Direct coculture of human pluripotent stem cell-derived cardiac progenitor cells with epicardial cells induces cardiomyocyte proliferation and reduces sarcomere organization.

Epicardial cells (EpiCs) are necessary for myocardium formation, yet little is known about crosstalk between EpiCs and cardiomyocytes (CMs) during development and the potential impact of EpiCs on CM maturation. To investigate the effects of EpiCs on CM commitment and maturation, we differentiated human pluripotent stem cells (hPSCs) to cardiac progenitor cells (CPCs) and EpiCs, and cocultured EpiCs and CPCs for two weeks. When EpiCs were allowed to form epicardial-derived cells, we observed increased expression of cTnI in developing CMs. In the presence of the TGFß inhibitor A83-01, EpiCs remained in the epicardial state and induced CM proliferation, increased MLC2v expression, and led to less organized sarcomeres. These effects were not observed if CPCs were treated with EpiC-conditioned medium or if CPCs were indirectly cocultured with EpiCs. Finally, single cell RNA sequencing identified that EpiC-CPC coculture had bi-directional effects on transcriptional programs in EpiCs and CMs, and biased EpiC lineages from a SFRP2-enriched population to a DLK1- or C3-enriched population. This work suggests important crosstalk between EpiCs and CMs during differentiation which can be used to influence cell fate and improve the ability to generate cardiac cells and tissues for in vitro models and development of cardiac cellular therapies.

Developmental lineage of human pluripotent stem cell-derived cardiac fibroblasts affects their functional phenotype.

Cardiac fibroblasts (CFBs) support heart function by secreting extracellular matrix (ECM) and paracrine factors, respond to stress associated with injury and disease, and therefore are an increasingly important therapeutic target. We describe how developmental lineage of human pluripotent stem cell-derived CFBs, epicardial (EpiC-FB), and second heart field (SHF-FB) impacts transcriptional and functional properties. Both EpiC-FBs and SHF-FBs exhibited CFB transcriptional programs and improved calcium handling in human pluripotent stem cell-derived cardiac tissues. We identified differences including in composition of ECM synthesized, secretion of growth and differentiation factors, and myofibroblast activation potential, with EpiC-FBs exhibiting higher stress-induced activation potential akin to myofibroblasts and SHF-FBs demonstrating higher calcification and mineralization potential. These phenotypic differences suggest that EpiC-FBs have utility in modeling fibrotic diseases while SHF-FBs are a promising source of cells for regenerative therapies. This work directly contrasts regional and developmental specificity of CFBs and informs CFB in vitro model selection.

Human pluripotent stem cell-derived cardiac stromal cells and their applications in regenerative medicine.

Coronary heart disease is one of the leading causes of death in the United States. Recent advances in stem cell biology have led to the development and engineering of human pluripotent stem cell (hPSC)-derived cardiac cells and tissues for application in cellular therapy and cardiotoxicity studies. Initial studies in this area have largely focused on improving differentiation efficiency and maturation states of cardiomyocytes. However, other cell types in the heart, including endothelial and stromal cells, play crucial roles in cardiac development, injury response, and cardiomyocyte function. This review discusses recent advances in differentiation of hPSCs to cardiac stromal cells, identification and classification of cardiac stromal cell types, and application of hPSC-derived cardiac stromal cells and tissues containing these cells in regenerative and drug development applications.