Table1_Dynamic Epicardial Contribution to Cardiac Interstitial c-Kit and Sca1 Cellular Fractions.docx

Background: The cardiac interstitial cellular fraction is composed of multiple cell types. Some of these cells are known to express some well-known stem cell markers such as c-Kit and Sca1, but they are no longer accepted to be true cardiac stem cells. Although their existence in the cardiac interstitium has not been disputed, their dynamic throughout development, specific embryonic origin, and potential heterogeneity remain unknown. In this study, we hypothesized that both c-KitPOS and Sca1POS cardiac interstitial cell (CIC) subpopulations are related to the Wilms’ tumor 1 (Wt1) epicardial lineage.
Methods: In this study, we have used genetic cell lineage tracing methods, immunohistochemistry, and FACS techniques to characterize cardiac c-KitPOS and Sca1POS cells.
Results: Our data show that approximately 50% of cardiac c-KitPOS cells are derived from the Wt1-lineage at E15.5. This subpopulation decreased along with embryonic development, disappearing from P7 onwards. We found that a large proportion of cardiac c-KitPOS cells express specific markers strongly suggesting they are blood-borne cells. On the contrary, the percentage of Sca1POS cells within the Wt1-lineage increases postnatally. In accordance with these findings, 90% of adult epicardial-derived endothelial cells and 60% of mEFSK4POS cardiac fibroblasts expressed Sca1.
Conclusion: Our study revealed a minor contribution of the Wt1-epicardial lineage to c-KitPOS CIC from embryonic stages to adulthood. Remarkably, a major part of the adult epicardial-derived cell fraction is enriched in Sca1, suggesting that this subpopulation of CICs is heterogeneous from their embryonic origin. The study of this heterogeneity can be instrumental to the development of diagnostic and prognostic tests for the evaluation of cardiac homeostasis and cardiac interstitium response to pathologic stimuli.

背景：心脏间质细胞组分由多种细胞类型构成。其中部分细胞可表达c-Kit、Sca1等经典干细胞标志物，但目前已不再被认定为真正的心脏干细胞。尽管这些细胞存在于心脏间质中这一点尚无争议，但其在发育过程中的动态变化、特定胚胎起源以及潜在的细胞异质性仍未明确。本研究提出假说：c-Kit阳性（c-KitPOS）与Sca1阳性（Sca1POS）心脏间质细胞（cardiac interstitial cell, CIC）亚群均与Wilms肿瘤1（Wilms’ tumor 1, Wt1）心外膜谱系相关。
方法：本研究采用遗传细胞谱系追踪技术、免疫组织化学法以及荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）对心脏c-KitPOS与Sca1POS细胞进行表征分析。
结果：本研究数据显示，在胚胎发育第15.5天（E15.5），约50%的心脏c-KitPOS细胞源自Wt1谱系。该亚群随胚胎发育进程逐渐减少，并于出生后第7天（P7）起完全消失。研究发现，大部分心脏c-KitPOS细胞可表达特定标志物，强烈提示其为血液来源的细胞。与之相反，Wt1谱系内的Sca1POS细胞占比在出生后呈上升趋势。与此结果一致的是，成年心外膜来源的内皮细胞中90%表达Sca1，而60%的mEFSK4阳性（mEFSK4POS）心脏成纤维细胞也表达Sca1。
结论：本研究揭示，从胚胎发育阶段至成年期，Wt1心外膜谱系对c-KitPOS心脏间质细胞（CIC）的贡献占比极低。值得注意的是，成年心外膜来源的细胞组分中，Sca1阳性亚群富集程度极高，这表明该类心脏间质细胞亚群在胚胎起源层面存在异质性。对该异质性的研究可为开发用于评估心脏稳态以及心脏间质对病理刺激应答的诊断与预后检测手段提供重要支撑。