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

 描述(申请人提供)：估计有8000万美国成年人(三分之一)患有一种或多种心血管疾病(CVD)；7900,000,000人有心肌梗塞病史(美国心脏协会)。因此，在美国，每34秒就有一个人患上心肌梗死或心脏病发作，仅在美国每年就有150万例(AHA)。心肌梗死(MI)是指心肌因长时间的缺血而发生的不可逆性坏死，因心律失常而导致心脏骤停。干细胞疗法是一种很有前途的心肌梗死修复方法，利用干细胞可以 修复受损的心脏现在是当前心脏研究的主流。不幸的是，到目前为止，将干细胞直接注射到梗死心脏纤维化区的成功有限，这可能是由于干细胞在坏死区的保留率和存活率较低，以及输送细胞的心源性分化和功能整合有限。 在宿主心脏组织内。我们的建议通过一种新的策略来解决这些限制，设计和优化一种组织工程心脏补片，用于输送自体成人干细胞来源的心脏和血管细胞，战略性地在水凝胶支架内分层和排列，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查以及安全有效的心脏疗法也是至关重要的，方法是使用人类干细胞工程的心血管片材进行测试。为了实现这一目标，我们将使用生物打印技术制造包含不同比例的人诱导多能干细胞(HiPSCs)或心肌细胞(CMS)、血管内皮细胞(ECs)和血管平滑肌细胞(SMC)的细胞片，用于将它们分层成心脏补片，以测试其体外功能和在体内整合Ono梗死心壁的能力。我们的假设是，通过改变细胞膜中CM和非CM细胞的比例，并有策略地将它们分层，将产生优化的功能心脏补片。具体地说，我们建议提高HiPSC分化为CMS和非CMS的效率，包括血管内皮细胞和SMC。这些分化的细胞将被生物打印，通过改变CMS和非CMS的细胞比例和分层排列，在体外设计出具有功能的心脏补片。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，以测试未来组织工程心脏补片的功能和体内整合。综上所述，拟议的项目将通过揭示细胞排列和组装的机制，为当前的心肌梗死干细胞治疗提供信息并加以改进，这对形成工程心脏补片至关重要。此外，这项工作承诺未来将通过生物打印其他分子来改进方法学，这些分子用于修复类似的软组织损伤的支架，使用自体成人来源的干细胞。

 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.