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

项目摘要/摘要 我们的长期研究目标是了解调控干细胞命运决定的机制。在这 首次续展申请此奖项，我们建议追求自我更新和世系潜力的调节 我们在当前奖项中发现的发育受限的造血干细胞(DrHSCs) 句号。尽管这一群体满足功能性HSC的最严格标准，但在正常情况下，他们的寿命 发展被限制在有限的发展窗口内。功能正常的HSC不会持续到 成年期以前从未被观察到，因此定义了一种新的决定性的造血波 截然不同的端点。在这里，我们重点了解drHSC自我更新和 多能性：在移植后，drHSC被诱导自我更新，而他们固有的谱系偏见是 保存完好。令人惊讶的是，后者-淋巴偏向和特殊的B1a重建潜力-保持不变 即使在连续移植的反复压力下，也会持续数月之久。相比之下，单一的短期 暴露在压力下会诱导drHSCs长期存在的能力。我们建议追寻表观遗传学 支配这一悖论的机制。我们将进行全面的分子和细胞比较 DrHSCs、共存的胎肝HSCs和成人HSCs，并在竞争中进行严格的功能分析 重建分析。重要的是，我们将把单细胞转录图谱与功能性HSC能力结合起来 在高效而严谨的体内分析中。使用CRISPRi/a介导的转录操作，我们将直接 在体内测试重新编程的HSC自我更新和谱系潜力的要求。我们的转基因模型 非常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- 是在开发过程中建立并终生维护的，我们很高兴能将这些强大的工具 致力于开创发展中的造血命运决定。

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.