Functional assessment of donor-host cell coupling

供体-宿主细胞偶联的功能评估

• 批准号: 6921124
• 负责人: MICHAEL RUBART-VAN DER LOHE
• 金额: $ 37.88万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-15 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6921124
• 关键词: cardiac myocytes; cell cell interaction; cell differentiation; confocal scanning microscopy; electrophysiology; embryonic stem cell; green fluorescent proteins; heart function; heart imaging /visualization /scanning; hematopoietic tissue transplantation; immunocytochemistry; laboratory mouse; molecular /cellular imaging; myocardial infarction; regeneration; stem cell transplantation

DESCRIPTION (provided by applicant): Cellular transplantation has emerged as a therapeutic approach to restore lost systolic function to the diseased heart. To date, cellular engraftment approaches have utilized a variety of donor cell types (namely, fetal cardiomyocytes, embryonic stem cell-derived cardiomyocytes, skeletal myoblasts, and adult stem cells). Although cell transfer resulted in an improvement of global cardiac function in a number of studies, in most instances the functional assays employed were unable to distinguish between direct contractions of the donor cells vs. a beneficial effect imparted upon the surviving host myocardium. Accordingly, we have implemented a novel, 2-photon laser scanning microscopy (TPLSM)-based imaging system to monitor cytosolic Ca2+ transients in donor and host cardiomyocytes in situ. This system allows us to assess the level of functional coupling of transplanted cells as well as their effects on the function of recipient cardiomyocytes. In this application we propose to investigate the functional consequences of engrafting 3 cell types that are currently being pursued experimentally and in some cases clinically. Aim 1 will test the ability of fetal cardiomyocytes transplanted at the infarct border zone to functionally couple with the surviving host myocardium. We have recently demonstrated that, using TPLSM imaging of enhanced green fluorescent protein (EGFP)-expressing donor cells, fetal cardiomyocytes transplanted into normal mouse hearts are able to functionally couple with the host myocardium. We will transplant EGFP-expressing fetal cardiomyocytes to the infarct border zone and use TPLSM to determine the degree to which they are able to couple with the host myocardium. Aim 2 will test the hypothesis that skeletal myoblasts can alter electrical properties of the bordering host cardiomyocytes following transplantation into normal or infarcted hearts. EGFP-expressing primary skeletal myoblasts will be transplanted and TPLSM will be used to determine if there is functional coupling between host cardiomyocytes and the skeletal myotubes, and if the presence of myotubes influences the function of bordering host cardiomyocytes. Aim 3 will test the hypothesis that transplanted or mobilized adult stem cells have limited cardiomyogenic differentiation, but can alter the electrical properties of the bordering host cardiomyocytes in normal or infarcted hearts. We will utilize TPLSM and EGFP-expressing bone marrow progenitor cells to monitor the degree of differentiation and functional coupling following transplantation into normal or injured hearts, as well as after mobilization of progenitors following stable bone marrow transplant. In all aims, parallel echocardiographic studies will examine whether functional donor-host coupling as assessed with TPLSM imaging translates into improved global contractile activity of infarcted hearts following cellular transplantation. Additional studies examining the structural consequences of cell transplantation (i.e., angiogenesis, post infarct remodeling, and connexin distribution) will also be performed. The studies proposed here will address fundamental hypotheses regarding the fate of donor cells following cellular transplantation into normal and injured hearts.

描述(由申请人提供)：细胞移植已经成为一种治疗方法，可以恢复患病心脏失去的收缩功能。到目前为止，细胞移植方法已经利用了各种类型的供体细胞(即胎儿心肌细胞、胚胎干细胞来源的心肌细胞、骨骼肌成肌细胞和成人干细胞)。尽管细胞移植在一些研究中导致了整体心功能的改善，但在大多数情况下，所采用的功能分析无法区分供体细胞的直接收缩和对存活的宿主心肌的有利影响。因此，我们实现了一种新型的、基于双光子激光扫描显微镜(TPLSM)的成像系统，用于原位监测供体和宿主心肌细胞的胞浆钙瞬变。该系统使我们能够评估移植细胞的功能偶联水平以及它们对受体心肌细胞功能的影响。在这一应用中，我们建议研究移植3种细胞类型的功能后果，目前正在进行实验和临床研究。目的1检测移植于梗死区的胎儿心肌细胞与存活的宿主心肌的功能偶联能力。我们最近已经证明，使用表达增强型绿色荧光蛋白(EGFP)的供体细胞的TPLSM成像，移植到正常小鼠心脏的胎儿心肌细胞能够与宿主心肌功能偶联。我们将表达EGFP的胎儿心肌细胞移植到梗死边缘区域，并使用TPLSM来确定它们与宿主心肌的偶联程度。目的2将验证这样的假设，即骨骼肌成肌细胞在移植到正常或梗死心脏后，可以改变邻近宿主心肌细胞的电特性。表达EGFP的原代骨骼肌成肌细胞将被移植，TPLSM将用于确定宿主心肌细胞和骨骼肌管之间是否存在功能偶联，以及肌管的存在是否影响宿主心肌细胞的功能。目的3将验证这样一种假设，即移植或动员的成体干细胞具有有限的心肌源性分化，但可以改变正常或梗死心脏中邻近宿主心肌细胞的电特性。我们将利用TPLSM和EGFP表达的骨髓前体细胞来监测移植到正常或受损心脏后的分化程度和功能偶联程度，以及稳定骨髓移植后祖细胞的动员情况。在所有AIMS中，平行的超声心动图研究将检验TPLSM成像评估的功能性供体-宿主偶联是否转化为细胞移植后梗死心脏整体收缩活动的改善。还将进行其他研究，以检验细胞移植的结构后果(即血管生成、梗死后重塑和连接蛋白分布)。这里提出的研究将解决有关细胞移植到正常和受损心脏后供体细胞命运的基本假设。