Generation, Characterization, and Application of Inducible Proliferative Adult Human Epicardium-Derived Cells

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.

Transgenic Immortalization of Human Dermal Fibroblasts Mediated Through the MicroRNA/SIRT1 Pathway

“…Senescence is a poorly understood phenomenon exhibited by non-immortal cells in culture, characterized by an exit from the cell cycle and shortening of telomere length (26). In this study, human dermal fibroblasts (HDFs) were immortalized by using hTERT and SV40 large T antigen, in which both factors could immortalize the cells by controlling the cell cycle and preventing telomere erosion (27)(28)(29). hTERT and hTERC protein direct the elongation and metabolism of telomere, and they are widely implemented to immortalize various types of human cell lines (27,30,31).…”

Conditional immortalization of human cardiomyocytes for translational in vitro modelling of cardiovascular disease

“…Lower panel, generation of human cardiovascular cell types using conditional immortalization technology for the creation of complex human heterocellular cardiac models. Currently, conditionally immortalized lines of human atrial cardiomyocytes, 3 human epicardial cells,9 and human preadipocytes 10 have been generated.…”