A Comparative Assessment of Marker Expression Between Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells and the Developing Pig Heart

The course of differentiation of pluripotent stem cells into cardiomyocytes and the intermediate cell types are characterized using molecular markers for different stages of development. These markers have been selected primarily from studies in the mouse and from a limited number of human studies. However, it is not clear how well mouse cardiogenesis compares with human cardiogenesis at the molecular level. We tackle this issue by analyzing and comparing the expression of common cardiomyogenesis markers (PDGFR-α, FLK1, ISL1, NKX2.5, CTNT, CX43 and MYHC-B) in the developing pig heart at embryonic day (E)15, E16, E18, E20, E22 and E24 and in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSC). We found that porcine expression of the mesoderm marker FLK1 and the cardiac progenitor marker ISL1 was in line with our differentiating hiPSC and reported murine expression. The cardiac lineage marker NKX2.5 was expressed at almost all stages in the pig and human iPSC, with an earlier onset in the hiPSC compared to reported murine expression. Markers of immature cardiomyocytes, CTNT and MYHC-B were consistently expressed throughout E16 - E70 in the pig, which is comparable with mouse development, whereas the markers increased over time in the hiPSC. However, the commonly used mature cardiomyocyte marker, CX43, should be used with caution, as it was also expressed in the pig mesoderm, as well as hiPSC immature cardiomyocytes, whilst this has not been reported in mice. Based on our observations in the various species, we suggest to use FLK1/PDGFR-α for identifying cardiac mesoderm, ISL1/NKX2.5 for cardiac progenitors and CTNT/MYHC-B for immature cardiomyocytes. Further, CTNT+/ISL1+ could mark immature cardiomyocytes and CTNT+/ISL1- mature cardiomyocytes. CX43 should be used together with sarcomeric proteins. This knowledge may help improving differentiation of hiPSC into more in vivo like cardiac tissue in the future.