Structural remodeling and functional maturation of stem cell-derived cardiomyocytes in novel 3D microprinted scaffolds.

新型 3D 微打印支架中干细胞来源的心肌细胞的结构重塑和功能成熟。

• 批准号: 417543243
• 负责人: Dr. Nina Ullrich, Ph.D.
• 金额: --
• 依托单位: Institut für Physiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/417543243?language=en
• 关键词: Structural; remodeling; functional; maturation; stem

Stem cell-derived cardiomyocytes raise new hope for myocardial repair and may serve to replace diseased cardiomyocytes to compensate for functional loss in the failing heart. At the current degree of differentiation, these cells present a rather immature phenotype, characterized by undefined cellular structure and disordered myofilament arrangement compared to adult cardiomyocytes, inefficient Ca handling and spontaneous contractile activity, strong hallmarks of immaturity, which lead to fragile and instable Ca signaling and favor the development of undesired electrical activities in these cells. The structural and functional deficiencies reminiscent of premature native myocytes limit successful integration into host myocardium and call for new strategies to enhance maturation before these cells may be used in cell therapeutic approaches. The great challenge is to identify the specific requirements and parameters of stem cell-derived cardiomyocytes that trigger further cardiogenic maturation. Here, we propose the hypothesis that myocyte shape influences function, thereby determining cardiac features. In analogy to the shape of adult cardiomyocytes, we have designed novel rectangular-shaped 3D scaffolds for single cell culture to evaluate the influence of a particular cell shape and geometry on cardiomyocyte function. In preliminary experiments, reshaping stem cell-derived cardiomyocytes in cuboid scaffolds induced significant structural reorganization of the myofilaments and enhanced the efficiency of Ca handling. Based on these exciting new data, we propose two causally linked experimental strategies to provide deep insight into the structural and functional changes occurring upon cell growth in specific shapes. In the first aim, we investigate the exact trigger mechanisms that lead to structural remodeling and try to answer to the question whether interaction with scaffold walls (cuboidal growth in 3D) or just cellular reorientation in a rectangular shape with precise long axis formation (growth in 2D) are required to initiate the observed strong remodeling processes. Outside-in signaling analysis may provide further insight into the genetic control of cell architecture and formation of cardiac-specific microdomains, such as t-tubules and intercalated disks. The second part of the project proposal aims to elucidate the mechanisms that lead to enhanced Ca handling by structural remodeling. In particular the hypothesis will be tested that changes in cell microarchitecture influence the interaction of the Ca influx and Ca release pathways that critically control excitation-contraction coupling in stem cell-derived cardiomyocytes. Overall, the major goal of this project is to define the mechanisms that lead to stable and efficient Ca signaling in order to reduce the risk of arrhythmogenic electrical activity in these novel cardiomyocytes and to make them better suited for future clinical applications.

干细胞来源的心肌细胞为心肌修复带来了新的希望，并可能用于取代患病的心肌细胞，以弥补衰竭心脏的功能丧失。在目前的分化程度下，这些细胞呈现出相当不成熟的表型，特征是与成年心肌细胞相比，细胞结构不明，肌丝排列紊乱，钙处理和自发收缩活动效率低下，这是不成熟的强烈特征，导致钙信号脆弱和不稳定，并有利于这些细胞发展不希望看到的电活动。结构和功能的缺陷使人联想到早熟的天然心肌细胞，限制了与宿主心肌的成功整合，并要求在这些细胞用于细胞治疗之前，采取新的策略来促进成熟。最大的挑战是确定干细胞来源的心肌细胞触发进一步心源性成熟的具体要求和参数。在这里，我们提出的假设是，心肌细胞形状影响功能，从而决定心脏特征。类似于成人心肌细胞的形状，我们设计了用于单细胞培养的新型矩形三维支架，以评估特定细胞形状和几何形状对心肌细胞功能的影响。在初步实验中，在立方体支架中重塑干细胞来源的心肌细胞诱导了显著的肌丝结构重组，并提高了钙处理的效率。基于这些令人兴奋的新数据，我们提出了两种因果联系的实验策略，以深入了解特定形状的细胞生长过程中发生的结构和功能变化。在第一个目标中，我们研究导致结构重塑的确切触发机制，并试图回答这样一个问题：启动所观察到的强烈重塑过程是否需要与支架壁(3D中的立方体生长)的相互作用，或者仅仅是具有精确长轴形成的矩形中的细胞重定向(2D中的生长)。从外到内的信号分析可能提供对细胞结构和心脏特异微域形成的遗传控制的进一步洞察，如T管和插入盘。项目建议书的第二部分旨在阐明通过结构重塑提高钙处理能力的机制。特别是，这一假说将被检验，即细胞微结构的变化影响钙内流和钙释放通路的相互作用，而钙内流和钙释放通路是关键控制干细胞来源的心肌细胞兴奋-收缩耦合的途径。总体而言，该项目的主要目标是确定导致稳定和有效的钙信号的机制，以降低这些新的心肌细胞引起心律失常的电活动的风险，并使它们更适合于未来的临床应用。