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（HSCs）的独特性质，以重建整个血液系统， 这些干细胞在临床上用于治疗血液疾病。培养的可能性和 体外扩增HSC将使基于造血干细胞移植（HSCT）的治疗更加有效。 可行然而，三十多年来，这一领域一直没有得到解决，因此有必要更仔细地审查 控制HSC出现的天然发育机制。多年的调查 揭示了HSC需要多个分子输入以进行适当的规范，包括Notch，一氧化氮， 氧化物（NO）、Wnt、FGF和BMP信号传导途径。此外，炎症信号传导（Tnfa，NF-kB，Tlr 4， 干扰素、IL 1b和炎性小体）最近被报道为一组新的HSC命运调节剂，然而 潜在的分子机制尚不清楚。解决这一知识差距对于帮助发展 用于产生患者特异性HSC的体外方案。这项提议的目的是在体内揭示 从生血内皮（HE）解锁HSC特化的炎症网络及其关系 Notch和一氧化氮通路的联系为实现这一目标，将努力实现以下三个具体目标： (1)明确Nod 1信号通路在HSC发育中的作用;（2）确定NF-kB介导的HSC分化的机制。 （3）分析NOD 1/RIPK 2/NF-kB炎性轴对人HSC表达的影响。 多能干细胞衍生的永久造血祖细胞。 由于造血发育在脊椎动物物种之间是高度保守的， 提供了一个独特的机会来规避子宫内实验的挑战， 避免由细胞应激引起的人为炎症的实验。为了实现这个应用程序的目标， 新的斑马鱼报告基因和突变株系的组合，进行表观基因组和 通过CUT& RUN测序和RNA测序对HE进行转录组学分析，HSC的实时成像 通过共聚焦和光片显微镜、qPCR、FACS分选和使用Cre- 将使用介导的报告系统。此外，为了将这些体内发现转化为人类健康， 一个建议将补充从人类多能干细胞造血分化的模型 （hPSC）。在成功完成拟议的研究后，以前未描述的炎症途径 影响HSC特化的主要分子机制， 炎性信号传导驱动HSC命运和对其它主要HSC诱导物的串扰。这些新发现可能 提供指导HSC命运所需的关键见解，为体外方法提供信息， 治疗血液疾病的前体。