Klein Alyssa, Bayrau Bethel, Miao Yifei, Gu Mingxia
Department of Pediatrics, Division of Cardiology, Stanford School of Medicine; Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford School of Medicine; Stanford Cardiovascular Institute, Stanford School of Medicine.
Department of Pediatrics, Division of Cardiology, Stanford School of Medicine; Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford School of Medicine; Stanford Cardiovascular Institute, Stanford School of Medicine; Division of Pulmonary Biology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center; Center for Stem Cell and Organoid Medicine, CuSTOM, Division of Developmental Biology, and Perinatal Institute, Cincinnati Children's Hospital Medical Center;
J Vis Exp. 2020 Apr 15(158). doi: 10.3791/61126.
It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.
已表明心内膜内皮细胞（EECs）和冠状动脉内皮细胞（CECs）在起源、发育、标志物和功能方面存在差异。因此，这两种细胞群在心脏疾病中发挥着独特作用。目前涉及分离内皮细胞的研究调查的是由EECs和CECs组成的细胞群。本方案概述了一种独立分离这两种细胞群以进行细胞特异性表征的方法。在收集左、右心室游离壁后，使用消化缓冲液分别从外表面和内表面分离内皮细胞。对外表面和内心内膜层的顺序消化保持了这两种内皮细胞群的分离。通过鉴定每个细胞群特有的标志物进一步验证了EECs和CECs的单独分离。基于先前发表的小鼠心脏单细胞RNA谱分析，Npr3、Hapln1和Cdh11基因表达是EECs特有的；而Fabp4、Mgll和Cd36基因表达是CECs特有的。qPCR数据显示这些特征性标志物在各自样本中表达富集，表明成功分离了EECs和CECs，以及维持了细胞表型，从而能够进行进一步的细胞特异性功能分析。
