Directed differentiation and long-term maintenance of epicardial cells derived from human pluripotent stem cells under fully defined conditions

Abstract

The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to self-renewing epicardial cells in a completely defined, xeno-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate differentiation stage-specific application of Gsk3 inhibitor, Wnt inhibitor, then Gsk3 inhibitor is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 days. Characterization of differentiated cells is performed via flow cytometry and immunostaining to assess quantitative expression and localization of epicardial cell-specific proteins. In vitro differentiation to fibroblasts and smooth muscle cells is also described. In addition, culture in the presence of TGFβ inhibitors allows long-term expansion of hPSC-derived epicardial cells for at least 25 population doublings. Functional human epicardial cells differentiated via this protocol may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications.

Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing

“…The antibiotic resistance PGK‐BSD cassette remained in the Cre reporter lines, which may in some cases (not observed here) affect the endogenous gene expression. To differentiate hPSCs into cardiomyocytes and epicardial cells , hPSCs were dissociated into single cells with Accutase (Innovative Cell Technologies, San Diego, CA, USA) at 37°C for 5 minutes, then seeded onto a Matrigel‐coated 12‐well plate in mTeSR1 with 5 μM Y‐27632 (Selleckchem, Houston, TX, USA) (day −3) for 24 hours. Cells were then cultured and expanded in mTeSR1 for another 2 days.…”

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.…”

Generation of Heart Organoids Modeling Early Human Cardiac Development Under Defined Conditions

“…To increase organoid complexity and produce more developmentally relevant structures, we decided to adapt methods that have been used successfully in monolayer hPSC differentiation (for specific induction of cardiac lineages other than cardiomyocytes) to our hHO system 41,42 . The method consists of a second activation of canonical Wnt signaling on differentiation days 7-9 to induce secondary cardiac lineages (epicardial cells, cardiac fibroblasts).…”