Isolation and Expansion of hMB-MSCs

A standard protocol for the isolation and expansion of hMB-MSCs was used as previously described [40 (link)]. Bone marrow aspirates were obtained by percutaneous direct aspiration from the iliac crest of 5 healthy volunteers at University Hospital Virgen de la Arrixaca (Murcia, Spain). Bone marrow was collected with 20 U/ml sodium heparin, followed by Ficoll density gradient-based separation by centrifugation at 540 g for 20 min. After, the mononuclear cell fraction was collected, washed twice with Ca²⁺/Mg²⁺-free phosphate buffered saline (PBS) (Gibco Invitrogen) and seeded into 175-cm2 culture flasks (Nunc, Thermo Fisher Scientific) at a cell density of 1.5 × 10⁵ cells/cm² in serum-containing media (designated as the basal media), composed of DMEM low glucose medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Lonza), 1% GlutaMAX (Thermo Fisher Scientific), nonessential amino acid solution (Sigma‒Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific). After 3 days of culture at 37 °C and 7% CO₂, nonadherent cells were removed, and fresh complete medium was added. Culture media were renewed every 2 days, and the isolated hMB-MSCs were passaged when cultures were 70–80% confluent. All studies were performed using hMB-MSCs expanded within culture passages 3–4.