Characterization of Cell Subpopulations Expressing Progenitor Cell Markers in Porcine Cardiac Valves

Valvular interstitial cells (VICs) are the main population of cells found in cardiac valves. These resident fibroblastic cells play important roles in maintaining proper valve function, and their dysregulation has been linked to disease progression in humans. Despite the critical functions of VICs, their cellular composition is still not well defined for humans and other mammals. Given the limited availability of healthy human valves and the similarity in valve structure and function between humans and pigs, we characterized porcine VICs (pVICs) based on expression of cell surface proteins and sorted a specific subpopulation of pVICs to study its functions. We found that small percentages of pVICs express the progenitor cell markers ABCG2 (~5%), NG2 (~5%) or SSEA-4 (~7%), whereas another subpopulation (~5%) expresses OB–CDH, a type of cadherin expressed by myofibroblasts or osteo-progenitors. pVICs isolated from either aortic or pulmonary valves express most of these protein markers at similar levels. Interestingly, OB–CDH, NG2 and SSEA-4 all label distinct valvular subpopulations relative to each other; however, NG2 and ABCG2 are co-expressed in the same cells. ABCG2+ cells were further characterized and found to deposit more calcified matrix than ABCG2- cells upon osteogenic induction, suggesting that they may be involved in the development of osteogenic VICs during valve pathology. Cell profiling based on flow cytometry and functional studies with sorted primary cells provide not only new and quantitative information about the cellular composition of porcine cardiac valves, but also contribute to our understanding of how a subpopulation of valvular cells (ABCG2+ cells) may participate in tissue repair and disease progression.瓣膜间质细胞（valvular interstitial cells, VICs）是心脏瓣膜中的主要细胞群。这类定居的成纤维样细胞在维持瓣膜正常功能中发挥关键作用，其功能失调与人类心脏瓣膜疾病的进展密切相关。尽管VICs具有如此关键的生理功能，但目前对于人类及其他哺乳动物的VICs细胞组成仍未得到明确界定。鉴于健康人类瓣膜组织的获取难度有限，且人类与猪的瓣膜结构与功能高度相似，本研究基于细胞表面蛋白表达特征对猪瓣膜间质细胞（porcine VICs, pVICs）进行了表型鉴定，并分选得到特定的pVICs亚群以研究其功能。研究发现，仅少量pVICs表达祖细胞标志物ABCG2（约5%）、NG2（约5%）或SSEA-4（约7%）；另有约5%的pVICs亚群表达OB-CDH——一种由肌成纤维细胞或骨祖细胞表达的钙粘蛋白。从主动脉瓣或肺动脉瓣分离得到的pVICs，其上述多数蛋白标志物的表达水平无显著差异。有趣的是，OB-CDH、NG2与SSEA-4各自标记的瓣膜细胞亚群互不重叠；但NG2与ABCG2可在同一细胞中共表达。本研究进一步对ABCG2阳性细胞进行表型鉴定，结果显示，在成骨诱导条件下，ABCG2阳性细胞较ABCG2阴性细胞沉积更多钙化基质，提示该亚群可能参与瓣膜病理过程中成骨样VICs的形成。本研究基于流式细胞术的细胞分型及原代分选细胞的功能实验，不仅为猪心脏瓣膜的细胞组成提供了全新的定量数据，同时也加深了我们对瓣膜细胞亚群（ABCG2阳性细胞）如何参与组织修复与疾病进展的理解。