Abstract
BackgroundThe epicardium, a cell layer covering the heart, plays an important role during cardiogenesis providing cardiovascular cell types and instructive signals, but becomes quiescent during adulthood. Upon cardiac injury the epicardium is activated, which includes induction of a developmental gene program, epithelial-to-mesenchymal transition (EMT) and migration. However, the response of the adult epicardium is suboptimal compared to the active contribution of the fetal epicardium to heart development. To understand the therapeutic value of epicardial-derived cells (EPDCs), a direct comparison of fetal and adult sources is paramount. Such analysis has been hampered by the lack of appropriate culture systems.MethodsHuman fetal and adult EPDCs were isolated from cardiac specimens obtained after informed consent. EPDCs were cultured in the presence of an inhibitor of the TGFβ receptor ALK5. EMT was induced by stimulation with 1 ng/ml TGFβ. PCR, immunofluorescent staining, scratch assay, tube formation assay and RT2-PCR for human EMT genes were performed to functionally characterize and compare fetal and adult EPDCs.ResultsIn this study, a novel protocol is presented that allows efficient isolation of human EPDCs from fetal and adult heart tissue. In vitro, EPDCs maintain epithelial characteristics and undergo EMT upon TGFβ stimulation. Although similar in several aspects, we observed important differences between fetal and adult EPDCs. Fetal and adult cells display equal migration abilities in their epithelial state. However, while TGFβ stimulation enhanced adult EPDC migration, it resulted in a reduced migration in fetal EPDCs. Matrigel assays revealed the ability of adult EPDCs to form tube-like structures, which was absent in fetal cells. Furthermore, we observed that fetal cells progress through EMT faster and undergo spontaneous EMT when TGFβ signaling is not suppressed, indicating that fetal EPDCs more rapidly respond to environmental changes.ConclusionsOur data suggest that fetal and adult EPDCs are in a different state of activation and that their phenotypic plasticity is determined by this activation state. This culture system allows us to establish the cues that determine epicardial activation, behavior, and plasticity and thereby optimize the adult response post-injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0434-9) contains supplementary material, which is available to authorized users.
Highlights
The epicardium, a cell layer covering the heart, plays an important role during cardiogenesis providing cardiovascular cell types and instructive signals, but becomes quiescent during adulthood
Fetal and adult human epicardial-derived cells (EPDCs) maintain their epicardial character in vitro The epicardium is located directly on the myocardium and can be separated from the underlying tissue using forceps (Additional file 1: Figure S1a)
The cells were cultured in medium containing SB431542 (SB) which is a selective inhibitor of the activin receptor-like kinase (ALK5), a type I receptor of transforming growth factor beta (TGFβ)
Summary
The epicardium, a cell layer covering the heart, plays an important role during cardiogenesis providing cardiovascular cell types and instructive signals, but becomes quiescent during adulthood. Upon cardiac injury the epicardium is activated, which includes induction of a developmental gene program, epithelial-tomesenchymal transition (EMT) and migration. To understand the therapeutic value of epicardial-derived cells (EPDCs), a direct comparison of fetal and adult sources is paramount. Such analysis has been hampered by the lack of appropriate culture systems. The epicardium is merely a cell layer covering the myocardium, it is increasingly gaining interest due to its contribution to cardiac development, as well as its potential role in cardiac repair. A subset of epicardial cells undergo epithelial-to-mesenchymal transition (EMT), thereby forming epicardial-derived cells (EPDCs) that migrate into the subepicardial space [1, 2]. Blocking epicardial migration into the myocardium during development leads to dysmorphic hearts, including absence of the apex and reduction of myocardial thickness [11]
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