Cell Surface Proteins in Human Cardiomyocytes

 描述（由申请人提供）：从混合细胞群中分离充分表征的人诱导多能干细胞衍生的心肌细胞（iCM）的能力是心脏再生医学、疾病建模和药物发现工作的基本和主要未实现的目标。本提案的总体目标是开发一种临床适用的方法，用于高通量分离纯化的心室iCM，这是最受欢迎的细胞之一。 用于翻译应用程序的类型。分离策略基于免疫表型，其中通过免疫细胞化学方法实现临床相关细胞的功能潜力、身份和分离。 识别暴露的表面标记的抗体组。我们的初步研究已经确定了超过40个信息细胞表面标志物，为此，我们已经开发了一种新的单克隆抗体，以前没有在心脏中描述的一种新的细胞表面蛋白。我们已经确定，这些标记物中的几个仅限于心脏中的特定腔室，这表明它们将为体外分选亚型特异性iCM提供高度信息。这项工作将在两个目标中进行，旨在测试细胞表面蛋白抗体可用于分离心室iCM（目标1）的假设，并确定其在心室心肌发生中的功能作用（目标2）。这项工作将有助于一个新的，非转基因为基础的高通量方法分离活心室心肌细胞，这是不可能的，通过目前的方法。该提议的意义在于技术发展所实现的功能结果，因为当开发用于分选阶段和亚型特异性细胞的信息标记物组时，这将使得能够可再现地分离适合于药物发现、治疗和疾病建模研究的心室iCM。

 DESCRIPTION (provided by applicant): The ability to isolate well-characterized human induced pluripotent stem cell derived cardiomyocytes (iCM) from mixed cell populations is a fundamental and major unfulfilled goal of cardiac regenerative medicine, disease modeling and drug discovery efforts. The overall goal of this proposal is to develop a clinically- applicable method for high-throughput isolation of purified ventricular iCM, one of the most sought after cell types for translational applications. The isolation strategy is based on immunophenotyping, wherein functional potential, identity, and isolation of clinically-relevant cells are achieved via panels of antibodies that recognize exposed surface markers. Our preliminary studies have identified more than 40 informative cell surface markers for this purpose and we have developed novel monoclonal antibodies to one novel cell surface protein not previously described in the heart. We have already determined that several of these markers are restricted to specific chambers in the heart, indicating they will be highly informative for sorting subtype-specific iCM in vitro. The work will be carried out in two Aims designed to test the hypothesis that antibodies to cell surface proteins can be used to isolate ventricular iCM (Aim 1) and to determine their functional role in ventricular cardiomyogenesis (Aim 2). This work will contribute a new, non-transgene based high-throughput method for isolating live ventricular cardiomyocytes, which is not possible by current methodologies. The significance of this proposal lies with the functional outcomes enabled by the technological developments, as when an informative marker panel for sorting stage and subtype specific cells is developed, this will enable the reproducible isolation of ventricular iCM suitable for drug-discovery, therapeutic, and disease modeling studies.