The role of co-culture in the differentiation of iPS cells to cardiomyocytes – the yin and yang of epicardial cells.

November's Hot Topic

Professor Carolyn Carr

It is well-established that iPSC-derived cardiomyocytes are immature, with poorly developed sarcomeres, calcium handling and susbtrate metabolism. Protocols for differentiating pluripotent stem cells to cardiomyocytes have been designed based on knowledge of signalling pathways in the developing heart, as reviewed in detail by Karbassi et al in 2020. Once differentiated, cells can by matured over long-term culture or by providing a more physiological environment such as by applying stretch, electrical pacing or modulating the culture medium. This can be further enhanced by culture in 3D such as in engineered heart tissue or in self-assembling cardiac organoids.

Recent studies have investigated the role of non-myocytes in inducing maturation of iPSC-CM and co-culture with iPS-derived epicardial cells (EpiCs) has been shown to increase expression of electrical, mechanical and sarcomeric proteins. EpiCs are present in the developing mouse heart and differentiate to form epicardial-derived cells (EPDCs) comprising smooth muscle cells, pericytes and fibroblasts. However, elegant work from the Palacek group has shown that the story is complicated (Floy et al 2022). They found that co-culture of differentiating iPS-derived cardiac progenitor cells (CPCs) with EpiCs induced increased expression of MLC2v in the CPCs, but only when EpiCs were held in the epicardial state. Furthermore, coculture with EpiCs induced cardiomyocyte proliferation, as the CPCs were smaller and had less organized sarcomeres. When EpiCs were able to differentiate to EPDCs, the CPCs in co-culture had a slight increase in cTnI+ cells but not cTnT+ or MLC2V+ cells, and no increase in cell proliferation. They found that full co-culture with the EpiCs was necessary as the same effect was not observed in CPCs treated with EpiC-conditioned medium or indirectly cocultured with EpiCs. In addition, using single cell RNA sequencing to compare CPCs and EpiCs in mono-culture or in co-culture, they found that the pathways enriched in the co-culture suggested that the effect of the EpiCs was dominant as they saw decreased structural maturation and increased glycolysis compared with CPCs in mono-culture. Interestingly, they also found that coculture with CPCs was affecting the lineage of the EPiCs.

Clearly further work is required to understand what drives these changes. Interestingly, the winner of the Bernard and Joan Marshall Young Investigator Award in 2017, Johannes Bargehr, presented work showing that co-transplantation of hESC-derived epicardial cells with hESC-cardiomyocytes doubled the proliferation rate of the transplanted cardiomyocytes, thereby increasing graft size, while also increasing graft and host vascularization (Bargehr 2019).


  • Karbassi E, Fenix A, Marchiano S, Muraoka N, Nakamura K, Yang X, Murry CE. Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine. Nat Rev Cardiol. 2020 Jun;17(6):341-359. doi: 10.1038/s41569-019-0331-x. Epub 2020 Feb 3. PMID: 32015528; PMCID: PMC7239749. [LINK]
  • Floy ME, Dunn KK, Mateyka TD, Reichardt IM, Steinberg AB, Palecek SP. Direct coculture of human pluripotent stem cell-derived cardiac progenitor cells with epicardial cells induces cardiomyocyte proliferation and reduces sarcomere organization. J Mol Cell Cardiol. 2022 Jan;162:144-157. doi: 10.1016/j.yjmcc.2021.09.009. Epub 2021 Sep 22. PMID: 34560089; PMCID: PMC8766908. [LINK]
  • Bargehr J, Ong LP, Colzani M, Davaapil H, Hofsteen P, Bhandari S, Gambardella L, Le Novère N, Iyer D, Sampaziotis F, Weinberger F, Bertero A, Leonard A, Bernard WG, Martinson A, Figg N, Regnier M, Bennett MR, Murry CE, Sinha S. Epicardial cells derived from human embryonic stem cells augment cardiomyocyte-driven heart regeneration. Nat Biotechnol. 2019 Aug;37(8):895-906. doi: 10.1038/s41587-019-0197-9. Epub 2019 Aug 2. PMID: 31375810; PMCID: PMC6824587. [LINK]