Adaptable tissue-specific endothelial cells for organ regeneration

项目摘要 我们提出的研究计划的总体目标是开发一个发现管道， 鉴定用于治疗的工程化组织特异性内皮细胞（EC）的转录代码 心脏、肺和血液的器官再生。器官再生疗法承诺无限制地获得 替换组织然而，尽管在揭示器官的分子基础方面取得了突破， 形态发生和类器官技术，将再生医学转化为临床面临着 障碍。这些瓶颈部分是由于缺乏对小生境细胞如何协调器官功能的理解。 修复.具体而言，供应再生信号的血管小生境细胞的贡献尚未被认识到。 该R35应用建立在器官损伤后愈合不良是由于 组织特异性EC的功能障碍和丧失。本方案提案审查了以下假设： 损伤器官中干细胞的重建依赖于所提供的促再生血管分泌信号 通过组织特异性血管生态位EC。我们已经证明，通过部署确定的血管分泌，器官型EC 因子支持肺、心脏、肝和造血再生。因此，EC作为动态的， 组织特异性小生境细胞对组织稳态和修复至关重要。为了测试这一点， 我们已经通过转导细胞因子基因工程改造了适应性强的小鼠、非人灵长类动物和人类EC。 将转导因子（TF）ETV 2转化成成体成熟EC（R-VEC）和分化人诱导多能 将干细胞（iPSC）转化为通用的胎儿样EC（iVEC），可以告知诱导 器官型转录因子这些适应性iVEC和R-VEC将与心脏、肺和血液类器官共培养 在进行器官修复的小鼠中进行体外或体内输注，以鉴定这些小鼠中器官型TF的诱导。 细胞将回收受教育的iVEC和R-VEC，并进行RNA分析和从头基序 发现以鉴定诱导的组织特异性TF。识别的TF将被过度表达或敲除 在EC中，以验证它们在维持器官型和血管分泌特征以用于器官修复中的功能。我们预计 器官型内皮细胞的移植将促进持久的组织修复而不会引起肿瘤发生 或纤维化。我们已经启动了FDA批准的人体临床试验，以检查 同种异体通用EC输注用于造血恢复。作为后续行动，我们打算评估 的R-VEC或iVEC-来源于非人灵长类动物的猪尾猕猴再生， 目的是将器官型EC的潜力转化为临床。拟议工作组的预期成果 研究是鉴定组织特异性血管和 血管分泌异质性该提案的目标符合NHLBI R35奖的使命，即开发创新的 再生发现管道，以促进心脏，肺和血液的安全有效治疗 疾病

PROJECT ABSTRACT The overarching goal of our proposed research program is to develop a discovery pipeline that will enable identification of transcriptional codes for engineering tissue-specific endothelial cells (ECs) for therapeutic organ regeneration of heart, lung and blood. Therapies for organ regeneration promises unlimited access to the replacement tissues. However, despite breakthroughs in uncovering the molecular underpinnings of organ morphogenesis and organoid technology, translation of regenerative medicine to the clinic has confronted with hurdles. These bottlenecks are in part due to the lack of understanding as to how niche cells coordinate organ repair. Specifically, contribution of vascular niche cells that supply regenerative signals has not been realized. This R35 application builds upon the novel proposition that poor healing after organ damage is due to the dysfunction and loss of the tissue-specific ECs. This programmatic proposal examines the hypothesis that reconstitution of stem cells in injured organs is dependent on the pro-regenerative angiocrine signals supplied by tissue-specific vascular niche ECs. We have shown that organotypic ECs by deploying defined angiocrine factors support lung, cardiac, hepatic and hematopoietic regeneration. Thus, ECs perform actively as dynamic, tissue-specified niche cells critical for tissue homeostasis and repair. To test this and to set up the stage for therapies, we have engineered adaptable mouse, nonhuman primate and human ECs by transducing the transduction factor (TF) ETV2 into adult mature ECs (R-VECs) and differentiating human induced pluripotent stem cells (iPSCs) into generic fetal-like ECs (iVECs) that could inform on the pathways that induce organotypic TFs. These adaptive iVECs and R-VECs will be cocultured with heart, lung, and blood organoids in vitro or infused in vivo in mice undergoing organ repair to identify the induction of organotypic TFs in these cells. The educated iVECs and R-VECs will be recovered and subjected to RNA profiling and de novo motif discovery to identify induced tissue-specific TF(s). The identified TFs will be overexpressed or knocked down in ECs, to validate their function in sustaining organotypic and angiocrine profile for organ repair. We anticipate that transplantation of organotypic ECs will promote long-lasting tissue repair without provoking tumorigenesis or fibrosis. We have initiated FDA-approved human clinical trials to examine the safety and efficacy of allogeneic generic EC infusion for hematopoietic recovery. As a follow up, we intend to assess the contribution of R-VECs or iVECs-derived from nonhuman primates to regeneration in the pigtail macaque monkeys with the intention of translating the potential of organotypic ECs to clinic. The expected outcomes of the proposed research are identification of molecular signals and transcriptional determinants of tissue-specific vascular and angiocrine heterogeneity. Goals of this proposal fit with the mission of NHLBI R35 award to develop innovative regenerative discovery pipeline to promote safe and efficacious treatments for cardiac, pulmonary and blood maladies.