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

The murine developing epicardium heterogeneously expresses the transcription factors TCF21 and WT1. Here, we show that this cell heterogeneity is conserved in human epicardium, regulated by BNC1 and associated with cell fate and function. Single cell RNA sequencing of epicardium derived from human pluripotent stem cells (hPSC-epi) revealed that distinct epicardial subpopulations are defined by high levels of expression for the transcription factors BNC1 or TCF21. WT1⁺ cells are included in the BNC1⁺ population, which was confirmed in human foetal hearts. THY1 emerged as a membrane marker of the TCF21 population. We show that THY1⁺ cells can differentiate into cardiac fibroblasts (CFs) and smooth muscle cells (SMCs), whereas THY1^- cells were predominantly restricted to SMCs. Knocking down BNC1 during the establishment of the epicardial populations resulted in a homogeneous, predominantly TCF21^high population. Network inference methods using transcriptomic data from the different cell lineages derived from the hPSC-epi delivered a core transcriptional network organised around WT1, TCF21 and BNC1. This study unveils a list of epicardial regulators and is a step towards engineering subpopulations of epicardial cells with selective biological activities.