Niche signals in HSC genesis

项目总结 造血干细胞(HSC)在治疗遗传性和非遗传性骨质疏松症方面已有广泛的临床应用。 获得性血液病。然而，它们的治疗潜力可以通过工程显著扩大。 从多能干细胞或直接重新编程的成体细胞产生HSC的新方法。 为了实现这一目标，我们建立了基于内皮细胞(EC)生态位的培养方法，为 支持胚胎血源造血干细胞分化和自我更新的必要条件 前体，以及最近来自成年内皮细胞的，使用转录因子(TF)介导的绕过 一种多能中间体。我们假设，重建信号是必要和充分的，以形成一个 对于体外培养HSC具有临床意义的系统将需要一种全面的系统方法来 解构利基提供了HSC规范和自我更新所需的信号。因此，总的目标是 这笔赠款是为了利用合作实验室的独特专业知识来阐明信号相互作用。 胚胎造血祖细胞或转铁蛋白重编程成体对HSC特性的调控及自我更新 欧共体在欧共体利基的背景下。我们的方法由三个相互重叠的目标组成。第一个目标是确定EC 利基-提供胚胎HSC规范和自我更新所需的信号。第二个目标是确定 由这些信号诱导的独特的HSC程序，调节从胚胎造血的转变 骨化物重新植入HSC的前身。第三个将确定监管过渡的可比计划 在转录因子介导的EC生态位重编程过程中，从成体EC到HSC。这些研究的关键将是创新 功能分析、转录分析方法和计算方法，使我们能够解决 NICE细胞的细胞复杂性及其与发育中的胚胎或重编程的HSC的相互作用 单细胞水平。确定的信号因素在特定阶段支持HSC规范中的作用将得到验证 并通过在生态位EC背景下的获得和丧失功能的研究在体外进一步提炼。此外，为了扩大 这些研究将基质无细胞系统作为迈向临床翻译的一步，我们还将测试其贡献 在HSC规范中识别的信号因素和在阶段特定调制的背景下的自我更新 使用工程设计的切槽激动剂激活切槽。为了实现这项建议的目标，我们制定了一项 多学科协作，涉及我们每个实验室的独特专业知识，包括基础HSC和 EC生态位细胞生物学，基于直接转铁蛋白的细胞转化，临床造血干细胞移植，全基因组 在单细胞分辨率下对稀有干细胞群体的评估，以及创新的计算方法 解构调控发育转变的核心信号通路。综上所述，我们预计建议的 研究最终将指导设计新的策略，在体外派生和扩增HSC用于治疗 申请。

PROJECT SUMMARY Hematopoietic stem cells (HSC) have well established clinical applications in the treatment of heritable and acquired blood disorders. However, their therapeutic potential could be significantly broadened by engineering novel methods to generate HSC de novo from pluripotent stem cells or from directly reprogrammed adult cells. Toward this goal, we have established endothelial cell (EC) niche based culture methods that provide the necessary conditions to support the specification and self-renewal of HSC from embryonic hemogenic precursors, and more recently, from adult ECs using transcription factor (TF)-mediated conversion that bypasses a pluripotent intermediate. We hypothesize that recreating the signals necessary and sufficient to develop a clinically meaningful system for HSC generation in vitro will necessitate a comprehensive, systems approach to deconstruct the niche provided signals required for HSC specification and self-renewal. Thus, the overall goal of this grant is to leverage unique expertise of the collaborating laboratories to elucidate the signaling interactions regulating HSC specification and self-renewal from embryonic hemogenic precursors or TF-reprogrammed adult EC in the context of the EC niche. Our approach consists of three overlapping aims. The first aim will identify EC niche-provided signals necessary for embryonic HSC specification and self-renewal. The second aim will identify the unique HSC programs induced by these signals that regulate the transition from embryonic hemogenic precursor to bone fide repopulating HSC. The third will identify comparable programs that regulate the transition from adult EC to HSC during TF-mediated reprogramming in the EC niche. Key to these studies will be innovative functional assays, transcriptional profiling methods, and computational approaches that will enable us to resolve cellular complexity of niche cells and their interactions with developing embryonic or reprogrammed HSC at the single cell level. The role of identified signal factors in stage-specific support of HSC specification will be validated and further refined in vitro by gain and loss of function studies in the context of niche EC. Furthermore, to extend these studies to stromal cell-free systems as a step toward clinical translation, we will also test the contribution of identified signal factors in HSC specification and self-renewal in the context of stage-specific modulation of Notch activation using engineered Notch agonists. To achieve the goals of this proposal, we have developed a multidisciplinary collaboration involving unique expertise in each of our laboratories, including basic HSC and EC niche cell biology, direct TF based cellular conversion, clinical HSC transplantation, genome wide assessment of rare stem cell populations at single cell resolution, and innovative computational approaches to deconstruct core signal pathways regulating developmental transitions. Altogether, we expect the proposed studies will ultimately guide the design of novel strategies for deriving and expanding HSC in vitro for therapeutic applications.