Stem cell therapy for cardiac repair is a field of regenerative cardiovascular medicine that is emerging as a promising therapeutic alternative to heart transplantation in patients suffering from chronic heart failure. Although promising, challenges to cell therapy for cardiac repair remain, including cell survival, maturation, graft size, revascularisation, and immunogenicity.

It has recently been shown that co-transplantation of human embryonic stem cell (hESC)-derived epicardial cells (EPI) with hESC-derived cardiomyocytes (CM) improves cardiac repair processes with respect to engraftment, cell maturation, and graft and host vascularisation. One plausible explanation for the observed benefits of co-transplantation is paracrine-mediated effects of the epicardium towards CMs and endothelial cells (EC). While RNA sequencing data has been used to identify putative players in hESC-EPIs, little is known about their involvement in mediating hESC-CM maturation and angiogenesis.

Here, the role of paracrine signalling in hESC-EPI-mediated promotion of hESC-CM maturation and angiogenesis is being investigated. To study the relative importance of secreted factors and extracellular vesicles (EVs) a protocol was established to split hESC-EPI conditioned medium into hESC-EPI-EVs and hESC-EPI-flowthrough, which is depleted in EVs.

HESC-EPI-EVs failed to confirm the benefits on CM maturation observed in preliminary studies. Both fractions independently promoted tube formation in HUVECs in vitro and hESC-EPI-flowthrough additionally promoted proliferation and migration. Both fractions improved branching in vivo in chick yolk-sac membrane (YSM) assays. Characterisation of the hESC-EPI-EV-cargo on the protein and miRNA level lead to the identification of pro-angiogenic candidate factors that were screened in vitro and validated in vivo.

Moreover, hESC-EPI-EVs reduced reactive oxygen species (ROS) in hESC-CMs and HUVECs in vitro. Viable mitochondria were transferred between hESC-EPIs and hESC-CMs and HUVECs, which was partly mediated by hESC-EPI-EVs.

Identification of the paracrine signalling factors involved in the hESC-EPI-mediated promotion of CM maturation and revascularisation would make it possible to effectively address these challenges in stem cell therapy for cardiac repair, resulting in new therapeutic approaches.