Dmem high glucose with stable glutamine and sodium pyruvate

MSCs were obtained from the Wharton Jelly of umbilical cords (WJ-MSCs) from term-gestation newborns after birth, having obtained consent from the parents (three different individuals, n = 3), as previously described [79 (link)]. Isolated WJ-MSCs were cultured, as reported previously [13 (link)], in Dulbecco's modified Eagle's medium DMEM high glucose with stable glutamine and sodium pyruvate (BioWest, Miami, FL, USA) plus 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in a humidified atmosphere of 5% CO_{2 (21% O2)}. Cells from all three donors were maintained in culture in order to produce early- (passage (p) < 14), middle- (15 < p < 40), and late-passage cells (p > 40) that were used for the experiments. The medium was changed twice a week and cells were subcultured when they reached 80% confluency at a ratio of 1:2 until they entered senescence at about 50 cumulative population doublings (PD). Cumulative PD was calculated with the formula PDL = (log (Nn/Nn-1))/log 2 (n: passage, N: cell number).