Stem cell fate and embryonic development

描述（由申请人提供）：我们研究项目的主要目标是剖析控制人类多能干细胞命运决定的分子机制，并利用这些知识促进早期发育，细胞治疗和药物发现的研究。人胚胎干细胞（hESCs）和人诱导多能干细胞（hiPSCs）可以作为未分化细胞生长，并且可以分化为体内几乎所有类型的细胞。这些人类多能干细胞被认为是治疗退行性、恶性或遗传性疾病以及炎症、感染和创伤引起的损伤的可能手段。同时，它们是模拟人类早期发育（包括正常和异常）的宝贵研究工具，也是开发和测试新药的平台。然而，为了充分发挥它们的潜力，必须更好地了解多能性和定向分化的因素和分子机制。本研究计划的目的是确定一种新发现的蛋白激酶在调节早期胚胎发育过程中骨形态发生蛋白（BMP）信号通路，特别是神经分化中的功能。在过去四年的研究中，我们发现了控制多能性和早期分化的几个关键调控分子和途径，建立了一种简单、经济高效的从hESCs和hiPSCs高效大规模生产神经干细胞的方法，探索了机械因素在调节细胞行为和命运决定中的作用，并开发了新的hESCs基因传递技术。最近，我们使用高通量筛选（HTS），基因组学和蛋白质组学方法进一步推进我们对干细胞命运控制机制的理解。通过筛选靶向人类kinome的small-hairpin (sh)RNAs库（约3500个shrna靶向约700个激酶），我们发现了一种功能未知的蛋白激酶，它是BMP信号传导的关键调节剂，BMP信号传导是控制人类多能干细胞命运和早期胚胎发育的最关键调控途径之一。我们的初步结果表明，该激酶通过蛋白酶体途径促进BMP -I型受体（BMPR-Is）的降解，从而负向调节BMP通路，是hESCs和非洲爪蟾神经发育所必需的。在本研究计划中，我们的目标是探索该激酶如何控制hESCs中BMPR-Is的蛋白酶体降解（aim 1）。此外，我们将评估该激酶如何调节hESCs的早期神经分化（Aim 2）和非洲爪蟾的神经发生（Aim 3）。这项研究的结果将为BMP信号的调控、人类多能干细胞的命运决定和早期胚胎发育提供新的机制见解，使我们能够设计新的定向分化策略，并促进hESCs和hiPSCs在细胞治疗和再生医学中的应用。

DESCRIPTION (provided by applicant):The broad goal of our research program is to dissect the molecular mechanisms governing the fate decisions of human pluripotent stem cells and use the knowledge to facilitate the study of early development, cell-based therapy and drug discovery. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can grow as undifferentiated cells and can differentiate into nearly all types of cells in the body. These human pluripotent stem cells have been hailed as a possible means for treating degenerative, malignant, or genetic diseases, as well as injuries due to inflammation, infection and trauma. Meanwhile, they are an invaluable research tool for modeling early human development (both normal and abnormal), and serve as a platform to develop and test new drugs. However, to fully realize their potential, a better understanding of the factors and molecular mechanisms for pluripotency and directed differentiation must be achieved. The objective of this research plan is to define the function of a newly identified protein kinase in regulating the bone morphogenetic protein (BMP) signaling pathway and specifically, neural differentiation during early embryonic development. Our research efforts in the past four years enabled us to identify several key regulatory molecules and pathways that control pluripotency and early differentiation, establish a simple and cost- effective method for highly-efficient large-scale production of neural stem cells from hESCs and hiPSCs, explore the roles of mechanical factors in regulating cellular behaviors and fate determination, and develop new gene-delivery techniques for hESCs. More recently, we have used high-throughput screening (HTS), genomics and proteomics approaches to further advance our understanding of the mechanisms governing stem cell fate. By screening a library of small-hairpin (sh)RNAs that target the human kinome (~3,500 shRNAs targeting ~700 kinases), we identified a protein kinase of previously unknown function as a key regulator of BMP signaling - one of the most critical regulatory pathways that control the fate of human pluripotent stem cells and early embryonic development. Our preliminary results suggest that the kinase promotes the degradation of BMP type I receptors (BMPR-Is) via the proteasome pathway, thereby negatively regulating the BMP pathway, and is necessary for neural development of hESCs and Xenopus laevis. In this research plan, we aim to explore how this kinase controls proteasomal degradation of BMPR-Is in hESCs (Aim 1). In addition, we will assess how the kinase regulates early neural differentiation in hESCs (Aim 2) and neurogenesis in Xenopus laevis (Aim 3). The results from the proposed study will lead to new mechanistic insights into the regulation of BMP signaling, fate determination of human pluripotent stem cells and early embryonic development, enable us to design new strategies for directed differentiation, and facilitate the utilization of hESCs and hiPSCs for cell-based therapy and regenerative medicine.