Molecular dissection of Hematopoietic Stem Cell specification triggered by inflammatory mediators

炎症介质触发造血干细胞规范的分子解剖

• 批准号: 10346708
• 负责人: Raquel Espín Palazón
• 金额: $ 39.34万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10346708
• 关键词: Address; Affect; Anemia; Binding; Bioinformatics; Blood; Cells; Cellular Stress; Chromatin; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Data; Development; Dissection; Epigenetic Process; FDA approved; Family member; Fibroblast Growth Factor; Generations; Genes; Genetic; Genetic Epistasis; Goals; Health; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Cell Production; Hematopoietic Stem Cell Specification; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemoglobinopathies; Human; Image; Immune system; In Vitro; Inflammasome; Inflammation; Inflammation Mediators; Inflammatory; Interferons; Investigation; Knowledge; Laboratories; Light; Mediating; Methods; Microscopy; Modeling; Molecular; NF-kappa B; Nitric Oxide; Nitric Oxide Pathway; Nucleotides; Organism; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphotransferases; Pluripotent Stem Cells; Property; Proteins; Protocols documentation; Public Health; RIPK2 gene; Reporter; Reporting; Research; Research Personnel; Role; Signal Pathway; Signal Transduction; Sorting - Cell Movement; Stem Cell Development; System; Techniques; To specify; Transgenic Organisms; Translating; Zebrafish; base; blood treatment; epigenomics; experimental study; hematopoietic differentiation; hematopoietic stem cell emergence; hematopoietic stem cell fate; hemogenic endothelium; human pluripotent stem cell; improved; in utero; in vivo; insight; leukemia; mutant; notch protein; novel; p65; receptor; reconstitution; recruit; stem cell technology; stem cells; success; tool; transcriptome sequencing; transcriptomics

Project Summary Due to the unique property of hematopoietic stem cells (HSCs) to reconstitute the entire blood system of the organism, these stem cells are utilized clinically to treat blood disorders. The possibility of culturing and expanding HSCs in vitro would make hematopoietic stem cell transplantation (HSCT)-based therapies more feasible. However, this has eluded the field for more than three decades, necessitating a closer examination of the native developmental mechanisms that govern the emergence of HSCs. Many years of investigation have revealed that HSCs require multiple molecular inputs for proper specification, including activity of the Notch, nitric oxide (NO), Wnt, FGF, and BMP signaling pathways. In addition, inflammatory signaling (Tnfa, NF-kB, Tlr4, interferons, Il1b and inflammasome) have been recently reported as a novel group of HSC fate modulators, yet the underlying molecular mechanisms are unclear. Addressing this knowledge gap will be critical to help develop in vitro protocols for the generation of patient-specific HSCs. The goal of this proposal is to reveal in vivo the inflammatory network that unlocks HSC specification from the hemogenic endothelium (HE), and its relationship with the Notch and nitric oxide pathways. To attain this goal, the following three specific aims will be pursued: (1) Identify the role of Nod1 signaling during HSC development; (2) determine the mechanism of NF-kB-directed HSC specification; and (3) analyze the impact of the NOD1/RIPK2/NF-kB inflammatory axis on human pluripotent stem cell-derived definitive hematopoietic progenitor cells. Since hematopoietic development is highly conserved between vertebrate species, the zebrafish model provides a unique opportunity to circumvent the challenges of in utero experimentation, permitting non-invasive experiments that avoid the artifactual inflammation caused by cellular stress. To achieve this application's goals, a combination of novel zebrafish reporter and mutant lines, new methods to perform epigenomic and transcriptomic profiling of the HE by CUT&RUN-sequencing and RNA-sequencing, live imaging of HSC development by confocal and light-sheet microscopy, qPCR, FACS-sorting, and lineage tracing using Cre- mediated reporter systems will be utilized. In addition, to translate these in vivo findings to human health, this proposal will be complemented with a model of hematopoietic differentiation from human pluripotent stem cells (hPSCs). Upon successful completion of the proposed research, a previously undescribed inflammatory pathway affecting HSC specification will be identified, in addition to the central molecular mechanism by which inflammatory signaling drives HSC fate and crosstalk to other main HSC inductors. These new findings could provide key insights needed to instruct HSC fate, informing in vitro approaches to generate HSCs from pluripotent precursors for the treatment of blood disorders.

项目摘要 由于造血干细胞（HSC）的独特特性，以重建整个血液系统 这些干细胞在临床上被用来治疗血液疾病。培养和 体外扩展HSC会使造血干细胞移植（HSCT）基于基于的疗法更多 可行的。但是，这已经避开了三十多年的领域，需要对 控制HSC出现的本地发展机制。多年的调查有 揭示了HSC需要多个分子输入以进行适当的规格，包括凹槽的活性，一氮 氧化物（NO），WNT，FGF和BMP信号通路。此外，炎症信号传导（TNFA，NF-KB，TLR4， 最近已经报道了干扰素，IL1B和炎性体）是一组新型的HSC命运调节剂 基本的分子机制尚不清楚。解决这一知识差距对于帮助发展至关重要 生成患者特异性HSC的体外方案。该提议的目的是在体内揭示 炎症网络可解锁HSC规格从血肿内皮（HE）及其关系 带有缺口和一氧化氮途径。为了实现这一目标，将实现以下三个具体目标： （1）确定NOD1信号在HSC开发过程中的作用； （2）确定NF-KB定向的机制 HSC规范； （3）分析NOD1/RIPK2/NF-KB炎症轴对人的影响 多能干细胞衍生的定性造血祖细胞。 由于造血性发育在脊椎动物物种之间高度保守，因此斑马鱼模型 提供了一个独特的机会来规避子宫实验的挑战，允许无创 避免由细胞应激引起的人为炎症的实验。为了实现该应用程序的目标， 新颖的斑马鱼记者和突变线的结合，进行表观基因组和的新方法 通过剪切和跑步和RNA测序，HSC的实时成像对HE的转录组分析 共聚焦和灯页显微镜，QPCR，FACS术和谱系跟踪的开发 将使用中介记者系统。此外，要将这些体内发现转化为人类健康，这 提案将与人类多能干细胞的造血分化模型相辅相成 （HPSC）。成功完成拟议的研究后，先前未描述的炎症途径 除了中央分子机制外，还将确定影响HSC规范 炎症信号传导将HSC命运和串扰驱动到其他主要HSC电感器。这些新发现可以 提供指导HSC命运所需的关键见解，告知体外方法以产生HSC。 治疗血液疾病的前体。