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.

项目概要 由于造血干细胞（HSC）具有重建整个血液系统的独特特性 在生物体中，这些干细胞在临床上用于治疗血液疾病。培养的可能性和 体外扩增造血干细胞将使基于造血干细胞移植（HSCT）的疗法更加有效 可行的。然而，这个领域已经回避了三十多年，需要更仔细的研究 控制 HSC 出现的天然发育机制。经过多年的调查研究 揭示 HSC 需要多种分子输入才能实现正确的规格，包括 Notch 的活性、硝酸 氧化物 (NO)、Wnt、FGF 和 BMP 信号通路。此外，炎症信号（Tnfa、NF-kB、Tlr4、 最近报道称，干扰素、Il1b 和炎症小体是一组新型 HSC 命运调节剂，但 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至关重要 用于生成患者特异性 HSC 的体外方案。该提案的目标是揭示体内 从造血内皮 (HE) 解锁 HSC 规范的炎症网络及其关系 与Notch和一氧化氮途径。为实现这一目标，将努力实现以下三个具体目标： (1) 明确Nod1信号在HSC发育过程中的作用； (2)确定NF-kB介导的机制 HSC规范； (3)分析NOD1/RIPK2/NF-kB炎症轴对人体的影响 多能干细胞衍生的定形造血祖细胞。 由于造血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内实验的挑战，允许非侵入性 避免细胞应激引起的人为炎症的实验。为了实现此应用程序的目标， 新颖的斑马鱼报告基因和突变株系的组合，进行表观基因组和突变的新方法 通过 CUT&RUN 测序和 RNA 测序对 HE 进行转录组分析，HSC 实时成像 通过共聚焦和光片显微镜、qPCR、FACS 分选和使用 Cre- 进行谱系追踪进行开发 将利用介导的报告系统。此外，为了将这些体内发现转化为人类健康，这 该提案将得到人类多能干细胞造血分化模型的补充 （hPSC）。成功完成拟议的研究后，以前未描述过的炎症途径 除了影响 HSC 规格的中心分子机制之外，还将确定 炎症信号驱动 HSC 命运以及与其他主要 HSC 诱导物的串扰。这些新发现可以 提供指导 HSC 命运所需的关键见解，为从多能生成 HSC 的体外方法提供信息 治疗血液疾病的先驱。