Megakaryocyte erythroid progenitor fate specification

巨核细胞红系祖细胞命运规范

• 批准号: 9363263
• 负责人: Diane S Krause
• 金额: $ 49.83万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9363263
• 关键词: Abnormal Platelet; Address; Affect; Anemia; Benign; Biological Assay; Blood Platelets; Bone Marrow; CDKN1C gene; Cell Cycle; Cell Cycle Kinetics; Cell Line; Cells; 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; Stem cells; Testing; Therapeutic; Thrombocytopenia; Transfusion; Validation; base; clinical application; clinically relevant; common treatment; deep sequencing; improved; in vivo; insight; iron deficiency; knock-down; novel; progenitor; small molecule; transcriptome sequencing

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.

项目概要 更深入地了解调节造血谱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疗法。 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）可以分化红细胞或巨核细胞谱系。整体 该提案的目标是确定这些双能细胞中决定谱系命运的机制 祖先。确定双能细胞如何定向的进展受到阻碍，部分原因是 缺乏识别和富集处于这一关键阶段的双能细胞的能力。克劳斯实验室有 最近通过增强并使用体外功能测定来解决这一进展障碍 个体双能 MEP 开发改进的方法来富集细胞。我们的初步数据使用 这些 MEP 强烈支持这样的假设：更快的细胞循环会导致 MEP 产生偏见 向巨核细胞谱系的红细胞方向移动。这些数据包括1）单细胞RNA深度测序 证明富集的细胞代表了一个独特的祖细胞群，该祖细胞群并未完全致力于其中任何一个 其潜在的下游命运，并提供有关 MEP 脂肪潜在机制的假设生成数据 决心; 2）影响命运决定的小分子的测定； 3) 验证方法 测试影响命运决定的特定基因（例如 MYB）的敲除； 4) CFSE 对变化的评估 增殖和伴随的细胞命运偏差； 5) 长期延时显微镜的验证 细胞集落形成以评估细胞周期时间和命运决定； 6）新型体内细胞的使用 循环计时器记者。基于这些广泛的初步数据，我们建议：1）检验细胞的假设 循环速度在 MEP 的命运决定中起着至关重要的作用； 2）剖析其背后的分子机制 欧洲议会命运决定； 3) 检验以下假设：服用铁剂后人类和小鼠的血小板计数会升高 缺乏性贫血是由于欧洲议会命运决定的偏见造成的。这些研究的结果将有助于我们 了解小鼠和健康人类捐赠者正常造血祖细胞的命运调控， 并将提供与常见难治性造血病发病机制相关的重要见解 铁疾病包括难治性缺铁性贫血和骨髓衰竭。临床应用还 包括增强我们在体外生产红细胞和血小板用于输血治疗贫血的能力，以及 血小板减少症。