*2.9. Cardiac Magnetic Resonance Analysis*

LVEF was assessed from the cine sequences using the freely available software Segment v2.0 R5165 (Medviso, Lund, Sweden) (http://segment.heiberg.se) [14]. The left ventricular (LV) endocardium in these slices was manually segmented to exclude the papillary muscles. The volumes of these segments were then integrated along the six planes of the LV and LVEF was then calculated from their summated end systolic and end diastolic volumes.

#### *2.10. Statistics*

All data are presented as mean values ± standard deviation (SD). Flow cytometric data and qPCR values are presented as mean values ± standard error of mean. Student's *t*-test was used for statistical analysis of parametric data and the Mann–Whitney test was used for nonparametric analysis of flow cytometric data. Values of *p* < 0.05 were considered statistically significant.

#### **3. Results**

#### *3.1. Cardiac Induced Cells Show Increased Cardiac Markers and Beating Activity During Di*ff*erentiation*

In order to ascertain the differentiation status of the cells, we examined the expression of various markers at the beginning and at day six of differentiation (Figure 3A). We observed the expression of early cardiac markers such as *MesP1*, *Nkx2.5*, and cardiac troponin (*cTnnt2*). They were increased several fold at day six, whereas the expression of the pluripotency marker, *Pou5f1* (also known as *Oct4*), was strongly decreased. This indicates the cardiac lineage commitment of the cells. We also investigated the beating activity of the cells along the differentiation process to determine their functional capability (Figure 3B). In accordance with the strong induction with the induction of the cardiac markers, the cells were robustly beating and the number of beating foci per EB consistently increases over time till day 30 of differentiation. Therefore, these cardiac-induced cells have committed to the cardiac lineage with the potential to form beating cells in the host after transplantation.

**Figure 3.** *Cont*.

*Cells* **2019**, *8*, 1613

**Figure 3.** Characterization of cardiac-induced cells: (**A**) Analysis of mRNA expression after six days of cardiac differentiation shows a decrease of the pluripotency marker *Oct4*, whereas cardiac markers are upregulated. The mRNA expression was normalized to the expression of the house keeping gene *hprt*. Values are expressed as fold increase as compared with day 0. Values are presented as mean ± SEM. (**B**) Starting at day 7 of the cardiac differentiation protocol, beating foci per embryoid bodies (EB) were counted until the end of differentiation (n = 3). Values are presented as mean ± SD. *p*-value was calculated using the student *t*-test.
