Investigating regulators controlling differentiation potential of ES cells

摘要 多能干细胞，如胚胎干细胞(ES)和诱导多能干细胞(IPS)可以 在体外无限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类型的细胞(多能性)。由于这样的特殊情况 特征、ES和iPS细胞已被广泛研究并用作了解 早期胚胎发育的分子基础，也是药物发现和开发的有用工具 建立各种疾病模型。要充分利用它们在治疗应用中的潜力，至关重要的是 为了完全了解这两个独特的特征是如何调制的。以前的研究基本上是这样的 重点了解自我更新，让我们更好地照亮调控机制 由关键转录因子(TF)、信号通路和其他相关基因组特征介导。 另一方面，理解从自我更新到细胞命运指定的退出机制， 而参与多能干细胞正确分化的因素还没有被系统地研究出来。 检查过了。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。在我们之前由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，它们影响着 胚胎干细胞的分化潜能。在这些因素中，我们发现YAP1，一个转录协同- 位于河马途径下游的调节子对于自我更新是不必要的，但对于 胚胎干细胞的分化。我们进一步揭示了YAP1在保护ES细胞免受 分化过程中细胞过度死亡。我们还观察到，细胞密度与 河马的信号活动，不仅显著影响全球基因表达程序的自我更新 ES细胞，更重要的是其分化潜能。然而，ES细胞的潜在机制 在细胞密度和生存与死亡决定方面的分化一直是难以捉摸的。致信地址 这一关键的知识差距，我们提案的目标将是1)在单细胞水平上确定， 当ES细胞分化时，如何做出生存和死亡的决定，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使用 分化，以及3)识别控制密度依赖的基因表达程序的效应器和 阐明它们的调控机制。从提案中获得的信息将提供新颖的 对细胞密度不一致引起的生物医学研究中的重复性问题的见解 在不同的实验技术之间。此外，这项提案的结果将提供一个 为操纵干细胞以控制细胞命运以达到所需谱系并有助于 以干细胞为基础的治疗进展。

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.