Population doubling

We measured the population doubling (PD) of BM-MSCs over four passages (Ps). Cells were seeded at 5 × 10³ cells/cm² in six-well plates with various prepared media. After reaching 80%–90% confluence, cells were detached with 0.05% trypsin-EDTA (Sigma, St. Louis, MO, USA) and harvested for counting. Ten microliters of the cells suspension were loaded in the well of the hemocytometer. The cells in the four sets of 16 squares were counted and then divided by 4 squares to get the mean. The mean was multiplied by 10⁴ as a concentration of cells in 1 mL. This concentration was multiplied by the total volume of cells harvested from the well to get the total number of cell (Nf). PD was assessed at the end of each P using the following formula: PD = (log10(Nf) – log10(Ni))/log10(2), where Nf denotes the number of cells retrieved at the end of the P, and Ni is the number of cells seeded at the beginning of the P. To calculate the cumulative PD, the number of PDs at the end of each P was added to the PD of the previous P.