Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells

Authors

  • Xiu Qin Xu,

    Corresponding author
    1. ES Cell International Pte Ltd., 60 Biopolis St #01-03 Genome, Singapore 138672, Republic of Singapore
      Tel: +65 6774 9533
      Fax: +65 6774 5077
    2. Institute of Medical Biology (IMB) 8A Biomedical Grove # 06-06 Immunos, Singapore 138648, Republic of Singapore
    Search for more papers by this author
  • Ralph Graichen,

    1. ES Cell International Pte Ltd., 60 Biopolis St #01-03 Genome, Singapore 138672, Republic of Singapore
      Tel: +65 6774 9533
      Fax: +65 6774 5077
    Search for more papers by this author
  • Set Yen Soo,

    1. ES Cell International Pte Ltd., 60 Biopolis St #01-03 Genome, Singapore 138672, Republic of Singapore
      Tel: +65 6774 9533
      Fax: +65 6774 5077
    2. Institute of Medical Biology (IMB) 8A Biomedical Grove # 06-06 Immunos, Singapore 138648, Republic of Singapore
    Search for more papers by this author
  • Thavamalar Balakrishnan,

    1. ES Cell International Pte Ltd., 60 Biopolis St #01-03 Genome, Singapore 138672, Republic of Singapore
      Tel: +65 6774 9533
      Fax: +65 6774 5077
    2. Institute of Medical Biology (IMB) 8A Biomedical Grove # 06-06 Immunos, Singapore 138648, Republic of Singapore
    Search for more papers by this author
  • Siti Norfiza Bte Rahmat,

    1. ES Cell International Pte Ltd., 60 Biopolis St #01-03 Genome, Singapore 138672, Republic of Singapore
      Tel: +65 6774 9533
      Fax: +65 6774 5077
    2. Institute of Medical Biology (IMB) 8A Biomedical Grove # 06-06 Immunos, Singapore 138648, Republic of Singapore
    Search for more papers by this author
  • Shirly Sieh,

    1. ES Cell International Pte Ltd., 60 Biopolis St #01-03 Genome, Singapore 138672, Republic of Singapore
      Tel: +65 6774 9533
      Fax: +65 6774 5077
    Search for more papers by this author
  • Su Chin Tham,

    1. ES Cell International Pte Ltd., 60 Biopolis St #01-03 Genome, Singapore 138672, Republic of Singapore
      Tel: +65 6774 9533
      Fax: +65 6774 5077
    Search for more papers by this author
  • Christian Freund,

    1. Hubrecht Institute, Uppsalalaan 8, Utrecht 3584CT, The Netherlands
    Search for more papers by this author
  • Jennifer Moore,

    1. Hubrecht Institute, Uppsalalaan 8, Utrecht 3584CT, The Netherlands
    Search for more papers by this author
  • Christine Mummery,

    1. Hubrecht Institute, Uppsalalaan 8, Utrecht 3584CT, The Netherlands
    Search for more papers by this author
  • Alan Colman,

    1. ES Cell International Pte Ltd., 60 Biopolis St #01-03 Genome, Singapore 138672, Republic of Singapore
      Tel: +65 6774 9533
      Fax: +65 6774 5077
    2. Institute of Medical Biology (IMB) 8A Biomedical Grove # 06-06 Immunos, Singapore 138648, Republic of Singapore
    Search for more papers by this author
  • Robert Zweigerdt,

    1. ES Cell International Pte Ltd., 60 Biopolis St #01-03 Genome, Singapore 138672, Republic of Singapore
      Tel: +65 6774 9533
      Fax: +65 6774 5077
    2. Institute of Medical Biology (IMB) 8A Biomedical Grove # 06-06 Immunos, Singapore 138648, Republic of Singapore
    Search for more papers by this author
  • Bruce P. Davidson

    1. ES Cell International Pte Ltd., 60 Biopolis St #01-03 Genome, Singapore 138672, Republic of Singapore
      Tel: +65 6774 9533
      Fax: +65 6774 5077
    Search for more papers by this author

✉ E-mail: joya.xu@imb.a-star.edu.sg

Abstract

Abstract Many applications of human embryonic stem cells (hESCs) will require fully defined growth and differentiation conditions including media devoid of fetal calf serum. To identify factors that control lineage differentiation we have analyzed a serum-free (SF) medium conditioned by the cell line END2, which efficiently induces hESCs to form cardiomyocytes. Firstly, we noted that insulin, a commonly used medium supplement, acted as a potent inhibitor of cardiomyogenesis in multiple hESC lines and was rapidly cleared by medium conditioning. In the presence of insulin or IGF-1, which also suppressed cardiomyocyte differentiation, the PI3/Akt pathway was activated in undifferentiated hESC, suggesting that insulin/IGF-1 effects were mediated by this signaling cascade. Time course analysis and quantitative RT-PCR revealed impaired expression of endoderm and mesoderm markers in the presence of insulin, particularly if added during early stages of hESC differentiation. Relatively high levels of the neural ectoderm marker Sox1 were expressed under these conditions. Secondly, comparative gene expression showed that two key enzymes in the prostaglandin I2 (PGI2) synthesis pathway were highly up-regulated in END2 cells compared with a related, but non-cardiogenic, cell line. Biochemical analysis confirmed 6–10-fold higher PGI2 levels in END2 cell-conditioned medium (END2-CM) vs. controls. Optimized concentrations of PGI2 in a fully synthetic, insulin-free medium resulted in a cardiogenic activity equivalent to END2-CM. Addition of the p38 mitogen-activated protein kinase-inhibitor SB203580, which we have shown previously to enhance hESC cardiomyogenesis, to these insulin-free and serum-free conditions resulted in a cardiomyocyte content of >10% in differentiated cultures without any preselection. This study represents a significant step toward developing scalable production for cardiomyocytes from hESC using clinically compliant reagents compatible with Good Manufacturing Practice.

Ancillary