Functional Maturation of Induced Pluripotent Stem Cell Cardiomyocytes (IPS-CMs) via Targeted Mechanical Conditioning and Work

 描述（由申请人提供）：由于缺乏相关的人类细胞类型，心血管研究和再生策略受到了极大的限制。分化为心肌细胞的人类胚胎干细胞（hESC）有可能解决这些缺点，但伦理问题阻碍了它们的广泛应用。最近在体细胞重编程以产生诱导多能干细胞（iPS）方面的突破为使用患者特异性细胞进行再生、研究和药理学测试提供了新的机会。iPS细胞衍生的心肌细胞（iPS cell derived cardiomyocytes，iPS CM）表达许多成体心肌细胞的形态和功能标志物，但由于分化不完全和表观遗传记忆残留，iPS CM的表型仍保持胚胎状态。毫不奇怪，使用iPS-CM的最大挑战是在培养中产生表型成熟的心肌细胞。心脏是一个高度适应性的器官，在发育过程中，协调传递机械应力，如压力，流动和拉伸，直接将单层细胞转化为复杂的4室器官。虽然iPS-CM已经被遗传重编程和分化，但它们在静态培养环境中培养。我们假设iPS-CM完全转化为功能性成年心肌细胞需要在机械应力可以从胚胎水平逐渐增加到成年水平的环境中分化和成熟，然后施加“工作”。为了实现这一点，我们将使用我们的心脏细胞培养模型（CCCM），该模型是作为左心室模拟物开发的，以使心脏细胞在生理相关的环境中培养。该模型代表了心脏细胞培养的最新技术水平，是目前唯一可用的模型，可以准确地模拟与胚胎水平到成人水平相关的机械应力，并能够对培养的细胞施加“功”。在这个提议中，我们寻求建立一个适当的调节机制，使表型成熟的iPS-CM在表型上仍然是胚胎，并通过适当的机械调节和工作施加将它们转化为成年心肌细胞。将广泛评价和表征在CCCM内分化和成熟的iPS-CM，以量化与未刺激的iPS-CM和来自成人肌细胞的公开数据相比的形态和功能差异。通过机械调节和工作施加产生的成人iPS-CM在心血管研究、没有免疫排斥风险的心脏组织再生以及患者特异性药物测试和治疗方面具有巨大潜力。

 DESCRIPTION (provided by applicant): Cardiovascular research and regenerative strategies have been significantly limited by the lack of relevant human cell types. Human embryonic stem cells (hESCs) differentiated into cardiomyocytes have potential to address these shortcomings but ethical concerns have hampered their widespread use. The recent breakthrough in somatic cell reprogramming to generate induced pluripotent stem (iPS) cells offers new opportunities to use patient specific cells for regeneration, research and pharmacological testing. Like cardiomyocytes generated from hESCs, iPS cell derived cardiomyocytes (iPS-CMs) express many morphological and functional markers of adult cardiomyocytes, however, they remain embryonic in phenotype due to incomplete differentiation and residual epigenetic memory. Not surprisingly, the most significant challenge in the use of iPS-CMs has been generating phenotypically mature cardiomyocytes in culture. The heart is a highly adaptive organ and during development, coordinated delivery of mechanical stresses like pressure, flow and stretch direct transformation of a monolayer of cells into a complex 4-chambered organ. While iPS-CMs have been genetically reprogrammed and differentiated, they are cultured in static culture environments. We hypothesize that complete transformation of iPS-CMs into functional adult cardiomyocytes requires differentiation and maturation in an environment where mechanical stresses can be gradually increased from embryonic levels to adult levels followed by imposition of 'work'. To accomplish this we will use our cardiac cell culture model (CCCM) which was developed as a left-ventricle mimic to enable culture of cardiac cells in a physiologically relevan environment. This model represents the state-of-the-art in cardiac cell culture and is the only model currently available that can accurately mimic mechanical stresses associated with embryonic levels to adult levels with the ability to impose 'work' on cultured cells. In this proposal we seek to establish an appropriate conditioning regime to phenotypically mature iPS-CMs that are still embryonic in phenotype and transform them into adult cardiomyocytes via appropriate mechanical conditioning and work imposition. iPS-CMs differentiated and matured within the CCCM will be extensively evaluated and characterized to quantify morphological and functional differences in comparison to both unstimulated iPS-CMs and published data from adult myocytes. Adult iPS-CMs generated via mechanical conditioning and work imposition has great potential for cardiovascular research, cardiac tissue regeneration without the risk of immune rejection, and patient specific drug testing and therapeutics.