Background: Reproducible and efficient differentiation of pluripotent stem cells (PSCs) to cardiomyocytes (CMs) is essential for their use in regenerative medicine, drug testing and disease modeling. The aim of this study was to evaluate the effect of some previously reported cardiogenic substances on cardiac differentiation of mouse PSCs. Methods: Differentiation was performed by embryoid body (EB)-based method using three different murine PSC lines. The differentiation efficiency was monitored by RT-qPCR, immunocytochemistry and flow cytometry, and the effect mechanistically evaluated by transcriptome analysis of treated EBs. Results: Among the five tested compounds (ascorbic acid, dorsomorphin, cyclic adenosine 3',5'-monophosphate, cardiogenol C, cyclosporin A) only ascorbic acid (AA) exerted a strong and reproducible cardiogenic effect in CGR8 cells which was less consistent in other two PSC lines. AA induced only minor changes in transcriptome of CGR8 cells after administration during the initial two days of differentiation. Cardiospecific genes and transcripts involved in angiogenesis, erythropoiesis and hematopoiesis were up-regulated on day 5 but not on days 2 or 3 of differentiation. The cardiac differentiation efficiency was improved when QS11, a small-molecule synergist of Wnt/β-catenin signaling pathway, was added to cultures after AA-treatment. Conclusion: This study demonstrates that only minor transcriptional changes are sufficient for enhancement of cardiogenesis of murine PSCs by AA and that AA and QS11 exhibit synergistic effects and enhance the efficiency of CM differentiation of murine PSCs.

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To find the relationship between the size of embryoid bodies and the efficiency of pluripotent stem cells differentiation into cardiomyocytes. Methods. Transgenic murine iPSC line AT25 and D3 ESC line αPIG (clone 44) were differentiated into cardiomyocytes in AggreWell plates containing microwells which cause the pluripotent stem cells to aggregate into EBs of an appropriate size. Both cell lines were genetically modified and expressed IRES-flanked enhanced green fluorescent protein (eGFP) under the control of cardiac alpha myosin heavy chain promoter. We applied flow cytometry and fluorescence microscopy to test the efficiency of the differentiation processes. Results. The efficiency of differentiation of embryoid bodies formed from iPSC line AT25 and containing 250 and 1000 cells was found to be lower as compared to embryoid bodies formed of 500 and 750 cells. The number of eGFP+ cells derived from embryoid bodies of 500 cells was 8.5 times higher compared to embryoid bodies of 250 cells (2.86 ± 0.30 % cardiomyocytes per embryoid bodies of 500 cells vs. only 0.34 % cardiomyocytes per embryoid bodies containing 250 cells); the difference was 4.7 times higher in comparison with embryoid bodies formed from 1000 cells. Conclusions. The size of embryoid bodies can affect differentiation of pluripotent stem cells into cardiomyocytes. Among the embryoid bodies formed from 250 to 2000 cells per embryoid body, the highest percentage of eGFP+ cells was obtained from 500-cell embryoid bodies. K e y w o r d s: pluripotent stem cells, induced pluripotent stem cells, embryoid bodies, cardiomyocyte, differentiation.