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.

项目摘要 造血干细胞（HSC）在整个生物的生命中产生所有血统，并且在临床上是 重要的是用来治疗血液学疾病的骨髓移植的治疗剂。然而， 移植疗法的可用性受到寻找免疫学兼容供体的困难的限制。 能够从诱导的多能干细胞（IPSC）体外产生大量自体HSC是一个 主要的生物医学目标，有可能消除细胞可用性问题。迄今为止，还没有 我们有可能产生具有较高植入潜力和多部门重建的真正的HSC。一 从IPSCS生成HSC的可能手段是重新构建全套HSC规格信号 但是，在体外，识别这些信号需要进一步询问控制的分子机制 HSC规范在体内。在脊椎动物胚胎中，HSC是由腹侧衬里的血液内皮（He）产生的 胚胎背主动脉（DA）的地板。我使用斑马鱼发展造血模型的工作 已经表明，躯干神经rest（NC）细胞为HES提供了所需的感性提示。到 确定新型的NC派生的HSC规范调节剂，目的是告知生成HSC的努力 体外，我进行了躯干NC和内皮细胞的转录分析屏幕。使用NC特定的目标 斑马鱼胚胎中基因的缺失，我将确定鉴定出候选者的分子机制 在屏幕中，指示HSC规范。此外，我已经确定了他表达的G蛋白耦合 受体GPR182和推定的共受体RAMP2，作为HSC规范的新调节剂。重要的是， GPR182或RAMP2的敲低直接的表观副本NC消融对HSC规范的影响， 表明GPR182可以介导NC衍生的HSC规范信号。使用针对性的敲门和 蛋白质组学，我将在HSC规范控制中表征GPR182和RAMP2。后备NC的衍生物， 特别是交感神经元和间充质干细胞，控制成人HSC的动员。 HSC规范中NC衍生物的要求表明这些细胞之间的终生信号传导关系 类型。我将采用永久的谱系跟踪方法来确定HSC规范和对照成人是否 HSC动员需要相同类型的NC衍生物。该提案的修改阶段将发生在 威尔逊·克莱门茨（Wilson Clements）领导下的圣裘德儿童研究医院 规范和关注独立阶段的突变/转基因斑马鱼线的产生。这 独立阶段将阐明新型调节器指导HSC规范的分子机制 以及NC衍生物的永久谱系跟踪。机构资源和学术环境以及 我的提案中概述的计划课程将确保我成功过渡到独立。