Defining the role of non-myocytes to achieve biologically relevant engineered myocardial tissues

项目摘要 迫切需要开发新的治疗心力衰竭的方法。新疗法的临床翻译 对于心脏病来说，合适的人体心脏模型的可获得性有限，这是一个障碍。对.的使用 人类工程心脏组织(HECT)可以弥补现有动物模型之间的差距， 提供了一种特定物种的人类心肌模型，并克服了2D培养的局限性 系统。随着新策略的提出，心脏组织工程领域正在不断发展。 用于制作结构和功能成熟的人心肌模型。这些技术 已被用于人类诱导多能干细胞分化的方法学优化所青睐 细胞(HiPSC)分化为心肌细胞和非心肌细胞类型。健康的心脏发育和功能是 由不同类型细胞之间相互作用的复杂网络支持，包括心肌细胞， 非心肌细胞和细胞外基质。这项研究的目标是利用 心肌细胞与非心肌细胞与基质聚合物混合构建工程化心肌(EngMyo) 具有天然人类心肌的功能和结构特性，以及更好的表达 它的细胞环境，长期目标是使用这些作为体外模型来测试新的疗法，可以 影响心脏功能。我们假设，在适当的比例中非心肌细胞的存在将导致 与CM构建的心肌组织相比，增强的功能和结构特征 单独，主要通过激活与心肌细胞发育和细胞相关的信号通路 营业额。我们将从两个方面阐述这一假说。在目标1中，我们将测试收缩能力的预测 在3D中生长的心肌细胞受到与非心肌细胞的串扰的影响。首先，我们将区分HiPSC CM、成纤维细胞、内皮细胞和平滑肌细胞，然后用流式细胞仪和 免疫荧光。然后我们将操作将被组合以制造的细胞的类型和数量 工程化心肌(EngMyo)，对于每个产生的EngMyo，我们将执行功能和结构 人物刻画。我们的中心假设预测，非心肌细胞的存在在适当的比例下，将 结果表明，与单纯使用HCM制备的EngMyo相比，EngMyo的收缩力有所增强。我们会确定哪一个是牢房 这一组合提供了最大的有效增强。在AIM2中，我们将确定分子途径 通过非心肌细胞的存在而激活。我们将在仅使用CM制造的EngMyo中执行RNAseq (对照)和由CMS和非心肌细胞组合而成的EngMyo。我们会调查哪一位 是差异表达的基因吗？从这个分析中，我们试图确定激活的信号通路 通过非心肌细胞的存在。这些发现将对更好地理解非政府组织的作用具有重要价值 为心肌细胞在心肌功能方面的研究提供了可靠、重复性好的配方 人体心肌模型。

Project Summary There is an urgent need to develop novel treatments for heart failure. The clinical translation of novel therapies for cardiac disease is hindered by the limited availability of suitable models of the human heart. The use of human engineered cardiac tissues (hECTs) can serve to bridge the gap between current animal models, providing a species-specific model of human myocardium, and also overcomes limitations of the 2D culture systems. The field of cardiac tissue engineering is constantly evolving, with the proposition of novel strategies for the fabrication of a structurally and functionally mature model of human myocardium. These technologies have been favored by the optimization of methodologies for the differentiation of human induced pluripotent stem cells (hiPSC) into cardiomyocytes and non-myocyte cell types. Healthy cardiac development and function is supported by a complex network of interactions between diverse cell types, including cardiomyocytes, nonmyocytes, and the extracellular matrix. The objective of this study is to utilize a combination of cardiomyocytes (CM) and nonmyocytes with a matrix polymer mix to fabricate engineered myocardium (EngMyo) with functional and structural properties of the native human myocardium, along with a better representation of its cellular milieu, with the long-term goal of using these as in vitro models to test novel therapies that could impact cardiac function. We hypothesize that the presence of non-myocytes in the proper ratios, will result in enhanced functional and structural characteristics when compared to myocardial tissues fabricated with CM alone, mainly through the activation of signaling pathways associated with cardiomyocyte development and cell turnover. We will address this hypothesis in two aims. In Aim 1, we will test the prediction that the contractility of cardiomyocytes grown in 3D is impacted by crosstalk with non-myocyte cells. First, we will differentiate hiPSC into CM, fibroblasts, endothelial, and smooth muscle cells, followed by characterization using flow cytometry and immunofluorescence. Then we will manipulate the type and number of cells that will be combined to fabricate engineered myocardium (EngMyo), and for each resulting EngMyo we will perform functional and structural characterization. Our central hypothesis predicts that the presence of non-myocytes in the proper ratios, will result in enhanced contractile force than EngMyo fabricated with hCM alone. We will determine which is the cell combination that provides the largest enhancement in force. In Aim2 we will identify the molecular pathways activated by the presence of non-myocytes. We will perform RNAseq in the EngMyo fabricated with CM-only (control) and EngMyo fabricated with combination of CMs and non-cardiomyocyte cells. We will investigate which are the differentially expressed genes and from this analysis we seek to identify the signaling pathways activated by the presence of non-myocytes. These findings will be of great value to better understand the role of non- myocytes in myocardial function and provide with the formulation for the fabrication of reliable and reproducible models of human myocardium.