Defining the self-renewal program in human hematopoietic stem cells

描述（由申请人提供）：不能从多能干细胞（PSC）或通过重编程产生自我更新的造血干细胞已被证明是抑制将体外工程化的造血细胞用于治疗目的的主要瓶颈。我们推测，在造血发育的关键阶段，调控HSC自我更新的正确转录网络的缺陷诱导阻止了体外环境中全功能HSC的出现。在转录谱的指导下 高纯度的人胎肝HSC，我们的目标是确定关键的转录调控，管理自我更新，在发展中的人HSC，并利用这些知识，开发新的策略，以改善在体外衍生的造血细胞的功能。为了确定自我更新调节剂的最佳候选者，我们利用了一种新的人类HSC表面标记物GPI 80来纯化在人类发育过程中高度富集真正自我更新HSC的群体。这种策略有助于鉴定一组转录调节因子，与其下游后代相比，这些转录调节因子对自我更新GPI 80 HSC是独特的，并且在PSC-HPC中也被抑制。利用我们新定义的HSC培养系统，该系统允许在体外研究人多谱系HSPC（造血干/祖细胞）等级，我们将首先在体外对这些候选HSC调节因子进行慢病毒敲低， 原代人胎肝GPI 80 + HSC以测试它们对于保护HSC自我更新和多能性的重要性。同时，我们还将研究这些候选自我更新调节剂的诱导在培养的HSC中延长HSC活性的程度，和/或甚至在非自我更新GPI 80-HPC中转换转录程序，使它们在功能上和分子上更接近胎肝HSC。最后，我们将通过分析参与维持HSC自我更新的个体转录调节因子的基因组结合位点和下游基因表达网络，来定义与HSC自我更新相关的转录网络的层次结构。鉴定能够在发展HSC中赋予自我更新的关键上游调控因子将增加我们对人类HSC基本调控机制的认识，并为开发用于治疗用途的HSC体外生成的新方法铺平道路。

DESCRIPTION (provided by applicant): Inability to generate self-renewing hematopoietic stem cells from pluripotent stem cells (PSC) or via reprogramming has proven to be a major bottleneck that inhibits the use of in vitro engineered hematopoietic cells for therapeutic purposes. We hypothesize that defective induction of the correct transcriptional networks governing HSC self-renewal during the critical stages of hematopoietic development prevents the emergence of fully functional HSC in an in vitro setting. Guided by the transcriptional profile of highly purified human fetal liver HSC, we aim to identify key transcriptional regulators that govern self-renewal in developing human HSC and use this knowledge to develop new strategies to improve the function of in vitro derived hematopoietic cells. To identify the best candidates for self-renewal regulators, we took advantage of a novel human HSC surface marker, GPI80, to purify a population that is highly enriched for the true self-renewing HSC during human development. This strategy facilitated the identification of a set of transcriptional regulators that are unique to the self-renewing GPI80 HSC as compared to their downstream progeny, and are also suppressed in PSC-HPC. Utilizing our newly defined HSC culture system, which allows the study of human multilineage HSPC (hematopoietic stem/progenitor cell) hierarchy in vitro, we will first perform lentiviral knockdown of these candidate HSC regulators in primary human fetal liver GPI80+ HSC to test their importance for protecting HSC self-renewal and multipotency. In parallel, we will also investigate the degree to which the induction of these candidate self-renewal regulators extends HSC activity in cultured HSC, and/or even converts the transcriptional program in non-self-renewing GPI80-HPC, bringing them functionally and molecularly closer to fetal liver HSC. Finally, we will define the hierarchy of transcriptional networks associated with HSC self-renewal by analyzing the genomic binding sites and downstream gene expression networks for the individual transcriptional regulators that are involved in maintaining HSC self-renewal. Identification of the key upstream regulators that can confer self-renewal in developing HSC will both increase our knowledge of the fundamental regulatory mechanisms governing human HSC as well as pave the way for developing novel approaches for the in vitro generation of HSC for therapeutic use.