Bioprinting of human iPSCs to facilitate their differentiation, recruitment and strategic assembly to form engineered cardiac patches

 描述（由申请人提供）：估计有80，000，000名美国成年人（三分之一）患有一种或多种心血管疾病（CVD）; 7，900，000人有心肌梗死病史（美国心脏协会）。因此，每34秒，美国就有一个人心肌梗塞或心脏病发作，仅在美国每年就有150万例（AHA）。心肌梗死（MI）是由于长时间缺血导致心律失常导致心脏骤停而导致的心肌不可逆坏死。干细胞治疗是一种有前途的心肌梗死修复方法， 修复受损的心脏现在是当前心脏研究的主流。不幸的是，迄今为止，将干细胞直接注射到梗死心脏的纤维化区域中的成功有限，这可能是由于干细胞在坏死区域中的低保留和存活，以及所递送细胞的有限的心源性分化和功能整合 在宿主的心脏组织中。我们的提议用新策略解决了这些限制，设计并优化了组织工程化心脏贴片，用于递送自体成人干细胞衍生的心脏和血管细胞，其在水凝胶支架内策略性地分层并对齐以修复受损的心肌。这项工作对于成功开发预测性药物和毒理学筛选以及通过使用人类干细胞工程心血管片测试安全有效的心脏疗法也至关重要。为了实现这一目标，我们将使用“生物打印”来制造含有不同比例的人类诱导多能干细胞（hiPSC）或心肌细胞（CM），血管内皮细胞（EC）和来自hiPSC的血管平滑肌细胞（SMC）的细胞片;用于将它们分层成心脏补丁，以测试它们的体外功能和在体内整合梗死心脏壁的能力。我们的假设是，通过改变细胞片内的CM和非CM细胞比例，并通过策略性地将它们分层，将产生优化的功能性心脏补丁。具体地，我们提出增加hiPSC分化成CM和非CM（包括血管EC和SMC）的效率。这些分化的细胞将被生物打印以通过改变CM和非CM的细胞比率和分层布置来体外工程化功能性心脏贴片。作为本项目的最后一部分，我们建议开发和优化大鼠左前降支（LAD）结扎后的补片植入方案，用于测试未来组织工程心脏补片的功能和体内整合。总的来说，拟议的项目将通过揭示细胞排列和组装的机制来告知和改善目前用于心肌梗死的干细胞治疗，这对于形成工程心脏补丁至关重要。此外，这一努力有望通过生物打印其他分子来改善未来的方法学，这些分子用于设计用于修复类似软组织损伤的支架，使用自体成体干细胞。

 DESCRIPTION (provided by applicant): An estimated 80,000,000 American adults (one in three) have one or more types of cardiovascular disease (CVD); 7,900,000 have a history of myocardial infarction (American Heart Association). So in every 34 seconds, someone in the United States has a myocardial infarction or a heart attack, accounting for 1.5 million cases annually in the States alone (AHA). Myocardial infarction (MI) is the irreversible necrosis of heart muscle, due to prolonged ischemia which leads to cardiac arrest due to arrhythmia. Stem cell therapy is a promising approach for myocardial infarction repair, and the use of stem cells to repair a damaged heart is now mainstream in current cardiac research. Unfortunately, thus far direct injection of stem cells into the fibrotic area of infarcted hearts has met with limited success, probably due to the low retention and survival of stem cells in the necrotic areas, together with the limited cardiogenic differentiation and functional integration of delivered cells within the host heart tissue. Our proposal addresses these limitations with a new strategy, to design and optimize a tissue- engineered cardiac patch for delivering autologous adult human stem cell derived cardiac and vascular cells strategically layered and aligned within hydrogel scaffolds to repair the damaged myocardium. This work is also critical for the successful development of predictive drug and toxicology screens as well as safe and efficient cardiac therapies by testing them on using human stem cell-engineered cardiovascular sheets. To achieve this aim, we will use 'bioprinting' to fabricate cell sheets containing either human induced pluripotent stem cells (hiPSCs) or cardiomyocytes (CMs), vascular endothelial cells (ECs), and vascular smooth muscle cells (SMCs) derived from hiPSCs in varying ratios; for layering them into cardiac patches to test their in-vitro functionality and ability to integrate ono infarcted heart walls in vivo. Our hypothesis is that by varying CM and non-CM cell ratios within the cell sheets, and by strategically layering them will yield an optimized functional cardiac patch. Specifically we propose to increase the efficiency of differentiation of hiPSC's into CMs and non-CMs including vascular ECs and SMCs. These differentiated cells will be bioprinted to engineer a functional cardiac patch in- vitro by varying the cell ratios and layering arrangements of CMs and non-CMs. As a final part of this project we propose to develop and optimize a patch implantation protocol after left anterior descending artery (LAD) ligation in rats, for testing functionality and in-vivo integration of tissue-engineered cardiac patches in future. Taken together, the proposed project will inform and improve current stem cell therapy for myocardial infarct by revealing mechanisms of cell alignment and assembly that is critical for formation of an engineered cardiac patch. In addition, this effort promises future methodological improvements by bioprinting other molecules for use in scaffolds designed to repair similar soft tissue injuries, with autologous adult derived stem cells.