*2.3. Phenotypical Characterization by Flow Cytometry*

Cell fractions were suspended in cold MACS-buffer containing PBS, 2 mM EDTA and 0.5% BSA. To reduce unspecific binding, FcR blocking reagent (Miltenyi Biotec) was added to all samples. Cells were stained using the mouse anti-human-CD133-phycoerythrin (PE; 293C2), -CD34-fluorescein isothiocyanate (FITC) and -CD271-allophycocyanine (all Miltenyi Biotec), -CD45-allophycocyanin-H7 as well as -CD45-Horizon-V500 (both BD Biosciences, Heidelberg, Germany) following incubation for 10 min in the dark at 4 ◦C. For optimal multicolor setting and correction of the spectral overlap single stained mouse isotype antibodies were considered as controls. The gating strategy was performed with matched isotype/fluorescence minus one control. After performing antibody staining 15 μM 4 ,6-Diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, Waltham, MA, USA.) was added, cells were incubated for 2 min and then immediately acquired by BD™ LSRII flow cytometer and data were analyzed using FACS-Diva software, version 6.1.2 (both Becton Dickinson, Franklin Lakes, NJ, USA). Purity and viability of all cell isolations were analyzed using near-IR live dead stain (Thermo Fisher).

#### *2.4. Angiogenesis Assay*

Freshly isolated stem cells were prepared at 4 ◦C in an extracellular growth matrix using 100 μL BD MatrigelTM Matrix (BD Biosciences) and 100 μL complete endothelial growth medium (EGM-2, Lonza, Basel, Switzerland) per well of a 24-well-plate (Greiner Bio-One, Kremsmünster, Austria). For the assay 100,000 CD133<sup>+</sup> or CD271<sup>+</sup> cells (single model) or both cell types in a ratio of 50,000 cells each (co-culture model) were seeded per well. After 20 min polymerization of the matrix at 37 ◦C the matrix 200 μL EGM-2was added carefully onto each well. The medium was changed every two days for another 14 days.

#### *2.5. Three Dimensional (3D) Microscopy Analysis*

After two weeks the tube formation assays were conducted according to the criteria of network length and count of nodal points. For this, z-stack images were acquired using the Zeiss high-resolution microscope ELYRA PS.1 LSM 780 confocal imaging system and corresponding Zen2011 software (both Carl Zeiss AG, Oberkochen, Germany). Z-Stack images are transversal slice images (two-dimensional) of the 3D assay and thus allow representative analysis of their structures. For each well, five z-stacks with ten images were taken and included into a final interpretation. The evaluation was performed using the ImageJ free software (NIH, Bethesda, MD, USA). For better illustration, the different levels of the z-stack were marked in different colors. Likewise, the amount of nodal points was counted, respectively. In total, 900 images (*n* = 6) were analyzed and measured toward network length and count of nodal points.

#### *2.6. Cell Tracking within Matrigel Matrix*

In order to further investigate the cell networks accomplished in Matrigel matrix, immunofluorescence staining was carried out on angiogenesis assay. For better discrimination and alterations within the matrix, freshly isolated CD133<sup>+</sup> cells were stained with the lipophilic cell permeable dye CFDA-SE as well as CD271<sup>+</sup> cells with the red fluorescent lipophilic tracer PKH26 (both Sigma-Aldrich, Saint Louis, MO, USA). Additionally, both cell types were stained for nuclei discrimination with Hoechst 33324 (Thermo Fisher). Acquisition and analyzes were performed using the Axiovert 40 CFL fluorescence microscope with Axio Cam MRm ZEN software (both Carl Zeiss AG).

#### *2.7. Immunofluorescence Staining within 3D Matrix*

Mouse anti-human-CD29 allophycocyanin as well as -CD73-phycoerythrin antibodies (both BD Biosciences) were diluted with EGM-2 in 1:10 ratio and incubated with the cells for 30 min. Afterwards, the assays were washed with EGM-2. For each marker an isotype control was applied in the same way in order to obtain a negative control. Additionally, both cell types were stained with Hoechst 33324. The analysis was performed by means of the Zeiss high-resolution microscope ELYRA PS.1 LSM 780 confocal imaging system and corresponding Zen2011 software Z-stack images were used for 3D reconstructions.

