*2.7. Statistical Analyses*

Results were expressed as mean ± SEM. Multiple group comparisons were performed by one-way analyses of variance (ANOVA) followed by the Bonferroni procedure for comparison of means. Comparisons between two groups were performed using the unpaired Student's *t*-test. Data were considered statistically significant at a value of *p* ≤ 0.05.

#### **3. Results**

#### *3.1. Improvement of Cardiac Function after Intramyocardial Injection of Human ECFCS into Ischemic Myocardium*

To investigate the regenerative potential of human ECFCs transplanted intramyocardially, 5 <sup>×</sup> 10<sup>5</sup> cells were injected directly after surgical occlusion of the LAD and functional parameters, and histological and immunohistochemical data were collected. To assess the functional parameters, pressure-volume relations of ECFC-treated and saline-treated hearts were measured at day 30 after MI using conductance catheters (Figure 1). Heart rates did not differ significantly between the groups, showing that experimental conditions such as anesthesia did not influence the measurements (data not shown). At day 30 after MI, we detected a significant improvement in cardiac contractility (Figure 1A; dPdt max: 5202.28 ± 316.68 mmHg/s vs. 3896.24 ± 534.95 mmHg/s, *p* = 0.03) in human ECFC-treated mice compared to saline-treated animals. Moreover, compared to saline-treated control animals, human ECFC-treated mice revealed significantly improved stroke work 30 days after MI (CO 3470.70 ± 254.44 μL/min vs. 2006.71 ± 243.18 μL/min; *p* < 0.001, Figure 1B–D). Accordingly, ECFC treatment was associated with improved LV ejection fraction (Figure 1E, LVEF 30.43 ± 1.20% vs. 22.61 ± 1.73%; *p* < 0.001).

**Figure 1.** *Cont.*

E

**Figure 1.** Improved myocardial function after human endothelial colony-forming cells (ECFC) transplantation into ischemic myocardium. Bar graphs representing cardiac output (**A**), contractility (**B**), and ejection fraction (**E**) in saline-treated animals (white bars) and ECFC-treated animals (black bars) 30 days after myocardial infarction (MI). Data represent mean ± SEM; n.s., not significant. Representative pressure volume loops of saline-treated animals (**C**) and ECFC-treated animals (**D**) 30 days after MI.

#### *3.2. Attenuated Infarct Remodeling after Transplantation of Human ECFCS into Ischemic Myocardium*

After functional profiling, we performed histological analysis of explanted hearts at day 30 following MI and cell transplantation. As reported in prior studies [15,19], permanent occlusion of the LAD artery resulted in a typical pattern of injury with transmural involvement of the myocardium in regions supplied by the main branches of the left coronary artery. Histological analyses revealed less pronounced thinning of the LV anterior wall after treatment with ECFCs (0.28 ± 0.08 mm vs. 0.20 ± 0.04 mm; not significant). However, this difference did not reach statistical significance. Infarction size was significantly diminished among human ECFC-treated animals compared to control animals 30 days after MI (50.3 ± 4.5% vs. 66.1 ± 4.3%, *p* < 0.05, Figure 2). Thirty days post-MI, as assessed by human-specific antibodies against HLA and vimentin, we were not able to detect any retained ECFCs (data not shown).

**Figure 2.** Attenuated infarct remodeling after human ECFC transplantation into ischemic myocardium. (**A**) Bar graphs representing the size of infarction (%) in saline-treated animals (white bar) and ECFC-treated animals (black bar) 30 days after MI. (**B**) Bar graphs representing anterior wall thickness (mm) in saline-treated animals (white bar) and ECFC-treated animals (black bar) 30 days after MI. Data represent mean ± SEM; n.s., not significant. (**C**) Representative Masson trichrome staining of infarcted hearts 30 days after MI.

#### *3.3. Increased Neovascularization after Intramyocardial Injection of Human ECFCS into Ischemic Myocardium*

We hypothesized that attenuated cardiac remodeling after transplantation of ECFCs might be a result of cell-induced enhanced neovascularization. Therefore, we performed anti-CD31 immunohistochemistry to analyze the extent of neovascularization in the border zone of animals treated with ECFCs and control animals. Consistent with the smaller infarct size after ECFC therapy, heart sections of these animals revealed a significantly increased capillary density at the infarct border zone compared to the control animals (8.6 ± 0.4 vs. 6.2 ± 0.5, *p* < 0.001, Figure 3) 30 days post-MI. In accordance to these data on enhanced neovascularization after cell transplantation in vivo, we detected increased expression of the proangiogenic transcription factors HIF-1alpha and MMP-2 in ECFCs compared to human umbilical vein endothelial cells (HUVEC) cells assessed by qPCR in vitro (Figure S1A,B).

