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.

项目摘要 对调节造血谱系规范的分子机制的更深入了解至关重要 用于开发改善影响红细胞和血小板异常的疾病的治疗剂。 目前，我们不知道影响巨核细胞 - 甲状腺素的命运决定的机制 祖细胞（MEP）可以区分红细胞或巨核细胞谱系。总体 该提案的目标是确定在这些二强中确定血统命运的机制 祖先。确定双能细胞如何犯规的进展部分受到了部分阻碍 缺乏识别和丰富在这个关键阶段的兼容细胞的能力。克劳斯实验室有 最近通过增强，然后使用一种体外功能分析来解决这一进步的障碍 单个双能MEP开发了改进的方法以富集细胞。我们的初步数据使用 这些MEP强烈支持以下假设，即更快的细胞循环会导致MEP变得偏见 朝向巨核细胞谱系上的红细胞。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一个独特的祖细胞种群，该人群尚未完全致力于任何一个 它的潜在下游命运，并提供有关MEP脂肪潜在机制的假设生成数据 决心; 2）确定影响命运决定的小分子； 3）验证方法 测试影响命运决策（例如MYB）的特定基因的敲除； 4）CFSE评估的变化 增殖和伴随的细胞命运偏见； 5）验证单个的长期时地解体显微镜 细胞形成菌落，以评估细胞周期的时间和命运确定； 6）使用小说中的体内细胞 循环计时器记者。基于这些广泛的初步数据，我们提出：1）检验细胞的假设 周期速度在MEP命运决策中起着至关重要的作用； 2）剖析依据的分子机制 MEP命运决定； 3）检验以下假设：人类和小鼠的血小板计数升高 缺乏贫血是由于MEP命运的偏见。这些研究的结果将有助于我们 了解小鼠和健康的人类供体中正常造血祖细胞细胞的命运调节， 并将提供与常见治疗难治性造血的发病机理有关的重要见解 包括铁不良铁缺乏贫血和骨髓衰竭在内的疾病。临床应用也是如此 包括增强我们在体外产生RBC和血小板的能力，以使贫血和 血小板减少症。