Human Cardiac Tissue Engineering for Stem Cell-Based Therapeutic Discovery

描述（由申请人提供）：心脏病是美国的主要死亡原因，主要是由于心脏在受伤后无法自我修复。最近的心脏病疗法已经研究了干细胞增强心脏修复和再生的潜力。特别地，间充质干细胞（MSC）已经在体外、体内和临床试验中显示出有益的效果。虽然这项工作的结果令人兴奋，但MSC增强心肌细胞功能的机制仍然未知。了解MSC如何介导其有益作用是开发更安全，更有效的治疗方法的关键。研究MSC生物学的一个挑战是缺乏受控的体外环境，其允许询问MSC生物学的关键机制组分，但仍保持用于临床相关性的高生物保真度。心脏组织工程为研究MSC生物学提供了一种新的方法，因为可以严格控制培养环境，同时保持天然心肌的基本三维结构。使用新生大鼠细胞的工程化心脏组织（ECT）为基础的方法，我们以前已经表明，MSC补充ECTs增强其功能。观察到的有益效果可以反映以下一种或两种：1）MSC通过直接细胞-细胞偶联对组织功能具有内在效果; 2）MSC通过释放有益的旁分泌因子对肌细胞功能具有外在效果。本F30重新提交提案的目的是研究1）MSC的内在和2）胞外作用对增强心肌细胞功能的相对贡献。为了增加临床相关性，所有提出的研究都专门利用来源于人胚胎干细胞（hCM）和人MSC（hMSC）的心肌细胞。将创建三种组织类型：仅hCM、hCM-hMSC杂交和仅hMSC。将通过免疫组织化学分析杂交组织中的闰盘蛋白连接蛋白-43和N-cadherin来检查内在效应。在鉴定细胞连接之后，在组织构建之前将针对两种蛋白质的shRNA转染到MSC中以抑制细胞-细胞连接。然后将评估组织的收缩和电功能以确定直接细胞接触在介导收缩中的贡献。 有益的MSC效应。此外，将在shRNA转染之前和之后评估MSC在hECT内排列细胞的能力。将使用仅含肌细胞的组织与杂合组织或仅含MSC的组织的新型并行培养系统检查外源性效应，以分离无直接细胞接触的旁分泌效应。将对来自混合组织或仅MSC组织的条件培养基进行生化和蛋白质组学分析，以确定负责介导MSC对肌细胞功能有益作用的因素，从而可能确定新型治疗方法。该项目的目的是在临床背景下进行研究，并为未来的临床科学家提供专业培训。

DESCRIPTION (provided by applicant): Cardiac disease is the leading cause of death in the United States, largely due to the inability of the heart to repair itself following injury. Recent therapies for heart disease have investigated the potential of stem cells to enhance cardiac repair and regeneration. In particular, mesenchymal stem cells (MSCs) have shown beneficial effects in vitro, in vivo and in clinical trials. While the results from this work are exciting, th mechanism by which MSCs enhance cardiomyocyte function remains unknown. Understanding how MSCs are mediating their beneficial effect is key to developing safer and more effective therapeutics. One challenge in studying MSC biology is the lack of a controlled in vitro environment that permits interrogation of key mechanistic components of MSC biology yet maintains high biofidelity for clinical relevance. Cardiac tissue engineering provides a novel approach to studying MSC biology since the culture environment can be tightly controlled yet the basic three-dimensional architecture of natural myocardium is maintained. Using an engineered cardiac tissue (ECT)-based approach with neonatal rat cells, we have previously shown that MSC- supplementation to ECTs enhances their function. The observed beneficial effects may reflect one or both of the following: 1) that MSCs are having an intrinsic effect on the tissue function via direct cell-cell coupling; 2) the MSCs are having an extrinsic effect on myocyte function via the release of beneficial paracrine factors. The aims of this F30 resubmission proposal are to investigate the relative contributions of 1) intrinsic and 2) extrinsi effects of MSCs to the enhancement of cardiomyocyte function. To increase clinical relevance, all proposed studies exclusively utilize cardiomyocytes derived from human embryonic stem cells (hCMs), and human MSCs (hMSCs). Three tissue types will be created: hCM-only, hCM-hMSC hybrid, and hMSC-only. Intrinsic effects will be examined via immunohistochemical analysis of the intercalated disk proteins connexin- 43 and N-cadherin in the hybrid tissues. Following identification of cell junctions, shRNA to both proteins will be transfected into MSCs prior to tissue construction to inhibit cell-cell connection. Contractile and electrical function o the tissues will then be assessed to determine the contribution of direct cell contact in mediating the beneficial MSC effects. Additionally, the ability of MSCs to align cells within the hECT will be assessed before and after shRNA transfection. Extrinsic effects will be examined using a novel side-by-side culture system of myocyte-only tissues with either the hybrid or MSC-only tissues to isolate paracrine effects without direct cell contact. Biochemical and proteomic analysis of conditioned media from either hybrid or MSC-only tissues will be performed to identify factors responsible for mediating the MSC beneficial effect on myocyte function, potentially identifying novel therapeutics. The project is designed to both frame the research within a clinical context and provide specialized training of a future clinician-scientist.