**Figure 3.** Increased neovascularization after transplantation of human ECFCs into ischemic myocardium. Histograms showing the numbers (**A**) and mean area (**B**) of CD31<sup>+</sup> capillaries at the infarct border zone of saline-treated control animals (white bars) and after ECFC transplantation (black bars) 30 days after MI. Data represent mean ± SEM. (**C**) Representative immunohistochemical staining of CD31 (brown) in infarcted hearts 30 days after MI.

### *3.4. No Enhanced Homing of BM-Derived Progenitor Cells after Human ECFC Injection into Ischemic Myocardium*

Because circulating BMCs cells are known carriers of angiogenic growth factors, we sought to address whether transplantation of ECFCs is able to attract BMCs from the peripheral blood to the ischemic myocardium, thereby facilitating augmented neovascularization. To address this question, we isolated a myocyte-depleted fraction of cardiac cells and performed flow cytometry respectively. In a first step, we were interested in the amount of CD45+/CD34<sup>+</sup> BMCs within the ischemic myocardium. Transplantation of ECFCs was associated with increased number of cardiac homing of CD45+/CD34<sup>+</sup> cells (1.1 ± 0.3% vs. 0.7 ± 0.1%), but the values did not reach statistical significance (Figure 4A,B). Next, we further characterized CD45+/CD34<sup>+</sup> cells utilizing the additional markers CD31, c-kit, Sca-1, CXCR4, Flk-1, LFA-1, and VLA-4. Compared to PBS treated controls, transplantation of ECFCs increased the number of all subfractions without reaching statistical significance (Figure 4C). Interestingly, among the different subfractions, the CD45+/CD34+/Sca-1 and CD45+/CD34+/CXCR4<sup>+</sup> showed the highest enrichment (CD45+/CD34+/Sca-1 <sup>+</sup> 0.8 <sup>±</sup> 0.1% vs. 0.5 <sup>±</sup> 0.1% and CD45+/CD34+/CXCR4<sup>+</sup> 0.6 <sup>±</sup> 0.1% vs. 0.4 ± 0.1%).

**Figure 4.** No enhanced homing of bone marrow (BM)-derived progenitor cells after human ECFC injection into ischemic myocardium. (**A**) Bar graphs representing the percentage of CD45+/CD34<sup>+</sup> stem cells in the ischemic hearts of control animals (white bar) and after ECFC transplantation (black bar) 2 days after MI. Data represent mean ± SEM; n.s., not significant. (**B**) Representative flow cytometry (FACS) analyses of CD45+/CD34<sup>+</sup> cells in the heart of control animals (left) and after ECFC transplantation (right) 2 days after MI. (**C**) Bar graphs representing the percentage of CD45+/CD34<sup>+</sup> subpopulations in the ischemic hearts of control animals (white bars) and after ECFC transplantation (black bars) 2 days after MI. Data represent mean ± SEM;n.s., not significant.

### *3.5. Increased Numbers of Sca-1 Positive Resident Cardiac Stem Cells after ECFCS Injection into Ischemic Myocardium*

In recent years, there has been emerging evidence that the heart contains a reservoir of resident cardiac progenitor cells [21]. These cells are positive for various markers, such as c-kit or Sca-1. We hypothesized that these resident cells may play a role in the repair of the injured heart, i.e., by secretion of angiogenic growth factors and contribution to improved neovascularization. To investigate these cells, we analyzed the fraction of CD45−/CD34− cells within the ischemic myocardium of control and ECFC-treated animals and further characterized cells that additionally expressed c-kit or Sca-1. Transplantation of ECFCs significantly increased the number of CD45−/CD34−/Sca-1<sup>+</sup> progenitor cells 2 days after myocardial ischemia compared to controls (11.70 ± 1.67% vs. 4.47 ± 1.71%, *p* < 0.05). In contrast, no difference in the number of CD45−/CD34−/c-kit<sup>+</sup> could be observed in ECFC-treated compared to control animals (0.33 <sup>±</sup> 0.11% vs. 0.40 ± 0.06%; not significant). Results are depicted in Figure 5.

**Figure 5.** Increased numbers of Sca-1<sup>+</sup> resident cardiac stem cells after ECFC injection into ischemic myocardium. (**A**) Bar graphs representing the percentage of Sca-1<sup>+</sup> resident cardiac stem cells in the ischemic hearts of control animals (white bar) and after ECFC transplantation (black bar) 2 days after MI. Data represent mean <sup>±</sup> SEM. (**B**) Representative FACS analyses of Sca-1<sup>+</sup> resident cardiac stem cells in the heart of control animals (left) and after ECFC transplantation (right) 2 days after MI. (**C**) Bar graphs representing the percentage of c-kit<sup>+</sup> resident cardiac stem cells in the ischemic hearts of control animals (white bar) and after ECFC transplantation (black bar) 2 days after MI. Data represent mean ± SEM; n.s., not significant.
