Niche signals in HSC genesis

HSC 发生中的生态位信号

• 批准号: 9547399
• 负责人: IRWIN D BERNSTEIN
• 金额: $ 64.43万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9547399
• 关键词: Address; Adult; Agonist; Algorithms; Aorta; Biological Assay; Blood Vessels; Bypass; Candidate Disease Gene; Cell Culture Techniques; Cell-Free System; Cells; Cellular biology; Clinical; Coculture Techniques; Development; Embryo; Embryonic Development; Endothelial Cells; Engineering; Gene Expression Profiling; Generations; Goals; Gonadal structure; Grant; Hematological Disease; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Heritability; Human; In Vitro; Interdisciplinary Study; Laboratories; Machine Learning; Mediating; Mesonephric structure; Methods; Molecular; Molecular Analysis; Pathway interactions; Physiological Processes; Pluripotent Stem Cells; Population Heterogeneity; Resolution; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Sorting - Cell Movement; Source; Stem Cell Development; Stromal Cells; System; Systems Development; Testing; Therapeutic; Transplantation; base; blastomere structure; bone; clinical application; clinical translation; design; embryonic stem cell; genome-wide; in vivo; indexing; innovation; insight; loss of function; notch protein; novel; novel strategies; programs; receptor; self-renewal; single-cell RNA sequencing; stem; stem cell fate specification; stem cell population; transcription factor

PROJECT SUMMARY Hematopoietic stem cells (HSC) have well established clinical applications in the treatment of heritable and acquired blood disorders. However, their therapeutic potential could be significantly broadened by engineering novel methods to generate HSC de novo from pluripotent stem cells or from directly reprogrammed adult cells. Toward this goal, we have established endothelial cell (EC) niche based culture methods that provide the necessary conditions to support the specification and self-renewal of HSC from embryonic hemogenic precursors, and more recently, from adult ECs using transcription factor (TF)-mediated conversion that bypasses a pluripotent intermediate. We hypothesize that recreating the signals necessary and sufficient to develop a clinically meaningful system for HSC generation in vitro will necessitate a comprehensive, systems approach to deconstruct the niche provided signals required for HSC specification and self-renewal. Thus, the overall goal of this grant is to leverage unique expertise of the collaborating laboratories to elucidate the signaling interactions regulating HSC specification and self-renewal from embryonic hemogenic precursors or TF-reprogrammed adult EC in the context of the EC niche. Our approach consists of three overlapping aims. The first aim will identify EC niche-provided signals necessary for embryonic HSC specification and self-renewal. The second aim will identify the unique HSC programs induced by these signals that regulate the transition from embryonic hemogenic precursor to bone fide repopulating HSC. The third will identify comparable programs that regulate the transition from adult EC to HSC during TF-mediated reprogramming in the EC niche. Key to these studies will be innovative functional assays, transcriptional profiling methods, and computational approaches that will enable us to resolve cellular complexity of niche cells and their interactions with developing embryonic or reprogrammed HSC at the single cell level. The role of identified signal factors in stage-specific support of HSC specification will be validated and further refined in vitro by gain and loss of function studies in the context of niche EC. Furthermore, to extend these studies to stromal cell-free systems as a step toward clinical translation, we will also test the contribution of identified signal factors in HSC specification and self-renewal in the context of stage-specific modulation of Notch activation using engineered Notch agonists. To achieve the goals of this proposal, we have developed a multidisciplinary collaboration involving unique expertise in each of our laboratories, including basic HSC and EC niche cell biology, direct TF based cellular conversion, clinical HSC transplantation, genome wide assessment of rare stem cell populations at single cell resolution, and innovative computational approaches to deconstruct core signal pathways regulating developmental transitions. Altogether, we expect the proposed studies will ultimately guide the design of novel strategies for deriving and expanding HSC in vitro for therapeutic applications.

项目概要 造血干细胞（HSC）在治疗遗传性和 获得性血液疾病。然而，通过工程设计可以显着扩大它们的治疗潜力 从多能干细胞或直接重编程的成体细胞中重新生成 HSC 的新方法。 为了实现这一目标，我们建立了基于内皮细胞（EC）生态位的培养方法，该方法提供了 支持胚胎造血HSC规范化和自我更新的必要条件 前体细胞，以及最近使用转录因子 (TF) 介导的转换绕过成人 EC 多能中间体。我们假设重新创建必要且充分的信号来开发 具有临床意义的 HSC 体外生成系统需要采用全面的系统方法 解构为 HSC 规范和自我更新提供所需信号的利基。因此，总体目标是 这笔赠款将利用合作实验室的独特专业知识来阐明信号相互作用 调节 HSC 规范和胚胎造血前体或 TF 重编程成人的自我更新 EC 利基背景下的 EC。我们的方法由三个重叠的目标组成。第一个目标是确定 EC 生态位提供胚胎 HSC 规范和自我更新所需的信号。第二个目标将确定 由这些信号诱导的独特 HSC 程序可调节从胚胎造血的转变 骨性再生HSC的前体。第三个将确定规范过渡的类似计划 在 EC 生态位中 TF 介导的重编程期间，从成人 EC 到 HSC。这些研究的关键是创新 功能分析、转录分析方法和计算方法将使我们能够解决 微环境细胞的细胞复杂性及其与发育中的胚胎或重编程的 HSC 的相互作用 单细胞水平。将验证已确定的信号因素在 HSC 规范的特定阶段支持中的作用 并通过利基 EC 背景下的功能获得和丧失研究在体外进一步完善。此外，为了延长 这些研究以无基质细胞系统作为临床转化的一步，我们还将测试其贡献 阶段特定调节背景下 HSC 规范和自我更新中已识别信号因子的研究 使用工程化的 Notch 激动剂激活 Notch。为了实现本提案的目标，我们制定了 多学科合作涉及我们每个实验室的独特专业知识，包括基础 HSC 和 EC 利基细胞生物学、直接基于 TF 的细胞转化、临床 HSC 移植、全基因组 以单细胞分辨率评估稀有干细胞群，以及创新的计算方法 解构调节发育转变的核心信号通路。总而言之，我们预计拟议的 研究最终将指导新策略的设计，用于体外衍生和扩展 HSC 用于治疗 应用程序。