Molecular dissection of Hematopoietic Stem Cell specification triggered by inflammatory mediators

项目摘要 由于造血干细胞(HSCs)具有重建整个血液系统的独特特性 这些干细胞在临床上被用来治疗血液疾病。培养和培养的可能性 体外扩增造血干细胞将使基于造血干细胞移植(HSCT)的疗法更多 可行。然而，这一点已经躲避了该领域30多年，需要更仔细地研究 管理造血干细胞出现的本地发育机制。经过多年的调查， 揭示了HSCs需要多个分子输入才能正确地指定，包括Notch、Nitroid的活性 一氧化氮(NO)、WNT、成纤维细胞生长因子和骨形态发生蛋白信号通路。此外，炎症信号(TNFa、NF-kB、Tlr4、 干扰素、IL-1b和炎症体)作为一组新的HSC命运调节剂最近被报道，但 其潜在的分子机制尚不清楚。解决这一知识差距将是帮助开发 用于产生患者特定的造血干细胞的体外方案。这项提议的目标是在体内揭示 从血源性内皮(HE)解锁HSC的炎性网络及其关系 通过Notch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信号在肝星状细胞发育中的作用；(2)确定核因子-kB的调控机制 HSC规范；以及(3)分析NOD1/RIPK2/NF-kB炎症轴对人类的影响 多能干细胞来源的最终造血祖细胞。 由于脊椎动物之间的造血发育是高度保守的，斑马鱼模型 提供了一个独特的机会来绕过宫内实验的挑战，允许无创 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， 一种新的斑马鱼报告和突变系的组合，执行表观基因组学和 用切割和Run测序、RNA测序和HSC的实时成像进行HE的转录图谱分析 通过共聚焦显微镜和薄片显微镜、定量聚合酶链式反应、流式细胞仪分选和使用Cre-CRE进行谱系追踪-- 将利用中介报道系统。此外，为了将这些体内发现转化为人类健康，这 该提案将与人类多能干细胞的造血分化模型相辅相成 (HPSCs)。在成功完成拟议的研究后，一种以前未描述的炎症途径 除了确定影响HSC规格的中心分子机制外，还将确定 炎症信号驱动HSC的命运和对其他主要HSC感应器的串扰。这些新发现可能 提供指导造血干细胞命运所需的关键见解，告知体外从多能干细胞生成造血干细胞的方法 治疗血液疾病的前体。

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.