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

项目摘要 迫切需要开发新的心力衰竭治疗方法。新疗法的临床翻译 由于人类心脏的合适模型的有限可用性，使用 人类工程心脏组织（hECT）可用于弥合当前动物模型之间的差距， 提供了人类心肌的物种特异性模型，并且还克服了2D培养的局限性 系统.心脏组织工程领域不断发展，提出了新的策略 用于制造结构和功能成熟的人类心肌模型。这些技术 已经受到用于人诱导多能干细胞分化的方法学优化的青睐， 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 由不同细胞类型，包括心肌细胞， 非肌细胞和细胞外基质本研究的目的是利用 心肌细胞（CM）和非心肌细胞与基质聚合物混合以制造工程化心肌（EngMyo） 具有天然人心肌的功能和结构特性，沿着更好地表示 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， 影响心脏功能。我们假设非肌细胞以适当的比例存在，将导致 与用CM制造的心肌组织相比，增强的功能和结构特征 单独使用，主要是通过激活与心肌细胞发育和细胞增殖相关的信号通路。 周转我们将在两个目标中讨论这个假设。在目标1中，我们将测试收缩性 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，然后使用流式细胞术进行表征， 免疫荧光然后，我们将操纵细胞的类型和数量，这些细胞将被组合以制造 工程心肌（EngMyo），并为每个产生EngMyo，我们将执行功能和结构 特征化我们的中心假设预测，非肌细胞以适当的比例存在， 导致比仅用hCM制造的EngMyo增强的收缩力。我们将确定哪个细胞 组合，提供最大的增强力。在Aim 2中，我们将确定 由非肌细胞的存在激活。我们将在仅用CM制造的EngMyo中进行RNAseq （对照）和用CM和非心肌细胞的组合制造的EngMyo。我们会调查 是差异表达的基因，从这个分析中，我们试图确定激活的信号通路， 非肌细胞的存在。这些发现对于更好地理解非- 心肌细胞的心肌功能，并提供了可靠的和可重复的制造配方 人类心肌模型。

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.