Human Cardiac Tissue Engineering for Stem Cell-Based Therapeutic Discovery

用于基于干细胞的治疗发现的人类心脏组织工程

• 批准号: 8925699
• 负责人: Timothy Cashman
• 金额: $ 3.37万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-02 至 2016-07-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8925699
• 关键词: 3-Dimensional; Achievement; Address; Adult; Apoptosis; Architecture; Biochemical; Biochemistry; Biomimetics; Bioreactors; Cardiac; Cardiac Myocytes; Cause of Death; Cell Communication; Cell Proliferation; Cells; Clinical; Clinical Trials; Collagen; Communication; Conditioned Culture Media; Connexin 43; Coronary heart disease; Coupling; Custom; Development; Devices; Drug Formulations; EGF gene; Engineering; Environment; Evaluation; Exhibits; Future; Heart; Heart Diseases; Histocompatibility Testing; Human; Human Engineering; Hybrids; In Vitro; Individual; Inflammation; Injury; Intercalated disc; Intercellular Junctions; Label; Lead; Life; Maps; Mechanics; Mediating; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Microarray Analysis; Modeling; Molecular; Muscle Cells; Muscle function; Myocardial Infarction; Myocardium; N-Cadherin; Natural regeneration; Neonatal; Optics; Paracrine Communication; Phase; Process; Property; Protein Analysis; Protein Microarray Assay; Proteins; Proteomics; Rattus; Regenerative Medicine; Relative (related person); Research; Rodent; Role; Scientist; Side; Signaling Protein; Stem cells; Supplementation; System; TNF gene; Testing; Therapeutic; Time; Tissue Engineering; Tissues; Training; Transfection; United States; Vascular Endothelial Growth Factors; Work; angiogenesis; base; cardiac regeneration; cardiac repair; clinically relevant; design; differential expression; enhancing factor; human embryonic stem cell; improved; in vitro Model; in vivo; knock-down; mortality; novel; novel strategies; novel therapeutic intervention; novel therapeutics; paracrine; polypeptide; public health relevance; release factor; repaired; small hairpin RNA; stem cell biology

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.

描述（由申请人提供）：在美国，心脏病是导致死亡的主要原因，主要是由于心脏损伤后无法自我修复。最近的心脏病治疗已经研究了干细胞增强心脏修复和再生的潜力。特别是，间充质干细胞（MSCs）在体外、体内和临床试验中都显示出有益的作用。虽然这项工作的结果令人兴奋，但MSCs增强心肌细胞功能的机制尚不清楚。了解间充质干细胞如何介导它们的有益作用是开发更安全、更有效的治疗方法的关键。研究间充质干细胞生物学的一个挑战是缺乏受控的体外环境，这种环境既允许对间充质干细胞生物学的关键机制成分进行研究，又能保持临床相关性的高生物保真度。心肌组织工程为研究MSC生物学提供了一种新的途径，因为培养环境可以被严格控制，但天然心肌的基本三维结构得以保持。利用基于工程心脏组织（ECT）的新生大鼠细胞方法，我们之前已经表明，补充MSC-可以增强ECT的功能。观察到的有益作用可能反映了以下一种或两种情况：1)MSCs通过直接的细胞-细胞偶联对组织功能具有内在影响；2) MSCs通过释放有益的旁分泌因子对肌细胞功能有外部影响。本F30再提交提案的目的是研究MSCs的1)内在和2)外在效应对增强心肌细胞功能的相对贡献。为了提高临床相关性，所有拟议的研究都只使用来自人胚胎干细胞（hCMs）和人间充质干细胞（hMSCs）的心肌细胞。将创建三种组织类型：仅hcm、hCM-hMSC杂交和仅hmsc。内在效应将通过杂交组织中插入盘蛋白连接蛋白- 43和n-钙粘蛋白的免疫组织化学分析来检验。在确定细胞连接后，在组织构建之前将这两种蛋白的shRNA转染到MSCs中以抑制细胞间连接。然后对组织的收缩和电功能进行评估，以确定细胞直接接触在介导损伤中的作用