Microfabricated coculture model: Myocyte rescue by TNT-transferred mitochondria

描述(由申请人提供)：除了少数例外，干细胞注入心脏可以在短期内改善心脏功能，但明显缺乏整合和分化。这种心功能的暂时改善可能是由于旁分泌因素，但其过程尚不清楚。最近，线粒体被观察到通过隧道纳米管(TNTs)转移到心肌细胞，这可能解释了干细胞注射在改善细胞功能方面的瞬时效应。了解干细胞和心肌细胞之间的线粒体转移可能通过直接转移功能性线粒体替代物来挽救衰竭的心肌细胞。值得注意的是，在儿科遗传性线粒体心肌病中，这种转移作为一种治疗方法可能特别有用。在许多环境中都观察到了TNT的形成；然而，使用当前可用的组织切片技术难以捕捉到如此小的结构，使得目前关于TNT形成和功能的知识严重依赖于细胞共培养模型。由于对TNT的形成和功能几乎没有系统的探索，对TNTs所知的只是它们被广泛观察到，而且它们似乎既运输细胞器又运输细胞质分子。系统的研究是困难的，因为在常规的细胞共培养中，在随机分布的细胞之间形成不同长度的多个TNTs。为了克服这一局限性，这里提出了一种微制造共培养模型，在该模型中，心肌细胞和干细胞分别沉积在一个房间的两侧，它们之间有穿孔屏障。由于几何限制，TNT的形成和特定细胞对之间的线粒体转移可以在长度和方向上定义。利用这样的模型，拟议的研究将探索干细胞和心肌细胞之间的TNT通讯过程，并测试模型系统，在该模型系统中，线粒体的交付将提供可衡量的结果改善。潜在的意义在于，基于TNT-线粒体转移的细胞营救是一个内在的靶向过程，而不是传统的基于旁分泌的营救机制。对基本原理的理解可能会导致制定战略，开发靶向疗法来挽救线粒体心肌病，或为衰竭的心脏提供额外的能量。这些研究的具体目的是：1)在微制室共培养模型中确定线粒体转移TNTs的个体发生；2)在体外存活模型中确定形成纳米管或通过纳米管转移线粒体是否介导了心肌细胞的存活功能；3)确定通过TNTs转移线粒体对线粒体遗传性心肌病的拯救作用；以及4)确定通过TNTs转移线粒体或其他材料对心肌梗死的拯救作用。这些目标的实现将为下列问题提供答案：1)所有的TNT是否都传递线粒体，或者是否有一个根据大小或结构可辨别的亚群，促进这种传递？2)TNT的形成是对心肌细胞压力的反应吗？3)线粒体、其他分子或细胞器的转移是共培养中增强心肌细胞存活的媒介吗？4)转移的线粒体是不同的，还是它们与宿主线粒体融合？6)线粒体转移是否利用微管运动分子？7)在线粒体心肌病的遗传模型中，正常的线粒体能否拯救心肌细胞的功能？8)在活体心肌梗死模型中，心功能是否可见相应的可测量变化的可辨别线粒体转移？这些答案将对干细胞疗法具有翻译意义，并将对特定方法的设计和成功提供信息。这些研究还将影响罕见但致命的儿童线粒体肌病。如果确定TN的形成具有功能优势，未来的研究可以解决TNT形成的机制和这一过程的优化，以便临床使用。

DESCRIPTION (provided by applicant): With few exceptions, stem cell injections into the heart produce short-term improvement in cardiac function but a notable lack of integration and differentiation. This temporal improvement in cardiac function may be due to paracrine factors, but the process is not well understood. Recently, mitochondria have been observed to be transferred through tunneling nanotubes (TNTs) into myocytes, which might explain the transient effects of stem cell injections on improved cellular function. An understanding of mitochondrial transfer between stem cells and myocytes might enable rescue of failing cardiomyocytes by directed transfer of functional mitochondrial replacements. Significantly, there are pediatric genetic mitochondrial cardiomyopathies that in which such transfer could be particularly useful as a therapy. TNT formation has been observed in many settings; however, difficulty in capturing such a small structure using currently available tissue-sectioning techniques has made current knowledge of TNT formation and function heavily dependent on cell co-culture models. As there has been little systematic exploration of TNT formation and function, all that is known about TNTs is that they are widely observed and that they appear to transport both organelles and cytoplasmic molecules. Systematic study is difficult because in conventional cell co-culture, multiple TNTs of various lengths form between randomly distributed cells. To overcome this limitation, here it is proposed to develop a microfabricated coculture model, in which cardiomyocytes and stem cells are deposited on respective sides of a chamber with a perforated barrier between them. Due to geometric confinement, TNT formation and mitochondrial transfer between specific cell pairs can be defined in length and orientation. Using such a model, the proposed studies will explore the process of TNT communication between stem cells and myocytes and test model systems in which delivery of mitochondria will provide measurable improvement in outcomes. Of potential significance is that TNT-mitochondria transfer-based cell rescue is an intrinsic targeting process as opposed to conventional paracrine-based rescue mechanisms. An understanding of the underlying principles might lead to the formulation of strategies to develop a targeting therapy to rescue mitochondrial cardiomyopathies or to provide additional energy to failing hearts. The specific aims are 1) Determine ontogeny of mitochondria-transferring TNTs in a microfabricated, compartmental coculture model; 2) Determine whether formation of a nanotube or transfer of mitochondria through the nanotube mediates a myocyte-survival function in an in vitro survival model; 3) Determine the rescue effect of mitochondrial transfer through TNTs on mitochondrial genetic cardiomyopathy; and 4) Determine the rescue effect on cardiac infarct of mitochondrial or other materials transfer through TNTs. Achievement of these aims will provide answers to the following questions: 1) Do all TNTs transmit mitochondria or is there a subset, discernible by size or structure, that facilitates this transfer? 2) Is TNT formation a response to myocyte stress? 3) Is the transfer of mitochondria, other molecules, or organelles the mediator of enhanced myocyte survival in coculture? 4) Do transferred mitochondria remain distinct or do they fuse with host mitochondria? 6) Does mitochondrial transfer utilize microtubular motor molecules? 7) Can normal mitochondria rescue myocyte function in a genetic model of mitochondrial cardiomyopathy? 8) In an in vivo infarct model, can discernible mitochondrial transfer with corresponding measurable changes in cardiac function be seen? These answers will have translational significance for stem cell therapies and will be informative as to design and success of specific approaches. These studies will also impact pediatric mitochondrial myopathies that are rare but fatal. If it is established that there is a functional advantage to TN formation, future studies can address the mechanisms of TNT formation and the optimization of this process for clinical utilization.