Abstract
Rationale: In recent decades, the great potential of human epicardium-derived cells (EPDCs) as an endogenous cell source for cardiac regeneration has been recognized. The limited availability and low proliferation capacity of primary human EPDCs and phenotypic differences between EPDCs obtained from different individuals hampers their reproducible use for experimental studies. Aim: To generate and characterize inducible proliferative adult human EPDCs for use in fundamental and applied research. Methods and results: Inducible proliferation of human EPDCs was achieved by doxycycline-controlled expression of simian virus 40 large T antigen (LT) with a repressor-based lentiviral Tet-On system. In the presence of doxycycline, these inducible EPDCs (iEPDCs) displayed high and long-term proliferation capacity. After doxycycline removal, LT expression ceased and the iEPDCs regained their cuboidal epithelial morphology. Similar to primary EPDCs, iEPDCs underwent an epithelial-to-mesenchymal transition (EMT) after stimulation with transforming growth factor β3. This was confirmed by reverse transcription-quantitative polymerase chain reaction analysis of epithelial and mesenchymal marker gene expression and (immuno) cytochemical staining. Collagen gel-based cell invasion assays demonstrated that mesenchymal iEPDCs, like primary EPDCs, possess increased invasion and migration capacities as compared to their epithelial counterparts. Mesenchymal iEPDCs co-cultured with sympathetic ganglia stimulated neurite outgrowth similarly to primary EPDCs. Conclusion: Using an inducible LT expression system, inducible proliferative adult human EPDCs were generated displaying high proliferative capacity in the presence of doxycycline. These iEPDCs maintain essential epicardial characteristics with respect to morphology, EMT ability, and paracrine signaling following doxycycline removal. This renders iEPDCs a highly useful new in vitro model for studying human epicardial properties.
Highlights
The cardiac outer layer, or epicardium, is composed of multifunctional and multipotent cells with important roles during fetal development [1,2,3]
Primary adult human epicardium-derived cells (EPDCs) at PD3 were transduced with an lentiviral vector (LV) mediating Doxdependent expression of wild type large T antigen (LT) to generate a polyclonal line of EPDCs with inducible proliferation capacity (Supplemental Figure S1)
Immunofluorescence staining of primary and transduced EPDCs that were cultured in the presence of Dox showed LT expression in the transduced EPDCs, while no LT was detected in the primary EPDCs (Figure 1A)
Summary
The cardiac outer layer, or epicardium, is composed of multifunctional and multipotent cells with important roles during fetal development [1,2,3]. Studies in animal models have shown that epicardial cells become activated in response to pathological triggers, such as myocardial infarction [4,5,6]. Epicardial cells adopt a cuboidal appearance, undergo epithelial-to-mesenchymal transition (EMT), migrate into the subepicardial space, and subsequently into the myocardium [4]. Epicardial cells are referred to as epicardium-derived cells (EPDCs). These EPDCs can differentiate into coronary smooth muscle cells and cardiac fibroblasts [5,7]. EPDCs have strong paracrine effects, and factors secreted by EPDCs have been shown to promote angiogenesis, reduce infarct size, and improve cardiac function [8]
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