Deciphering Mechanisms of Early Human Hematoendothelial Development.

破译人类早期血液内皮发育的机制。

• 批准号: 8977219
• 负责人: Mathew Angelos
• 金额: $ 3.6万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-18 至 2018-08-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8977219
• 关键词: AHR gene; Adult; Affect; Agonist; Animal Model; Aorta; Aryl Hydrocarbon Receptor; Biological Assay; Blood Cells; CD34 gene; Cell Lineage; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Consensus; Data; Development; Disease; Dorsal; Embryo; Embryonic Development; Endothelial Cells; Engraftment; Failure; Fostering; Genes; Genetic; Genomics; Globin; Goals; Growth; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Hemoglobinopathies; Human; Human Development; Immunodeficient Mouse; Immunophenotyping; In Vitro; Individual; Knock-out; Learning; Mentors; Modeling; Pathway interactions; Patients; Phase; Phenotype; Physicians; Pluripotent Stem Cells; Population; Process; Production; RNA Sequences; Receptor Signaling; Reporter; Reporting; Role; Scientist; Signal Pathway; Signal Transduction; Staging; Stem Cell Development; Stem cells; Surface Antigens; Testing; Thalassemia; Time; Transplantation; Undifferentiated; base; career; clinical application; endonuclease; gene correction; human embryonic stem cell; improved; in vivo; induced pluripotent stem cell; novel; public health relevance; receptor expression; small molecule; stem; transcriptome sequencing

 DESCRIPTION (provided by applicant): Hemogenic endothelial cells (HE) represent a rare cell population that gives rise to hematopoietic stem cells (HSCs) through a process referred to as endothelial-to-hematopoietic transition (EHT). EHT is a key developmental stage required to produce the mature, adult hematopoietic system. The underlying mechanisms that regulate human definitive hematopoiesis remain poorly elucidated. Human embryonic stem cells (hESCs) provide a well-defined cellular platform that can be used to study these mechanisms and to improve the in vitro production of HSCs potentially suitable for clinical applications. Recent studies suggest the aryl hydrocarbon receptor (AHR) is a critical regulator of HSC development. However, it is unknown whether AHR signaling is required for definitive hematopoietic induction from human HE. My preliminary studies suggest that AHR is expressed in low levels in undifferentiated hESCs and becomes upregulated immediately after hematopoietic induction. Based on these studies in hESCs and other developmental models, I hypothesize AHR negatively regulates early hematoendothelial development from human HE. I will test this central hypothesis in two specific aims. In Specific Aim 1, I will determine the rol of AHR in definitive hematopoietic development from hESCs. I hypothesize blocking or eliminating AHR signaling will enhance definitive hematopoietic development. Our lab has formuated well-defined conditions to support the differentiation of hematopoietic cells from hESCs. Here, I will utilize small molecule antagonists and agonists of the AHR as well as CRISPR/Cas9 endonuclease gene editing to assess the effects AHR has in generating definitive hematopoietic cells. In Specific Aim 2, I will perform single-cell genomic analysis to characterize human HE and non-HE subpopulations derived from hESCs. Previous studies are confounding since there is no universally accepted definition of HE and most studies examine a heterogenous population of cells. My preliminary studies have utilized a combination of genetic expression with RUNX1c, a key EHT gene, with a CD34+CD144+CD41-CD43- phenotype that allows us to isolate a putative HE population derived from hESCs. Using these cells, I hypothesize a distinct HE population with a unique genetic signature can be identified in cells derived from hESCs. This signature will further allow us to determine how key hematopoietic and endothelial genes are regulated by the AHR signaling pathway. Collectively, these studies are significant in that they will provide novel information on the identity of human HE and how the AHR pathway contributes to EHT. The results of these studies will be broadly applicable in improving the production of patient-specific HSCs suitable for transplantation in individuals with various hematological diseases. Moreover, this application provides a rigorous, yet defined scientific and mentoring framework to foster my career goals of becoming a successful academic physician-scientist.

 描述（由适用提供）：血液生成内皮细胞（HE）代表稀有细胞群，通过称为内皮到山莫托伊特型过渡（EHT）的过程会引起造血干细胞（HSC）。 EHT是产生成熟的成人造血系统所需的关键发展阶段。调节人类确定造血的基本机制仍然很差。人类胚胎干细胞（HESC）提供了一个定义明确的细胞平台，可用于研究这些机制并改善可能适合临床应用的HSC的体外生产。最近的研究表明，芳基烃受体（AHR）是HSC发育的关键调节剂。但是，未知是否需要AHR信号传导才能从HE HE中进行确定的造血诱导。我的初步研究表明，AHR在未分化的hESC中以低水平表达，并在造血诱导后立即上调。基于在hESC和其他发展模型中的这些研究，我假设AHR对人类HE产生了早期的血红素内皮发育。我将以两个具体的目的来检验这一中心假设。在特定的目标1中，我将确定hESC的确定造血发育中AHR的ROL。我假设阻断或消除AHR信号传导将增强明确的造血发育。我们的实验室已经制定了明确的条件，以支持造血细胞与hESC的分化。在这里，我将利用AHR的小分子拮抗剂和激动剂以及CRISPR/CAS9核酸内切酶基因编辑来评估AHR在产生明确造血细胞的影响。在特定的目标2中，我将进行单细胞基因组分析，以表征人类和非he子群的人类群体。先前的研究令人困惑，因为没有普遍接受的HE定义，并且大多数研究检查了细胞的异源群体。我的初步研究利用了遗传表达与关键EHT基因Runx1c和CD34+CD144+CD41-CD43-表型的组合，使我们能够隔离从HESC衍生的假定人群。使用这些细胞，我假设一个具有独特遗传特征的独特的HE种群可以在hESC的细胞中鉴定出来。该签名将进一步使我们能够确定关键的造血基因和内皮基因如何受AHR信号通路调节。总的来说，这些研究很重要，因为它们将提供有关人类身份以及AHR途径如何对EHT贡献的新颖信息。这些研究的结果将广泛地用于改善适用于各种血液学疾病个体移植的患者特异性HSC的产生。此外，该应用程序提供了一个严格但定义的科学和心理框架，以促进我成为成功的学术身体科学家的职业目标。