Bioengineering a Living Tissue Conductor

 描述（由申请人提供）：尽管干细胞衍生的心肌细胞用于治疗心脏病非常有前途，但仍面临许多挑战。特别地，涉及多能干细胞的疗法缺乏在体外选择期望的心脏电表型和产生成熟心肌细胞以及在体内控制移植细胞的分化命运及其与宿主心肌细胞的相互作用的方法。总之，这些限制可能使基于干细胞的心脏疗法不仅效率低下，而且致瘤性和致炎性。类似地，最近开发的将心脏成纤维细胞直接转化为心肌细胞的重编程技术存在效率低和可重复性差的问题，并且即使在培养数周后也无法获得人细胞的功能表型。理想情况下，安全有效的基于心肌细胞的治疗应该包括植入同质细胞或工程组织移植物，其功能特性稳定且与周围成年心肌细胞的功能特性相似。因此，我们建议开发一种新的基于细胞的心脏修复的生物工程策略，该策略不依赖于使用干细胞或直接将成纤维细胞重编程为心肌细胞。相反，基于我们对永生化人类细胞系的概念验证研究，我们建议快速有效地将原代人类皮肤成纤维细胞转化为能够与心肌细胞进行动作电位传导和功能耦合的电活性细胞。具体而言，我们建议：1）开发遗传工程算法以稳定地将成人成纤维细胞转化为具有类似于新生或成年大鼠心肌细胞的定制电生理表型的导电细胞，和2）评估注射的电活性成纤维细胞和由这些细胞制成的组织贴片在心肌梗死大鼠模型中的治疗潜力。成功的竞争所提出的研究将使我们能够评估潜在的工程可兴奋体细胞的未来使用在体外实验研究和细胞为基础的心脏治疗在体内。该项目的成果也可能促进心脏病新基因疗法的发展，其中内源性心脏成纤维细胞选择性原位转化为电兴奋和传导细胞可以显着改善受损的心脏功能。

 DESCRIPTION (provided by applicant): Although highly promising, the use of stem cell-derived cardiomyocytes for treatment of heart disease faces a number of challenges. In particular, therapies involving pluripotent stem cells suffer from the lack of methods to select a desired cardiac electrical phenotype and generate mature cardiomyocytes in vitro, as well as control differentiation fate of transplanted cells and their interactions with host cardiomyocytes n vivo. Together, these limitations can render stem cell-based cardiac therapies not only inefficient, but also tumorigenic and arrhythmogenic. Similarly, recently developed reprogramming techniques to directly convert cardiac fibroblasts to cardiomyocytes suffer from low efficiency and reproducibility, as well as the inability to obtain functional phenotype with human cells even after several weeks of culture. Ideally, a safe and efficient cardiac cell-based therapy should involve the implantation of homogeneous cells or engineered tissue grafts with the functional properties that are stable and similar to those of the surrounding adult cardiomyocytes. Thus, we propose to develop a new bioengineering strategy for cell-based cardiac repair that does not rely on the use of stem cells or direct reprogramming of fibroblasts to cardiomyocytes. Rather, building on our proof-of-concept studies with immortalized human cell lines, we propose to rapidly and efficiently convert primary human dermal fibroblasts into electrically active cells capable of action potential conduction and functional coupling with cardiomyocytes. Specifically, we propose to: 1) develop genetic engineering algorithm to stably convert adult human fibroblasts into electrically conducting cells with tailored electrophysiological phenotype resembling that of neonatal or adult rat cardiomyocytes, and 2) evaluate the therapeutic potential of injected electrically active fibroblasts and tissue patches made of these cells in a rat model of myocardial infarction. Successful competition of the proposed studies will allow us to evaluate the potential of engineered excitable somatic cells for future use in experimental studies in vitro and cell-based cardiac therapies in vivo. The outcomes of this project may also promote the development of new gene therapies for heart disease where selective in situ conversion of endogenous cardiac fibroblasts into electrically excitable and conducting cells could significantly improve compromised heart function.