Efficient Differentiation of TBX18⁺/WT1⁺Epicardial-Like Cells from Human Pluripotent Stem Cells Using Small Molecular Compounds

Abstract

The epicardium promotes neovascularization and cardiomyocyte regeneration by generating vascular smooth muscle cells (SMCs) and producing regenerative factors after adult heart infarction. It is therefore a potential cell resource for repair of the injured heart. However, the epicardium also participates in fibrosis and scarring of the injured heart, complicating its use in regenerative medicine. In this study, we report coexpression of TBX18 and WT1 in the majority of epicardial cells during mouse embryonic epicardial development. Furthermore, we describe a convenient chemically defined, immunogen-free, small molecule-based method for generating TBX18+/WT1+ epicardial-like cell populations with 80% homogeneity from human pluripotent stem cells by modulation of the WNT and retinoic acid signaling pathways. These epicardial-like cells exhibited characteristic epicardial cell morphology following passaging and differentiation into functional SMCs or cardiac fibroblast-like cells. Our findings add to existing understanding of human epicardial development and provide an efficient and stable method for generating both human epicardial-like cells and SMCs.

BNC1 regulates cell heterogeneity in human pluripotent stem cell-derived epicardium

“…Identifying the molecular signatures of the various epicardial cells may unveil their different functions. Because a better understanding of the epicardium could pave the way to treating the adult heart following injury, we and others have recently developed and validated protocols to generate in vitro models of human developing epicardium from human pluripotent stem cells (hPSC-epi) (Witty et al, 2014;Iyer et al, 2015;Bao et al, 2017;Guadix et al, 2017;Zhao et al, 2017). We hypothesised that analysing gene expression at the single cell level in our system will provide key insights into the molecular and functional regulation of the different human epicardial cell populations.…”

Engineering Myocardium for Heart Regeneration—Advancements, Considerations, and Future Directions

“…This is likely due to the longstanding difficulty of attaining endocardial and epicardial cells. However, the increasing number of protocols for the differentiation of iPSC-derived endocardial (59) and epicardial (60) cells may open the door for studies investigating their inclusion in tissue engineered myocardium. Recapitulation of this architecture through inclusion of an engineered endocardium and epicardium using iPSC-derived cells and a trilayer scaffold could significantly improve the viability, integration, and growth of implanted TEMPs.…”

From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges

“…Orlova and colleagues [ 55 ] also described a method to obtain pericytes after vascular progenitor population specification, by culturing the CD31 − fraction, after sorting, in endothelial cell basal medium followed by medium supplementation with TGFβ3 and PDGF-BB for pericyte commitment. Regarding the generation of vSMCs from pro-epicardial cells ( Figure 4 C), the majority of the protocols are based on the use of a combination of TGFβ1 and FGF2 [ 58 , 59 , 60 ], or TGFβ1 and PDGF-BB [ 61 ], to generate a highly pure population of ACTA2 + /CNN1 + /TAGLN + vSMCs.…”

“…After generating mesoderm progenitor cells (Bry + /CD90 + ) by Wnt signaling activation, this population was exposed to FGF2 for 18 days, thus generating more than 70% CFs (TE-7 + ) ( Figure 4 B). Indirect differentiation protocols, which comprise a pre-differentiation stage of hPSCs into epicardial cells followed by a stage of differentiation into CFs, have been also explored to generate these types of cardiac cells [ 58 , 59 , 60 , 61 ] ( Figure 4 C). For this purpose, the hPSC-derived epicardial cells (>90% WT1 + cells) are exposed to FGF2 for 6–14 days to generate an almost pure population of POSTN + CF-like cells.…”