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For the differentiation of human pluripotent stem cells we used the human embryonic stem cell line HES3-GFP, ubiquitously expressing GFP. Previously, we have shown that co-culture of human embryonic stem cells with a mouse endoderm cell line, END-2, lead to beating cardiomyocytes within 12 days. END-2 cells were treated with mitocmycin C to block proliferation. In general, using this differentiation procedure (in the absence of serum) beating clusters contained 25 % cardiomyocytes[2], [3]. END-2 cells are cultured as a monolayer, whereas differentiated stem cells are grown on top as three-dimensional structures, which allows separation of human embryonic stem cell-derived populations. From undifferentiated human embryonic stem cells and every day during cardiomyocyte differentiation until day 12 samples were collected and used for RNA isolation (n=3).
References:
[2] R. Passier, D. W.-V. Oostwaard, J. Snapper, J. Kloots, R. J. Hassink, E. Kuijk, B. Roelen, A. B. de la Riviere, and C. Mummery, “Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures.,” Stem Cells, vol. 23, no. 6, pp. 772–780, Jun. 2005.
[3] C. Mummery, D. Ward-van Oostwaard, P. Doevendans, R. Spijker, S. van den Brink, R. Hassink, M. van der Heyden, T. Opthof, M. Pera, A. B. de la Riviere, R. Passier, and L. Tertoolen, “Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells.,” Circulation, vol. 107, no. 21, pp. 2733–2740, Jun. 2003.
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