**2.1. Cell isolation and culturing**

MSCs of the first passage were obtained from the Faculty of Basic Medicine at Lomonosov Moscow State University. All procedures that involved human participants were performed in accordance with the ethical standards approved by the Bioethical Committee of the Faculty based on the 1964 Helsinki declaration and its later amendments. The study involved 21 healthy (not suffered from infectious or systemic diseases and malignancies) individuals from 21 to 55 years old, and informed consent was obtained from each participant.

Cells were isolated from subcutaneous fat tissue of healthy donors using enzymatic digestion as previously described [24]. Briefly, the adipose tissue was extensively washed with two volumes of Hank's Balanced Salt Solution (HBSS) containing 5% antibiotic/antimycotic solution (10,000 units of penicillin, 10,000 μg of streptomycin, and 25 μg of Amphotericin B per mL; HyClone), fragmented, and then digested at 37°C for 1 h in the presence of collagenase (200 U/ml, Sigma-Aldrich) and dispase (10 U/ml, BD Biosciences). Enzymatic activity was neutralized by adding an equal volume of culture medium (HyClone™ AdvanceSTEM™ Mesenchymal Stem Cell Basal Medium for human undifferentiated mesenchymal stem cells containing 10% of HyClone™ AdvanceSTEM™ Mesenchymal Stem Cell Growth Supplement (CGS), 1% antibiotic/antimycotic solution (HyClone) and centrifuged at 200 g for 10 min. This led to the sedimentation of diverse cells, including MSCs, macrophages, lymphocytes, and erythrocytes, unlike adipocytes that remained floating. After removal of supernatant, a lysis solution (154 mM NH4 Cl, 10 mM KHCO3 , and 0.1 mM EDTA) was added to a cell pellet to lyse erythrocytes, and cell suspension was centrifuged at 200 g for 10 min. Sedimented cells were resuspended in the MSC culture medium and filtered through a 100-μm nylon cell strainer (BD Biosciences). As indicated by flow [24], after isolation and overnight preplating, the obtained cell population contained not only MSC cells that basically represented the most abundant subgroup but also admixed macrophages and lymphocytes. The two last cell subgroups were dramatically depleted by culturing for a week in the MSC culture medium and humidified atmosphere (5% CO<sup>2</sup> ) at 37°C. The obtained MSC population was maintained at a subconfluent level (~80% confluency) and passaged using HyQTase (HyClone). By using the methodology described previously [25], cultured cells were demonstrated to differentiate into the osteogenic, chondrogenic, and adipogenic directions, the finding confirming their multipotency. In experiments, MSCs of the second to fourth passages were usually used.
