In vitro organization of human induced pluripotent stem cell-derived cardiovascular cells into vascularized cardiac tissue

心脏组织工程策略可能为心血管疾病的治疗提供新的治疗选择，患者特异性干细胞衍生的心血管细胞是理想的细胞来源。对于体外生成功能性心脏组织，内皮细胞和平滑肌细胞的充分血管化是一个关键方面，此外还有心肌细胞的收缩力和成纤维细胞的机械稳定。最近的发现为从多能干细胞中产生所有类型的心脏细胞提供了独特的机会。本研究的总体目标是研究干细胞衍生的人类血管化生物人工心脏组织在体外形成和自组织的机制及其在体内的再生潜力。我们假设1)心血管细胞类型可以从人类诱导多能干细胞（iPSCs）这一单一细胞来源中获得；2)这些细胞能够在体外自组织形成完全自体的血管化心肌组织用于治疗；周细胞可能在支持血管功能结构的形成中起重要作用。因此，具体目标包括：-优化分化和/或选择策略，从单一干细胞来源（人类多能干细胞）产生天然心脏组织中存在的五种不同类型的细胞（心肌细胞、内皮细胞、平滑肌细胞、周细胞和成纤维细胞）。到目前为止，还没有单一的分化方案可用于同时在一锅反应中生成心脏所有心血管细胞类型，产生所需细胞类型的定义和适当比例。因此，使用单独的方案来指导各自细胞类型的分化似乎更有希望。-实现所有心血管细胞类型的体外生成功能心肌组织。组织形成机制、最佳细胞类型化学计量和空间组织以及进一步原位成熟的必要性将被研究。-研究周细胞在体外生物人工心脏组织形成模型中血管形成的作用，包括分析周细胞衍生的促血管生成因子的表达和其他心血管细胞类型的细胞信号通路的激活。-在豚鼠心肌梗死小动物模型中研究血管化生物人工心脏组织的体内再生潜能，并与非血管化组织进行直接比较。随着患者特异性iPSC生成及其临床应用的进展，本项目所获得的知识将有助于从人类iPSC中生成自体生物人工血管化心脏组织，用于未来受损和病变心肌的组织替代治疗。

Cardiac tissue engineering strategies may provide new therapeutic options for the treatment of cardiovascular disease, with patient-specific stem-cell derived cardiovascular cells as an ideal cell source. For the in vitro generation of functional heart tissue, sufficient vascularization by endothelial cells and smooth muscle cells is a critical aspect in addition to the contractile force of cardiomyocytes and mechanical stabilization by fibroblasts. Recent findings offer the unique opportunity to generate all cell types of the heart from pluripotent stem cells. The overall objective of the proposed research is the investigation of mechanisms of stem cell-derived human vascularized bioartificial cardiac tissue formation and self-organization in vitro and its regenerative potential in vivo. We hypothesize that 1) cardiovascular cell types can be derived from a single cell source of human induced pluripotent stem cells (iPSCs); and 2) these cells are able to self-organize in vitro to form fully autologous vascularized myocardial tissue for therapeutic application; and 3) pericytes might play an important role to support the formation of functional vascular structures. Consequently, the specific objectives include:- Optimization of differentiation and/or selection strategies to generate from a single stem cell source (human pluripotent stem cells) five different cell types present in the native heart tissue (cardiomyocytes, endothelial cells, smooth muscle cells, pericytes, and fibroblasts). So far, no single differentiation protocol is available for the generation of all cardiovascular cell types of the heart in a simultaneous one pot reaction, yielding defined and appropriate proportions of the desired cell types. Therefore, the use of individual protocols for the directed differentiation of the respective cell types seems more promising. - Implementation of all cardiovascular cell types for the in vitro generation of functional myocardial tissue. Mechanisms of tissue formation, optimal cell type stoichiometry and spatial organization as well as the need for further in situ maturation will be investigated.- Investigation of the role of pericytes for vascularization in the in vitro model of bioartificial cardiac tissue formation, including analysis of pericyte-derived pro-angiogenic factor expression and activation of cell signaling pathways in other cardiovascular cell types. - Investigation of the regenerative potential of vascularized bioartificial cardiac tissue in vivo in a small animal model of myocardial infarction in guinea-pigs with a direct comparison to non-vascularized tissue.Together with progress concerning patient-specific iPSC generation and their clinical applicability, knowledge gained in this project would facilitate the generation of autologous bioartificial vascularized cardiac tissue from human iPSCs for future tissue replacement therapy of damaged and diseased myocardium.