Niche signals in HSC genesis

HSC 发生中的生态位信号

• 批准号: 10224676
• 负责人: IRWIN D BERNSTEIN
• 金额: $ 34.07万
• 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 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10224676
• 关键词: Address; Adult; Agonist; 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 Specification; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Heritability; Human; In Vitro; Interdisciplinary Study; Laboratories; 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; hematopoietic stem cell expansion; hematopoietic stem cell self-renewal; in vivo; indexing; innovation; insight; loss of function; machine learning algorithm; notch protein; novel; novel strategies; programs; receptor; self-renewal; single-cell RNA sequencing; stem; 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.

项目摘要 造血干细胞（HSC）在治疗可遗传和 获得的血液疾病。但是，通过工程学可以大大扩展其治疗潜力 从多能干细胞或直接重编程的成年细胞中产生HSC DE的新方法。 为了实现这一目标，我们已经建立了基于内皮细胞（EC）的基于基于的培养方法 支持胚胎血液中HSC规范和自我更新的必要条件 前体，以及最近使用转录因子（TF）介导的转换的成人EC的前体 多能中间体。我们假设重新创建所需和足够的信号以开发 HSC在体外产生的临床意义有意义的系统将需要采取全面的系统方法 解构利基提供了HSC规范和自我更新所需的信号。因此，总体目标 这笔赠款是为了利用合作实验室的独特专业知识来阐明信号交互 调节HSC规范和自我更新，从胚胎血液生成前体或TF重编程成人 EC在ec the Eche的背景下。我们的方法包括三个重叠的目标。第一个目标将确定EC 胚胎HSC规范和自我更新所需的利基提供信号。第二个目标将确定 这些信号引起的独特的HSC程序，这些信号调节了胚胎血源的过渡 骨fIDE重新流动HSC的前体。第三个将确定调节过渡的可比较程序 从成人EC到HSC，在TF介导的EC利基市场中进行了重编程。这些研究的关键将是创新的 功能测定，转录分析方法和计算方法，使我们能够解决 利基细胞的细胞复杂性及其与在胚胎或重编程的HSC中的相互作用 单细胞水平。确定信号因子在特定阶段的HSC规范支持中的作用将得到验证 并通过在利基EC的背景下通过功能研究的增益和丧失功能研究进一步完善。此外，要扩展 这些对基质细胞系统的研究是迈向临床翻译的一步，我们还将测试贡献 在HSC规范中确定的信号因子和在特定于阶段的调制中的自我更新 使用工程凹槽激动剂激活Notch。为了实现该提议的目标，我们已经开发了 多学科合作涉及我们每个实验室中的独特专业知识，包括基本HSC和 EC细胞生物学，基于TF的直接细胞转换，临床HSC移植，基因组宽 在单细胞分辨率下评估稀有干细胞群，以及创新的计算方法 解构调节发育过渡的核心信号路径。总之，我们期望提议 研究最终将指导在体外得出和扩展HSC的新型策略的设计 申请。