Characterizing Novel Neural Crest Derived Regulators of Hematopoietic Stem Cell Specification

表征造血干细胞规范的新型神经嵴衍生调节因子

• 批准号: 10058881
• 负责人: Erich William Damm
• 金额: $ 24.9万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10058881
• 关键词: Ablation; Adult; Alleles; Aorta; Autologous; Blood; Blood Circulation; Bone Marrow; Bone Marrow Transplantation; CRISPR/Cas technology; Candidate Disease Gene; Cell Transplantation; Cells; Clinical; Competence; Cues; Data; Development; Disease; Dorsal; Embryo; Embryonic Development; Endothelial Cells; Endothelium; Engraftment; Ensure; Environment; Floor; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Gene Deletion; Gene Expression Profiling; Generations; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cell Specification; Hematopoietic System; Hematopoietic stem cells; Immunologics; In Vitro; Knock-out; Knowledge; Label; Life; Malignant - descriptor; Mediating; Mentors; Mesenchymal Stem Cells; Modeling; Molecular; Mutagenesis; Neural Crest; Neural Crest Cell; Neurons; Non-Malignant; Organ; Organism; Pattern; Phase; Phenocopy; Population; Proteomics; Protocols documentation; Regulation; Resources; Role; Saint Jude Children' s Research Hospital; Signal Transduction; Therapeutic Agents; Tissue Expansion; Tissues; Transgenic Organisms; Transplantation; Work; Zebrafish; cell type; gene therapy; hemogenic endothelium; in vivo; induced pluripotent stem cell; knock-down; loss of function; mutant; novel; programs; receptor; reconstitution; self renewing cell; vertebrate embryos

Project Summary Hematopoietic stem cells (HSCs) generate all blood lineages throughout the life of an organism and are clinically important as the therapeutic agents of bone marrow transplants used to treat hematological disorders. However, the availability of transplant therapy is limited by difficulty in finding immunologically compatible donors. The ability to generate large numbers of autologous HSCs in vitro from induced pluripotent stem cells (iPSCs) is a major biomedical objective and has the potential to eliminate problems of cell availability. To date, it has not been possible to generate bona fide HSCs with high engraftment potential and multi-lineage reconstitution. One possible means of generating HSCs from iPSCs would be to reconstitute the full set of HSC specification signals in vitro, however identifying these signals requires further interrogation of the molecular mechanisms controlling HSC specification in vivo. In vertebrate embryos, HSCs arise from hemogenic endothelium (HE) lining the ventral floor of the embryonic dorsal aorta (DA). My work using the zebrafish model of developmental hematopoiesis has shown that trunk neural crest (NC) cells provide required inductive cues for HSC specification to the HE. To identify novel NC derived regulators of HSC specification with the goal of informing efforts to generate HSCs in vitro, I conducted a transcriptional profiling screen of trunk NC and endothelial cells. Using NC specific targeted deletion of genes in zebrafish embryos, I will determine the molecular mechanisms by which candidates identified in the screen instruct HSC specification. Additionally, I have identified the HE expressed G-protein coupled receptor Gpr182 and putative co-receptor Ramp2, as novel regulators of HSC specification. Importantly, knockdown of either gpr182 or ramp2 directly phenocopy the effect of NC ablation on HSC specification, suggesting that Gpr182 could mediating a NC derived HSC specification signal. Using targeted knock out and proteomics, I will characterize Gpr182 and Ramp2 in the control of HSC specification. Derivatives of trunk NC, specifically sympathetic neurons and mesenchymal stem cells, control mobilization of adult HSCs. The requirement for NC derivatives in HSC specification suggests a lifelong signaling relationship between these cell types. I will employ permanent lineage tracing approaches to determine if HSC specification and the control adult HSC mobilization require the same types of NC derivatives. The mentored phase of this proposal will occur at St. Jude Children’s Research Hospital, under Dr. Wilson Clements and will confirm candidate regulators of HSC specification and focus on generation of mutant/transgenic zebrafish lines for the independent phase. The independent phase will elucidate the molecular mechanisms by which novel regulators instruct HSC specification and on permanent lineage tracing of NC derivatives. The institutional resources and academic environment and the planned courses outlined in my proposal will ensure my successful transition to independence.

项目概要 造血干细胞（HSC）在生物体的整个生命周期中产生所有血统，并且在临床上 作为用于治疗血液疾病的骨髓移植的治疗剂很重要。 由于难以找到免疫相容的供体，移植治疗的可用性受到限制。 体外从诱导多能干细胞 (iPSC) 产生大量自体 HSC 的能力是 主要的生物医学目标，并且有可能消除细胞可用性的问题。 能够产生具有高植入潜力和多谱系重建的真正的 HSC。 从 iPSC 生成 HSC 的可能方法是重构全套 HSC 规范信号 在体外，然而识别这些信号需要进一步询问控制的分子机制 在脊椎动物胚胎中，HSC 源自腹侧的造血内皮 (HE)。 我的工作使用发育造血的斑马鱼模型。 研究表明，躯干神经嵴 (NC) 细胞为 HSC 向 HE 的规范提供所需的诱导线索。 识别 HSC 规范的新型 NC 衍生调节器，目的是为在以下领域生成 HSC 的工作提供信息 在体外，我使用 NC 特异性靶向细胞对躯干 NC 和内皮细胞进行了转录谱筛选。 删除斑马鱼胚胎中的基因，我将确定候选者识别的分子机制 此外，在屏幕指导 HSC 规范中，我还鉴定了 HE 表达的 G 蛋白偶联。 受体 Gpr182 和假定的辅助受体 Ramp2，作为 HSC 规范的新型调节剂。 gpr182或ramp2的敲低直接表型复制NC消融对HSC规范的影响， 表明 Gpr182 可以通过靶向敲除和介导 NC 衍生的 HSC 规范信号。 蛋白质组学，我将表征 Gpr182 和 Ramp2 在 HSC 规范的控制中的衍生物， 特别是交感神经元和间充质干细胞，控制成体 HSC 的动员。 HSC 规范中对 NC 衍生物的要求表明这些细胞之间存在终生信号传导关系 我将采用永久谱系追踪方法来确定 HSC 规格和对照成人是否相同。 HSC 动员需要相同类型的 NC 衍生品。该提案的指导阶段将在以下时间进行。 Wilson Clements 博士领导下的 St. Jude 儿童研究医院将确认 HSC 的候选监管机构 规范并专注于独立阶段突变/转基因斑马鱼品系的生成。 独立阶段将阐明新型调节剂指导 HSC 规范的分子机制 以及NC衍生品的沿袭溯源的机构资源和常设学术环境。 我的提案中概述的计划课程将确保我成功过渡到独立。