*2.2. Animal Model*

For this study, we used 8–12 weeks old male severe combined immunodeficiency (SCID)beige mice (Charles River, Wilmington, MA, USA), which were kept under pathogen-free conditions. MI was induced by surgical occlusion of the left anterior descending artery (LAD) through a left anterolateral approach as described previously [15,16]. Briefly, animals were anesthetized with a mixture of 100 mg/kg ketamine (Sigma, St. Louis, MO, USA) and 5 mg/kg xylazine (Sigma) intraperitoneally. Subsequently, they were intubated and artificially ventilated with room air at 200 breaths/min using a mouse ventilator (HUGO SACHS, March-Hugstetten, Germany). A left anterolateral thoracotomy was performed, and MI was induced by surgical occlusion of the left anterior descending artery (LAD) with an 8-0 Prolene suture. Animal care and all experimental procedures were performed in strict accordance with the German and National Institutes of Health animal legislation guidelines and were approved by the local animal care committees (AZ 209.1/211-2531-117/02). The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

#### *2.3. Cell Delivery*

For cell delivery, a 15 <sup>μ</sup>L suspension containing 5 <sup>×</sup> <sup>10</sup><sup>5</sup> human ECFCs or a 15 <sup>μ</sup>L saline solution was administered directly after LAD ligation by two injections in the border zone of the infarcted myocardium using a 10 μL 32G Hamilton syringe (Reno, NV, USA). One injection was performed on the medial and one at the lateral side of the infarcted area.

#### *2.4. Invasive Evaluation of Cardiac Function*

For evaluation of myocardial function, mice of the previously described groups underwent impedance-micromanometer catheterization. The method as well as data analyses were performed as previously described in the literature [17,18]. Briefly, the animals were anesthetized with thiopental (100 mg/kg intraperitoneal) and ventilated using a mouse ventilator (HUGO SACHS). After that, a 1.4 French impedance-micromanometer catheter (Millar Instruments, Houston, TX, USA) was introduced into the left ventricle retrogradely via the right carotid artery, and pressure—volume loops were recorded. The method was based on measuring the time-varying electrical conductance signal of two segments of blood in the left ventricle from which total volume is calculated. Raw conductance volumes were corrected for parallel conductance by the hypertonic saline dilution method [15].

#### *2.5. Flow Cytometry of Nonmyocyte Cardiac Cells*

We previously hypothesized that EPC may not be responsible only for the formation of new vessels but may also recruit local cells [13]. Therefore, we performed flow cytometry in order to evaluate the effects of cell transplantation on proangiogenic cardiac cell populations.

Hearts of the mice were investigated by flow cytometry (FACS) as described previously [19]. Briefly, for cardiac FACS analyses, infarcted hearts of the mice were explanted at day 2 and retrogradely perfused with saline (0.9% NaCl) to wash out circulating blood cells. Thereafter, a "myocyte-depleted" cardiac cell suspension was prepared, incubating minced myocardium in 0.1% collagenase IV (Gibco, Co Dublin, Ireland) for 30 min at 37 ◦C, lethal to most adult mouse cardiomyocytes. Cells from peripheral blood and hearts were incubated for 40 min in the dark at 4 ◦C with the following fluoresceinisothiocyanate (FITC), phycoerythrin (PE), and peridininchlorophyll-protein (PerCP) conjugated monoclonal antibodies: CD45-PerCP, CD34-FITC, and CXCR4-PE (all from BD Pharmingen). A matching isotype antibody served as control. Cells were analyzed by 3-color flow cytometry using a Coulter® Epics® XL-MCLTM flow cytometer (Beckman Coulter, Brea, USA). Each analysis included 50,000 events.

#### *2.6. Histology and Immunohistochemical Analyses*

Infarct size was calculated as the average of four coronal sections sampled at 2 mm intervals from the apex to the base using the following Equation (1) developed by Pfeffer et al. [20]:

$$\text{Infract Size} = \frac{\text{Coromial Infract Perimeter} \left(\text{Epicardal} + \text{Endocradial}\right)}{\text{TotalCoromial Perimeter} \left(\text{Epicardal} + \text{Endocradial}\right)} \times 100\tag{1}$$

infarct size <sup>1</sup> <sup>4</sup> [coronal infarct perimeter (epicardial plus endocardial)/total coronal perimeter (epicardial plus endocardial)] × 100. Infarct wall thickness was measured in Masson's trichrome stained sections by taking the average length of five segments along evenly spaced radii from the centre of the LV through the infarcted free LV wall [15]. To assess the incorporation and phenotype of injected EPCs in infarcted myocardium, we performed standard histological procedures (hematoxylin/eosin and Masson Trichrome) and immunostaining, which was performed as follows:

For immunohistochemical analyses, hearts were fixed in 4% phosphate-buffered formalin overnight and embedded in paraffin as described previously [15]. Before immunostaining, mounted tissue sections were deparaffinized by rinsing 3× for 5 min in Xylene followed by 2× for 5 min 100%, 2× for 5 min 96%, and 2× for 5 min 70% ethanol rinses. Endogenous peroxidases were quenched in 7.5% H2O2 in distilled water for 10 min. Following that, slides were rinsed in distilled water for 10 min and twice in TRISbuffer (pH 7.5) for 5 min. Finally, sections were incubated at room temperature for 60 min with either a primary antibody detecting vimentin (monoclonal mouse anti-human; Dako, Glostrup, Denmark) or class I human leukocyte antigen (HLA) (monoclonal mouse anti-human HLA-A, B, C; WAK-Chemie, Steinbach, Germany). Pretreatment was performed for 30 min (microwave 750 W) using citrate buffer (10 mM, pH 6.0) for vimentin or a target retrieval solution (Dako) for HLA-A,B,C, respectively. The detection system for vimentin and HLA-A, B, C was Dako REAL and APAAP mouse.
