Microfabricated coculture model: Myocyte rescue by TNT-transferred mitochondria

描述（由申请人提供）：除了少数例外，干细胞注射到心脏中产生心脏功能的短期改善，但明显缺乏整合和分化。这种心脏功能的暂时改善可能是由于旁分泌因素，但这一过程尚未得到很好的理解。最近，已经观察到线粒体通过隧道纳米管（TNT）转移到肌细胞中，这可能解释了干细胞注射对改善细胞功能的瞬时作用。理解干细胞和心肌细胞之间的线粒体转移可能通过功能性线粒体替代物的定向转移来拯救衰竭的心肌细胞。值得注意的是，有儿科遗传线粒体心肌病，其中这种转移可能特别有用的治疗。TNT的形成已被观察到在许多设置;然而，在捕获这样一个小的结构，使用目前可用的组织切片技术的困难，使目前的知识TNT的形成和功能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，关于TNT的所有已知的是，它们被广泛观察到，并且它们似乎可以运输细胞器和细胞质分子。系统研究是困难的，因为在常规细胞共培养中，在随机分布的细胞之间形成多种不同长度的TNT。为了克服这一限制，这里提出了开发一种微制造的共培养模型，其中心肌细胞和干细胞沉积在腔室的相应侧上，在它们之间具有穿孔屏障。由于几何限制，特定细胞对之间的TNT形成和线粒体转移可以在长度和方向上定义。使用这样的模型，拟议的研究将探索干细胞和肌细胞之间的TNT通信过程，并测试模型系统，其中线粒体的递送将提供可测量的结果改善。潜在的意义在于，基于TNT-线粒体转移的细胞拯救是一种内在的靶向过程，而不是传统的基于旁分泌的拯救机制。对基本原理的理解可能会导致制定策略，以开发靶向治疗来挽救线粒体心肌病或为衰竭的心脏提供额外的能量。具体目的是：1）在微制造的隔室共培养模型中确定线粒体转移TNT的个体发生; 2）在体外存活模型中确定纳米管的形成或线粒体通过纳米管的转移是否介导肌细胞存活功能; 3）确定线粒体通过TNT转移对线粒体遗传性心肌病的拯救作用;（4）确定线粒体或其他物质通过TNTs转移对心肌梗死的挽救作用。这些目标的实现将提供以下问题的答案：1）是否所有的TNT传输线粒体或是否有一个子集，可辨别的大小或结构，促进这种转移？2)TNT的形成是对肌细胞应激的反应吗？3)线粒体、其他分子或细胞器的转移是否是共培养中增强心肌细胞存活的介质？4)转移的线粒体保持独特还是与宿主线粒体融合？6)线粒体转移利用微管马达分子吗？7)线粒体心肌病遗传模型中正常线粒体能挽救心肌细胞功能吗？8)在体内梗死模型中，是否可以观察到可辨别的线粒体转移以及相应的可测量的心脏功能变化？这些答案将对干细胞疗法具有转化意义，并将为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科线粒体肌病。如果确定TN形成具有功能优势，则未来的研究可以解决TNT形成的机制以及该过程的优化以供临床使用。

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.