#### *2.8. Gene Expression Analysis by Quantitative Real-Time-PCR*

Cells derived from the single and co-culture models were collected after the termination of the angiogenesis assay and have undergone lysis in TRIzol® reagent (Thermo Fisher). RNA was extracted following the manufacturer's instructions. For reverse transcription of total RNA amount (2 μg) and cDNA synthesis, SuperScript® III Reverse Transcriptase (Thermo Fisher) and oligo (dT)15 Primers (Promega, Fitchburg, WI, USA) were used. Quantitative real time-PCR was performed with StepOnePlusTM Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) in TaqMan® Universal Master Mix (Thermo Fisher) according to the instructions of the manufacturer. The expression of the housekeeping gene ribosomal protein, large, P0 (human RPLP0, TaqManTM VIC® Endogenous Control 4310879E) was used on each cell type. Similarly, human *ACTA* (alpha smooth muscle actin) (TaqMan® Assay ID: Hs00909449\_m1, FAM-MGB), *NGFR* (nerve growth factor receptor) (TaqMan® Assay ID: Hs00609976\_m1, FAM-MGB) and *vWF* (von Willebrand factor) (TaqMan® Assay ID: Hs01109446\_m1, FAM-MGB, all Thermo Fisher) were analyzed in duplicates and normalized to RPLP0. Negative controls were included in each assay. Cycle thresholds (CT) for single reactions were determined with StepOne™ Software 2.0 (formula: ΔCT mean = CT mean − CT mean RPLP0).

#### *2.9. Animals*

All animal procedures were in conformity with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. The federal animal care committee of LALLF Mecklenburg-Vorpommern (Germany) approved the study protocol (approval number LALLF M-V/TSD/7221.3-1.1-088/11). Severe Combined Immunodeficiency beige mice (SCID *beige*; strain CB17.Cg-*PrkdcscidLystbg-J*/Crl, female, 22 ± 2 g, Charles River, Sulzfeld, Germany) were randomly assigned to 4 groups: Healthy control (SHAM, *n* = 3), two MI groups with implanted human stem cells of the respective source (MI133, MI271 each *n* = 3) and untreated MI control group (MIC *n* = 3).

### *2.10. Generation of Reperfused MI in Mice and Stem Cell Implantation*

Mice were anesthetized with 50mg/kg Pentobarbital (Vetmedica GmbH, Ingelheim, Germany) intraperitoneal injection. After thoracotomy and preparation, the left anterior descending coronary artery (LAD) was ligated. After 45 min each mouse received an intramyocardial cell injection. For cell treatment, 100,000 stem cells were suspended in 10 μL PBS containing 0.5% BSA and 2 mM EDTA and mixed with an equal amount of reduced growth-factor BD MatrigelTM Matrix. The same experimental

set-up was applied to control groups using cell suspension buffer and MatrigelTM for application. Four injections, 5 μL each were given along the border of the blanched myocardium. Subsequently, the ligation was removed. Healthy control (SHAM)-operated mice underwent identical surgical procedures without left anterior descending coronary artery (LAD)-ligation.

#### *2.11. Organ Harvesting*

Mice underwent euthanization 48 h after intervention by cervical dislocation. Each heart was removed, embedded in O.C.T.TM Compound (Sakura Finetek, Alphen aan den Rijn, Nederlands) and snap-frozen in liquid nitrogen. For further examinations of the infarction area [13] and RT-PCR, the heart tissue was divided into four horizontal levels from the apex to the base and cut into slices.

### *2.12. Immunofluorescence Staining within Tissue Sections*

For immunofluorescence staining slices of cryosectioned hearts were fixed with 4% paraformaldehyde (Sigma Aldrich) for 20 min at room temperature following treatment with M.O.M.TM Mouse Ig Blocking Reagent and M.O.M.TM protein concentrate according to the instructions of Vector® M.O.M.TM Immunodetection Kit (LINARIS GmbH, Mannheim, Germany). To identify human cells, monoclonal anti-human-nuclei (Merck Millipore, Darmstadt, Germany) primary antibody as first and anti-mouse Alexa-Fluor® 594 as secondary antibody was used. Nuclei were stained with DAPI and the tissue was mounted using coverslip and Dako mounting medium (Dako, Santa Clara, CA, USA).
