Microfabricated coculture model: Myocyte rescue by TNT-transferred mitochondria

描述（由申请人提供）：除了少数例外，将干细胞注射到心脏可以短期改善心脏功能，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）转移到肌细胞中，这可能解释了干细胞注射对细胞功能改善的短暂效应。了解干细胞和心肌细胞之间的线粒体转移，可以通过定向转移功能性线粒体替代物来拯救衰竭的心肌细胞。值得注意的是，在儿童遗传性线粒体心肌病中，这种转移可能特别有用。在许多环境中都观察到TNT的形成；然而，利用现有的组织切片技术很难捕获如此小的结构，这使得目前对TNT形成和功能的了解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，所有关于TNT的了解都是它们被广泛观察到，并且它们似乎运输细胞器和细胞质分子。由于在常规细胞共培养中，随机分布的细胞之间会形成多个不同长度的tnt，因此很难进行系统的研究。为了克服这一限制，本文提出了一种微制造共培养模型，其中心肌细胞和干细胞分别沉积在腔室的两侧，它们之间有穿孔屏障。由于几何约束，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通讯过程，并测试模型系统，其中线粒体的递送将提供可测量的改善结果。潜在的重要意义是，基于tnt -线粒体转移的细胞救援是一个内在的靶向过程，而不是传统的基于旁分泌的救援机制。对潜在原理的理解可能会导致制定策略，以开发靶向治疗来拯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)确定线粒体转移tnt在微制造的细胞间共培养模型中的个体发生；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心肌细胞存活功能；3)确定通过tnt进行线粒体转移对线粒体遗传性心肌病的拯救作用；4)确定线粒体或其他经tnt转运的物质对心肌梗死的抢救作用。实现这些目标将提供以下问题的答案：1)是否所有的tnt都传递线粒体，或者是否有一个子集，通过大小或结构可识别，促进这种转移？2) TNT的形成是对肌细胞应激的反应吗？3)在共培养中，线粒体、其他分子或细胞器的转移是增强肌细胞存活的介质吗？4)转移的线粒体是保持独立还是与宿主线粒体融合？6)线粒体转移是否利用微管运动分子？在线粒体心肌病遗传模型中，正常线粒体能拯救心肌细胞功能吗？8)在体内梗死模型中，是否可以看到明显的线粒体转移和相应的心功能可测量的变化？这些答案将对干细胞治疗具有翻译意义，并将为特定方法的设计和成功提供信息。这些研究也将影响罕见但致命的儿科线粒体肌病。如果能确定TNT的形成具有功能优势，未来的研究可以解决TNT形成的机制，并优化这一过程，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.