*3.1. Immunophenotypic Analysis of CD271*<sup>+</sup> *Mesenchymal Stem Cells*

We isolated CD271<sup>+</sup> stem cells according to our established protocol which yields all mesenchymal colony forming progenitors [17,23]. Again, flow cytometric analysis confirmed a mesenchymal phenotype, reflected by significant overexpression of CD73 and CD105 markers in the isolated CD271<sup>+</sup> cell fraction as compared with the entire BM-MNC fraction (9.2% ± 2.8% vs. 0.6% ± 0.2% for CD73 and 8.3% ± 2.8% vs. 0.5% ± 0.2% for CD105, respectively, Figure 2). Moreover, only a subfraction of CD271<sup>+</sup> cells coexpressed all of the mesenchymal markers while being CD45<sup>−</sup> (1.2% <sup>±</sup> 0.3% CD271+CD44+CD73+CD105<sup>+</sup> of CD271<sup>+</sup> cells).

#### *3.2. Induced Ventricular Arrhythmias*

Aiming to develop a murine in vivo model to study human stem cell transplantation, we found that VA are only detectable in the first day after induction of MI. To study engraftment and any alteration in the development of transplanted human cells in a murine model it is necessary to use an immunodeficient mouse strain. The trial of testing the potential antiarrhythmic effects of CD271<sup>+</sup> MSC through catheter-based transjugular intracardial burst stimulation for induction of VA, a described

method by Roell et al. [4], was not promising. We tested six immune compromised mice in which three mice underwent MI and three were healthy animals. All mice of the infarction group and the healthy group developed VA. This observation did not allow for the creation of a proper control group for comparison. Therefore, burst stimulation does not appear to be a suitable approach when using this mouse strain. Consequently, we developed a new mouse model in which we re-induce VA one week after a first ischemia reperfusion by performing a second permanent ligation. This served to simulate the situation in patients where the affected coronary vessel tends to reclose after successful initial recanalization.

In total, one animal (URI group) out of 22 mice died one-hour post induction of the second infarction, due to development of sinus, and, subsequently, third degree heart block. Consequently, this specimen was excluded from the statistical analysis. No other complications were observed after subcutaneous implantation of the telemeter and first LAD ligation, as well as the second LAD ligation.

**Figure 2.** Flow cytometric analysis of MACS-isolated human BM CD271<sup>+</sup> stem cells. The freshly isolated BM derived CD271<sup>+</sup> stem cells showed a mesenchymal identity by a predominant expression of CD73 and CD105 MSC markers as compared with the entire MNC fraction. Mean ± SD, \* *p* ≤ 0.015 (Mann–Whitney *U* Test).

#### *3.3. Induction of Ventricular Arrhythmias after the Reinfarction*

To simplify the study of the mechanisms underlying the developed VA, the ECG analyses were subdivided into an acute phase until 15 min and 15 to 45 min post intervention, as well as a delayed phase after 12 h (Table 1), as previously classified by [24]. There were no significant differences in any of the evolved arrhythmias between the groups after the first infarction. Likewise, quantitatively, there was no observed difference in the frequency of the development of VPB, ventricular salvos, and BG/TG 12 h after the first myocardial insult (Figure 3A).

The quantitative assessment of VPB events within 12 h following the second LAD ligation (URI) revealed a significant difference as compared with the control group MIC (1105.0 ± 1146.72 vs. 7.5 ± 8.98, respectively, Figure 3B). This significant difference is also evident in the time frame between 45 min and 12 h after the permanent infarction (URI 1082.2 ± 1127.77 vs. MIC 3.3 ± 2.42, Figure 3C). There was no significant difference in the occurrence of VPB after the first LAD ligation of both groups (URI 60.1 ± 42.19 vs. MIC 259.0 ± 457.69) for the first 12 h and at the time point between 45 min and 12 h (URI 54.5 ± 39.85 vs. MIC 246.8 ± 440.84).


**Table 1.** Developed ventricular arrhythmias at various time points.

Interestingly, while a significant occurrence of VT after performing the second LAD ligation was identified for URI (32.6 ± 52.5), one week after the onset of the first ligation, none of that was observed in the MIC. This occurred during the first 12 h after the second infarction (Figure 3B) and also in the time frame between 45 min and 12 h after the second infarction (Figure 3C). There was no significant difference in terms of the development of VT after the first infarction (URI 1 ± 2.6 vs. MIC 1.6 ± 1.8).
