Megakaryocyte erythroid progenitor fate specification

项目摘要 更深入地理解调节造血系规范的分子机制至关重要。 为影响红细胞和血小板异常的疾病开发改进的治疗方法。 目前，我们还不知道影响巨核细胞-红系细胞命运决定的机制 祖细胞(MEP)可以向下分化为红血球或巨核细胞。整体而言 这项提议的目标是确定决定这些二能者血统命运的机制。 祖先。在确定双能细胞如何定位方面的进展受到阻碍，部分原因是 缺乏识别和丰富处于这一关键阶段的双能细胞的能力。克劳斯实验室已经 最近解决了这一障碍，通过加强，然后使用，在体外功能测试 个体双能MEP开发改进的方法来为细胞进行浓缩。我们的初步数据使用 这些MEP强烈支持这样的假设，即更快的细胞周期会导致MEP变得有偏见 更倾向于红系而不是巨核系。这些数据包括1)单细胞RNA深度测序 为了证明丰富的细胞代表了一个独特的祖细胞群体，而这个群体并不完全致力于 其潜在的下游命运，并提供关于MEP脂肪潜在机制的假说生成数据 测定；2)测定影响命运决定的小分子；3)验证一种方法 测试影响命运决定的特定基因的敲除(例如MYB)；4)CFSE评估 增殖和伴随的细胞命运偏差；5)长期时间流逝显微镜的验证 细胞克隆形成以评估细胞周期计时和命运决定；以及6)使用一种新的体内细胞 自行车计时器报道员。基于这些广泛的初步数据，我们建议：1)检验细胞 循环速度在MEP命运决定中起着关键作用；2)剖析 MEP命运的决定；以及3)检验服用铁的人和小鼠的血小板计数升高的假设 缺乏性贫血是由于MEP命运决定有偏见所致。这些研究的结果将有助于我们 了解正常造血祖细胞在小鼠和健康献血者体内的命运调控， 这将为常见治疗-难治性造血的发病机制提供重要的见解 疾病包括铁难治性缺铁性贫血和骨髓衰竭。临床应用也 包括提高我们在体外生产红细胞和血小板用于输血治疗贫血能力，以及 血小板减少症。

Project Summary A deeper understanding of the molecular mechanisms regulating hematopoietic lineage specification is critical for developing improved therapeutics for disorders that affect red blood cell and platelet abnormalities. Currently, we do not know the mechanisms that influence the fate decisions of the megakaryocyte-eryhtroid progenitors (MEP) that can differentiate down either the red blood cell or megakaryocyte lineage. The overall goal of this proposal is to identify the mechanisms by which the lineage fate is determined in these bipotent progenitors. Progress in determining how bipotent cells become committed has been hampered in part due to lack of ability to identify and enrich for bipotent cells that are at this critical stage. The Krause laboratory has recently addressed this barrier to progress by enhancing, and then using, an in vitro functional assay for individual bipotent MEP to develop improved approaches to enrich for the cells. Our preliminary data using these MEP strongly support the hypothesis that more rapid cell cycling causes an MEP to become biased toward the erythroid over the megakaryocytic lineage. These data include 1) single cell RNA deep sequencing to prove that the enriched cells represent a unique progenitor population that is not fully committed to either of its potential downstream fates and to provide hypothesis-generating data on potential mechanisms of MEP fat determination; 2) determination of small molecules that influence fate decisions; 3) validation of an approach to test knockout of specific genes that affect the fate decision (e.g. MYB); 4) CFSE assessment of changes in proliferation and accompanying cell fate biases; 5) validation of longterm timelapse microscopy from single cells to colony formation to assess cell cycle timing and fate determination; and 6) use of a novel in vivo cell cycle timer reporter. Based on these extensive preliminary data, we propose to: 1) test the hypothesis that cell cycle speed plays a critical role in the MEP fate decision; 2) dissect the molecular mechanisms underlying the MEP fate decision; and 3) test the hypothesis that the elevated platelet counts in humans and mice with iron deficiency anemia are due to a biased MEP fate decision. The results of these studies will contribute to our understanding of fate regulation of normal hematopoietic progenitor cells in mice and healthy human donors, and will provide important insights relevant to the pathogenesis of common treatment-refractory hematopoietic diseases including iron refractory iron deficiency anemia and bone marrow failure. Clinical applications also include enhancement of our ability to produce RBCs and platelets in vitro for transfusion for anemia and thrombocytopenia.