Defining the self-renewal program in human hematopoietic stem cells

描述(申请人提供)：无法从多能干细胞(PSC)或通过重新编程生成自我更新的造血干细胞已被证明是阻碍体外工程造血细胞用于治疗目的的主要瓶颈。我们假设，在造血发育的关键阶段，控制HSC自我更新的正确转录网络的缺陷诱导阻止了体外环境下全功能HSC的出现。由转录配置文件指导 为了获得高纯度的人胎肝HSC，我们的目标是找出在人HSC发育过程中控制自我更新的关键转录调控因子，并利用这一知识开发新的策略来改善体外来源的造血细胞的功能。为了确定自我更新调控因子的最佳候选者，我们利用一种新的人类HSC表面标记GPI80来纯化在人类发育过程中真正自我更新的HSC高度浓缩的种群。这一策略有助于识别一组转录调控因子，这些转录调控因子与其下游后代相比是自我更新的GPI80 HSC所特有的，并且在PSC-HPC中也受到抑制。利用我们新定义的HSC培养系统，该系统允许在体外研究人类多系HSPC(造血干细胞/祖细胞)层次结构，我们将首先在 原代人胎肝GPI80+HSC检测其对保护HSC自我更新和多能性的作用。同时，我们还将研究这些候选自我更新调节因子的诱导在多大程度上延长了培养的HSC的活性，和/或甚至转换了非自我更新的GPI80-HPC的转录程序，使它们在功能和分子上更接近胎肝HSC。最后，我们将通过分析参与维持HSC自我更新的单个转录调控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。识别在HSC发育过程中能够自我更新的关键上游调控因子将增加我们对人类HSC基本调控机制的了解，并为开发用于治疗的体外培养HSC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.