Megakaryocyte erythroid progenitor fate specification

巨核细胞红系祖细胞命运规范

• 批准号: 10001510
• 负责人: Diane S Krause
• 金额: $ 48.06万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001510
• 关键词: Abnormal Platelet; Address; Affect; Anemia; Benign; Biological Assay; Blood Cell Count; Blood Platelets; Bone Marrow; CDKN1C gene; Cell Cycle; Cell Cycle Kinetics; Cell Line; Cells; Cellular Assay; Cellular biology; Data; Development; Disease; Erythrocytes; Erythroid; Fatty acid glycerol esters; Genes; Goals; Hematopoietic; Hematopoietic stem cells; Human; Hypoxia; In Vitro; Individual; Iron; Iron deficiency anemia; Knock-out; Laboratories; Link; Malignant - descriptor; Megakaryocytes; Megakaryocytopoieses; Microscopy; Modification; Molecular; Mus; Normal Cell; Pancytopenia; Pathogenesis; Pathway interactions; Patients; Platelet Count measurement; Platelet Transfusion; Play; Population; Positioning Attribute; Process; Production; Proteins; Publishing; RNA; Refractory; Regulation; Reporter; Risk; Role; Speed; Testing; Therapeutic; Thrombocytopenia; Transfusion; Validation; base; clinical application; clinically relevant; common treatment; deep sequencing; improved; in vivo; insight; iron deficiency; knock-down; novel; progenitor; single-cell RNA sequencing; small molecule; stem cells

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.

项目摘要 更深入地了解调节造血谱系特化的分子机制至关重要 用于开发用于影响红细胞和血小板异常的疾病的改进的治疗剂。 目前，我们还不知道影响巨核红细胞命运决定的机制 祖细胞（MEP），其可以向下分化为红细胞或巨核细胞谱系。整体 该建议的目的是确定在这些双能性动物中决定谱系命运的机制。 祖先在确定双能细胞如何定型方面的进展受到阻碍，部分原因是 缺乏鉴定和富集处于这一关键阶段的双能细胞的能力。克劳斯实验室 最近通过增强并使用一种体外功能测定来解决这一进展障碍， 个体双能MEP来开发用于富集细胞的改进方法。我们的初步数据使用 这些MEP强有力地支持了细胞周期更快导致MEP偏向的假设 向红细胞系转移而不是巨核细胞系。这些数据包括1）单细胞RNA深度测序 为了证明富集的细胞代表了一种独特的祖细胞群体，该祖细胞群体不完全定型为以下任一种 其潜在的下游命运，并提供关于MEP脂肪的潜在机制的假设生成数据 确定; 2）确定影响最终决定的小分子; 3）验证 测试影响命运决定的特定基因（例如MYB）的敲除; 4）CFSE评估 增殖和伴随的细胞命运偏差; 5）从单个细胞中验证长期时移显微镜检查 细胞到集落形成以评估细胞周期定时和命运确定;和6）使用新的体内细胞 循环计时器记者。基于这些广泛的初步数据，我们提出：1）测试假设，细胞 循环速度在MEP命运决定中起着关键作用; 2）剖析MEP的分子机制， MEP的命运决定;和3）测试的假设，在人类和小鼠的血小板计数升高与铁 缺乏性贫血是由于有偏见的MEP命运决定。这些研究的结果将有助于我们 了解小鼠和健康人供体中正常造血祖细胞的命运调控， 并将提供重要的见解有关的发病机制，常见的治疗难治性造血干细胞， 包括铁难治性缺铁性贫血和骨髓衰竭的疾病。临床应用也 包括增强我们在体外产生红细胞和血小板以用于贫血输血能力， 血小板减少症