Functional assessment of donor-host cell coupling

描述（由申请人提供）：细胞移植已成为一种治疗方法来恢复失去的收缩功能的患病心脏。迄今为止，细胞移植方法已经利用了各种供体细胞类型（即胎儿心肌细胞、胚胎干细胞衍生的心肌细胞、成骨肌细胞和成体干细胞）。尽管在许多研究中，细胞移植导致了整体心功能的改善，但在大多数情况下，所采用的功能测定无法区分供体细胞的直接收缩与对存活的宿主心肌的有益影响。因此，我们已经实现了一种新的，基于双光子激光扫描显微镜（TPLSM）的成像系统来监测供体和宿主心肌细胞的细胞质Ca2+瞬态。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。在这个应用中，我们建议研究移植3种细胞类型的功能后果，这些细胞类型目前正在实验中进行，在某些情况下也在临床中进行。目的1将测试在梗死边界区移植的胎儿心肌细胞与存活的宿主心肌功能偶联的能力。我们最近证明，使用TPLSM成像增强绿色荧光蛋白（EGFP）表达供体细胞，胚胎心肌细胞移植到正常小鼠心脏能够与宿主心肌功能偶联。我们将把表达egfp的胎儿心肌细胞移植到梗死边界区，并使用TPLSM来确定它们能够与宿主心肌偶联的程度。目的2将验证骨骼肌母细胞在移植到正常或梗死心脏后可以改变宿主心肌细胞电特性的假设。将移植表达egfp的原代骨骼肌母细胞，并使用TPLSM来确定宿主心肌细胞与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将验证移植或动员的成体干细胞具有有限的心肌分化，但可以改变正常或梗死心脏中邻近宿主心肌细胞的电学特性的假设。我们将利用表达TPLSM和egfp的骨髓祖细胞来监测移植到正常或损伤心脏后的分化程度和功能偶联，以及稳定骨髓移植后祖细胞的动员情况。在所有目的中，平行超声心动图研究将检查TPLSM成像评估的功能性供体-宿主耦合是否转化为细胞移植后梗死心脏整体收缩活性的改善。还将进行进一步的研究，检查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关于供体细胞移植到正常和受伤心脏后的命运的基本假设。

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.