Cardiac differentiation at an initial low density of human-induced pluripotent stem cells

  • Minh Nguyen Tuyet Le
  • Mika Takahi
  • Kenshiro Maruyama
  • Akira Kurisaki
  • Kiyoshi OhnumaEmail author


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.


Human-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.

Author contributions

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.

Supplementary material

11626_2018_276_MOESM1_ESM.docx (514 kb)
ESM 1 (DOCX 513 kb)
Video S1

Application of F7-based media from day 1 to day 3 allowed cardiac expression at the protein level associated with contractile cardiomyocytes. (MP4 3436 kb)

11626_2018_276_MOESM3_ESM.mp4 (7.5 mb)
Video S2 Transient calcium concentration in cardiac differentiation at a low cell density on day 14 was recorded. (MP4 7685 kb)


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Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  1. 1.Department of BioengineeringNagaoka University of TechnologyNagaokaJapan
  2. 2.Department of Science of Technology InnovationNagaoka University of TechnologyNagaokaJapan
  3. 3.Graduate School of Biological SciencesNara Institute of Science and TechnologyIkomaJapan
  4. 4.Biotechnology Research Institute for Drug DiscoveryNational Institute of Advanced Industrial Science and TechnologyIbarakiJapan

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