Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSC) possess a high potential for regenerative treatment of cardiovascular diseases as well as for drug screening and drug safety tests in pharmaceutical industry. However, one of the main obstacles to overcome is the low efficiency of in vitro cardiac differentiation of human induced pluripotent stem (hiPS) or human embryonic stem (hES) cells. Previous publications provided evidence that transcription factors (TFs) known to be

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... ential for cardiac development could increase cardiomyogenesis upon constitutive (over)expression in mouse ES cells [1], preformed mouse mesoderm [2] and also hES cells [3]. After failure to establish transgenic clones stably expressing multiple TFs, we tested a set of TFs alone and in combination for induction of CMs from hPSCs in a transient approach. Based on transfection by electroporation followed by a serum-free differentiation protocol, our monolayer-based system revealed Baf60c, Gata4 and Mesp1 (BGM) to be the most effective TF combination in hiPS derived from human cord blood endothelial cells (hcBEC-iPS) [4]. While there were no CMs in untransfected or control transfected cells, up to 60 cell clusters per well of BGM transfected cells stained positively for cardiac Troponin T (cTnT), alpha Myosin heavy chain (aMHC) and sarcomeric alpha-actinin although the clusters did not start beating. Removal of Baf60c from the transfection cocktail diminished cardiomyogenesis only marginally while removal of Gata4 or Mesp1 abolished CM formation completely. Each TF alone did not produce any CMs either. Whilst these results are encouraging we would like to note that our experiments revealed a strong sensitivity of the system regarding the changeability of cell densities i.e. induced by variable cell survival post-transfection, subsequent proliferation rates during differentiation, as well as local cell assembly. Especially where cells accumulated at the rim of wells cardiomyogenesis was observed even in controls. Dependence of CM formation on the starting cell number has also been reported in several differentiation systems based on embryoid bodies[5] and monolayers on matrigel [3] highlighting the importance of cell density for paracrine mesoderm and cardiac specification. Taken together, these results highlight the necessity for caution when assessing effects of TFs on directed CM generation in vitro in "monolayer protocols" as demonstrated by us and others.