures and enhancer usage during ES cell self-renewal and differentiation, and 3) to identify effectors controlling density-dependent gene expression programs and elucidate their regulatory mechanisms. The information obtained from the proposal will provide novel insights into the reproducibility issues in biomedical studies caused by inconsistencies in cell density between different experimental techniques. Furthermore, outcomes of this proposal will provide a foundation for manipulation of stem cells to control cell fates towards desired lineages and contribute to the advances in stem cell-based cell therapies.