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The expressions of antigens associated with hematopoietic, cardiomyocyte, and endothelial lineages were evaluated. Human control and differentiated CD34⁺ cells were dissociated with 0.25% Trypsin-EDTA (Life Technologies, Courtaboeuf, France) for 4 min at 37 °C, washed, counted, suspended in PBS, and stained for 15 min at room temperature with different combinations of the following mouse anti-human monoclonal antibody surface markers: FITC-CD34 (130-113-178), APC-CD309 (KDR) (130-093-601), CD73-PE (130-112-060), CD133-PE (130-098-826), CD90-PE (130-117-537) (Miltenyi Biotec, Bergisch Gladbach, Germany), CD106-APC (305809) (Biolegend, Amsterdam, The Netherlands), CD105-VioBlue (130-112-320), CD117-PE-Vio770 (130-111-672), CD172a-PE-Vio770 (130-099-793), CD146-PE-Vio770 (130-099-957), CD344-PE-Vio770 (130-106-572), and CD31-APC (130-110-808) (Miltenyi Biotec). The cells were washed and suspended in PBS, and 7AAD was used to discriminate between living and dead cells. The different samples were then analyzed using a FacsCanto II instrument (Becton Dickinson Biosciences, Le Pont de Claix, France).

Immunophenotypic analyses with flow cytometry were performed according to manufacturer’s recommendations. Briefly, 1 × 10⁶ cells per sample (Passage 3–4) were centrifuged at 300× g and resuspended in 100 µl of buffer. Afterwards, 10 µl of the respective antibodies, CD44-VioBlue, CD73-APC, CD90-PE, CD105-FITC, CD146-PE-Vio770 and CD271-APC-Vio770 (Miltenyi Biotec, Bergisch Gladbach, Germany) and a cocktail of CD14/CD20/CD34/CD45-PerCP (Miltenyi Biotec, Bergisch Gladbach, Germany), was added to cell suspension and incubated for 10 min in the dark in the refrigerator. Then, cells were washed with 2 mL of buffer and centrifuged. Supernatant was aspirated, and the final sediment was resuspended in buffer for flow cytometry analysis. Similarly, respective iso-types controls were used to assess background fluorescence and non-specific binding of anti-bodies to cells. All data were acquired using a MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) and further analyzed by MACS Quantify software (Miltenyi Biotec, Bergisch Gladbach, Germany).

To determine the origin of the obtained mesenchymal stem cells, isolated cells were immunostained with antibody against STRO-1 (1:200, Novus Biologicals, Littleton, CO, USA) and their surface markers were measured by flow cytometry. Cells were harvested and incubated with fluorochrome-conjugated rabbit anti-human antibodies: CD34-FITC, CD45-PerCP, CD90-PE, CD105-APC, CD146-APC, CD73-PE (Miltenyi, Bergisch Gladbach, Germany) for 20 minutes at room temperature in the dark. Stained cells were washed twice with 0.01 M phosphate buffer solution (PBS) and analyzed by BD FACSCalibur (BD Biosciences, San Jose, CA, USA).

STRO-1 is a protein-tagged gene of MSCs, which is the first isolated monoclonal antibody to identify MSCs. Cultured cells (3 days) were subjected to immunofluorescence staining with an antibody of STRO-1 (1:200, Novus Biologicals, Littleton, CO, USA), followed by determination of positive expression of STRO-1. Meanwhile, cells were incubated with CD34-FITC, CD45-PerCP, CD90-PE, CD105-APC, and CD73-PE (Miltenyi, Bergisch Gladbach, Germany) and subjected to FCM analysis (BD Biosciences, CA, USA).