Isolation and Expansion of Human MSCs

All BMA samples (5 mL) were processed as previously described [21 (link)] Following the lysis of RBCs using ammonium chloride, and after washing the cell pellet twice with 10 mL of PBS, the nucleated cells were re-suspended in complete MSC media (StemMACS™ MSC Expansion media, Miltenyi Biotec, Bisley, UK) in a volume equivalent to the original BMA, then seeded in a 6-well plate (Corning, New York, NY, USA ) at 750 µL/well containing 1 mL of StemMACS™ media to enable MSC attachment. After 48 h, the media were changed to low glucose DMEM supplemented with antibiotics and 10% human serum (Sigma-Aldrich, Dramstadt, Germany) for MSC expansion to passage 1 (p1). We have previously shown that the expansion of MSCs, particularly in FCS-containing media, affects their colony characteristics and gene expression profiles, while the use of human serum leads to a better preservation of their native characteristics [22 (link),23 (link)]. Half media changes were performed twice weekly, and the cells were trypsinised and expanded when approximately 60–70% confluent. P1 MSCs were then frozen at −80 °C in aliquots and used for further analysis, as described below.