Cardiac differentiation at an initial low density of human-induced pluripotent stem cells
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A high density of human-induced pluripotent stem cells (hiPSCs) improves the efficiency of cardiac differentiation, suggesting the existence of indispensable cell-cell interaction signals. The complexity of interactions among cells at high density hinders the understanding of the roles of cell signals. In this study, we determined the minimum cell density that can initiate differentiation to facilitate cell-cell interaction studies. First, we co-induced cardiac differentiation in the presence of the glycogen synthase kinase-3β inhibitor CHIR99021 and activin A at various cell densities. At an initial low density, cells died within a few days in RPMI-based medium. We then investigated the culture conditions required to maintain cell viability. We used a basal medium excluding important components for the maintenance of hiPSC pluripotency, including activin A, basic fibroblast growth factor, and insulin. Supplementation of the basal medium with Rho-associated protein kinase inhibitor and insulin improved cell viability. Interestingly, addition of basic fibroblast growth factor enabled the expression of cardiac markers at the mRNA level but not the protein level. After further modification of the culture conditions, 10% of the cells expressed the cardiac troponin T protein, which is associated with cell contraction. The novel protocol for cardiac differentiation at an initial low cell density can also be used to evaluate high cell density conditions. The findings will facilitate the identification of cell signals required for cardiomyocyte formation.
KeywordsHuman-induced pluripotent stem cells Cardiac differentiation Low cell density Cell-cell interaction
This research was supported by AMED under Grant Number JP17bk0104011h0005 (to A.K. and K.O.) and Japanese government scholarship for foreign students who study in higher education institutions (to M.L.). The funding bodies had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.
M.L. and K.O. designed the project. A.K. established and provided the cell line for the experiments. M.L. performed all experiments. T.M. and K.M. assisted with the experiments. M.L. and K.O. wrote the manuscript, and all authors reviewed the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
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