Probes and sequences of primers used in quantitative RT-PCR.

<p>Probes and sequences of primers used in quantitative RT-PCR.</p

Electrophysiological characteristics of differentiated cardiomyocytes.

<p>Beating frequency (A) and duration of peak width (B) analyses by Ca²⁺ flux in formed 5EB and 8EB cardiomyocytes on day 20 of differentiation. Data are expressed as mean ± SEM (n = a minimum of 4). Differences between samples were analyzed by paired T-test and considered statistically significant for p ≤ 0.05; they are marked with asterisks. Representative action potential recordings from cells stimulated with suprathreshold current pulses (0.5 ms, 4–10 nA) at 1 Hz; a cells with atrial (C) and ventricular (D) action potential morphology. In the majority of cells, we did not observe any spontaneous electrical activity during 20-s recordings (E; upper panel); occasionally, a single spontaneous AP appeared during this period (E; lower panel); <i>V</i>_m−membrane voltage. Whole cell membrane current during a 5-s ramp pulse at voltages between -110 and +40 mV (F); <i>I</i>_m−membrane current.</p

The acceleration of cardiomyogenesis in embryonic stem cells <i>in vitro</i> by serum depletion does not increase the number of developed cardiomyocytes - Fig 2

<p>Gene expressions of transcriptional factor Nkx2.5, Actn2, Myh6, Myh7, Myl2 and Myl7, recognized as markers of cardiomyocyte differentiation in mouse ES R1 cells that were differentiated for 20 days <i>in vitro</i> and primarily cultivated in the form of EBs under non-adherent conditions for 5 days or 8 days (A). The ratios of Myls expression to Nkx2.5 expression are also shown (B). Data are expressed as mean ± SEM (minimum n = 4). Differences between samples were analyzed by paired T-test and considered statistically significant for p ≤ 0.05; they are marked with asterisks.</p

The acceleration of cardiomyogenesis in embryonic stem cells <i>in vitro</i> by serum depletion does not increase the number of developed cardiomyocytes - Fig 4

<p>A number of viable cardiomyocytes determined as the level of ATP in the HG8 clone of the R1 ES cell (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173140#sec002" target="_blank">methods</a>) ratio in the culture derived from 5- and 8-day old EBs (A and B). Selection of cardiomyocytes by G418 antibiotics started on days 14 (Days of selection: 14–20) and 20 (Days of selection: 20–26) of differentiation, and continued for 6 days in both cases. Data are expressed as mean ± SEM (n = 4). Differences between samples were analyzed by paired T-test and considered statistically significant for p ≤ 0.05; they are marked with asterisks. The purity of the selected cardiomyocytes is also shown. Representative picture of non-selected (C, D) and selected (E, F) cardiomyocytes stained using the anti-MHC antibody MF20 (C, E) on day 20 of differentiation. Cell nuclei are counterstained by DAPI (D, F). Scale bar = 100 μm.</p

Flow-cytometric analysis of the Nkx2.5-GFP positive cell ratio in cultures derived from 5- and 8-day-old EBs.

<p>Samples were analyzed on days 8, 15 and 20 of differentiation (A). Number of cardiomyocytes formed per one EB (400 cells per EB were seeded) on days 15 and 20 of differentiation (B, C). Data are expressed as mean ± SEM (n = 4). Differences between samples were analyzed by ANOVA with Bonferroni's Multiple Comparison Test (A) and paired T-test (B, C) and considered statistically significant for p ≤ 0.05; they are marked with asterisks.</p

The experimental setup employed for comparison of the effects of adhesion, FBS supplementation, and NAC supplementation analyzed by determination of the expression of cardiomyogenic markers at the end of the differentiation experiment (8 days).

<p>Supplementation was altered for the last 3 days of cultivation under adherent or non-adherent conditions for EBs cultivation, after 5 days of cultivation in the form of EBs in the presence of FBS (A). Gene expressions of transcriptional factor Nkx2.5, Actn2, Myh6, and Myh7, recognized as markers of cardiomyocyte differentiation–data are presented as the effect of individual variables: the adhesion of EBs on days 5 or 8 of differentiation (B), the presence (S) or absence (SF) of FBS (C), and the presence ((+)NAC) or absence ((-)NAC) of N-Acetyl Cysteine (D). Data are expressed as mean ± SEM (n = 4). Differences between samples were analyzed by Kruskal-Wallis test and post-hoc Dunns test and considered statistically significant for p ≤ 0.05; they are marked with asterisks.</p

Sequence of primers used in quantitative RT-PCR.

<p>Sequence of primers used in quantitative RT-PCR.</p