Niche signals in HSC genesis

项目摘要 造血干细胞（HSC）在治疗遗传性和非遗传性疾病中具有良好的临床应用。 获得性血液病然而，通过工程改造， 从多能干细胞或直接重编程的成体细胞从头产生HSC的新方法。 为了实现这一目标，我们已经建立了基于内皮细胞（EC）小生境的培养方法， 胚胎造血干细胞分化和自我更新的必要条件 前体，以及最近使用转录因子（TF）介导的绕过 多能性中间体。我们假设，重新创造必要和足够的信号，以发展一个 临床上有意义的系统HSC体外生成将需要一个全面的，系统的方法， 解构小生境提供的HSC规格和自我更新所需的信号。因此， 该基金将利用合作实验室的独特专业知识来阐明信号相互作用 调节HSC特化和来自胚胎生血前体或TF重编程成人的自我更新 在欧共体利基环境下的欧共体。我们的方法包括三个相互重叠的目标。第一个目标将确定EC 胚胎HSC特化和自我更新所必需的小生境提供的信号。第二个目标是确定 由这些信号诱导的独特的HSC程序，调节胚胎造血细胞从胚胎造血细胞向造血细胞的转变， 造血干细胞再生的前体第三个将确定规范过渡的可比计划 在TF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 微生态位细胞的细胞复杂性及其与发育中的胚胎或重编程HSC的相互作用 单细胞水平。将验证所识别的信号因子在HSC特异性阶段支持中的作用 并通过在生态位EC背景下的功能获得和丧失研究在体外进一步完善。此外，为了扩展 这些研究将基质无细胞系统作为临床转化的一步，我们还将测试 HSC特化和自我更新的背景下，特定阶段的调制， 使用工程化Notch激动剂的Notch活化。为了实现这一建议的目标，我们制定了一个 多学科合作，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TF的直接细胞转化，临床HSC移植，全基因组 以单细胞分辨率评估罕见干细胞群体，以及创新的计算方法， 解构调节发育转变的核心信号通路。总的来说，我们希望拟议的 研究将最终指导设计用于体外衍生和扩增HSC的新策略， 应用.

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.