Role and Regulation of a Novel, Developmentally Restricted Hematopoietic Stem Cell

项目总结/摘要 我们的长期研究目标是了解调节干细胞命运决定的机制。在这 第一次更新申请这个奖项，我们建议追求自我更新和血统潜力的调节 我们在当前奖项中发现的发育受限造血干细胞（drHSC） 期尽管这一人群符合功能性HSC的最严格标准，但他们在正常发育期间的寿命 发展被限制在有限的发展窗口内。一个功能性的HSC，不持续到 成年期以前从未观察到过，因此定义了一种新的确定性造血波， 不同的终点。在这里，我们重点了解drHSC自我更新的矛盾调节， 多能性：在移植时，诱导drHSC自我更新，而它们的内在谱系偏好是 保存。令人惊讶的是，后者-淋巴偏向和特殊的B1 a重建潜力-得以维持 甚至在连续移植的反复压力下也能存活数月。相比之下，一个单一的，短期的 暴露于应激诱导了drHSC长期存活的能力。我们建议从表观遗传学的角度 控制这一矛盾的机制。我们将进行全面的分子和细胞比较， drHSC、共存的胎肝HSC和成人HSC，并在竞争性研究中进行严格的功能分析。 重构测定。重要的是，我们将单细胞转录谱与功能性HSC能力相结合， 在有效但严格的体内测定中。使用CRISPRi/a介导的转录操纵，我们将直接 测试体内重编程HSC自我更新和谱系潜力的要求。我们的转基因模型 是唯一适合于理解造血干细胞的核心特性-自我更新和多谱系潜力- 在开发过程中建立并终身维护，我们很高兴能够将这些强大的工具 致力于开创造血发育的命运决定。

PROJECT SUMMARY/ABSTRACT Our long-term research goals are to understand the mechanisms that regulate stem cell fate decisions. In this first renewal application of this award, we propose to pursue the regulation of self-renewal and lineage potential of the developmentally restricted hematopoietic stem cells (drHSCs) that we discovered in the current award period. Although this population fulfills the most stringent criteria for functional HSC, their life-span during normal development is restricted to a limited developmental window. A functional HSC that does not persist into adulthood had never been observed before and therefore defines a novel wave of definitive hematopoiesis with a distinct endpoint. Here, we focus on understanding the contradictory regulation of drHSC self-renewal and multipotency: upon transplantation, drHSC self-renewal is induced, whereas their intrinsic lineage bias is preserved. Amazingly, the latter – lymphoid bias and exceptional B1a reconstitution potential – is maintained over many months even upon the repeated stress of serial transplantation. In contrast, a single, short-term exposure to stress induces the ability of drHSCs to persist long-term. We propose to pursue the epigenetic mechanisms governing this paradox. We will perform comprehensive molecular and cellular comparisons of drHSCs, co-existing fetal liver HSCs, and adult HSCs, and pursue rigorous functional analysis in competitive reconstitution assays. Importantly, we will couple single-cell transcriptional profiles with functional HSC capacity in efficient yet rigorous in vivo assays. Using CRISPRi/a-mediated transcriptional manipulation, we will directly test the requirements for reprogramming HSC self-renewal and lineage potential in vivo. Our transgenic models are uniquely suited for understanding how the core properties of HSCs – self-renewal and multilineage potential - are established during development and maintained for life and we are excited to put these powerful tools to work to pioneer developmental hematopoietic fate decisions.