Population Doubling and Differentiation assays

Healthy, ischemic (ISC), or ischemic and diabetic (ISC + DM) MSCs were plated at 1000 MSCs/cm² and serially passaged with either 5% PL or 5% FBS. Total cell count was recorded every 7 days, and population doubling times were calculated by dividing the number of days over which the cells were allowed cells to grow (7) by the doubling time calculated as published¹⁷. Cumulative population doublings were determined by the sum of populations up to and including each week, which was synonymous with passage number (week 4=passage 4). Colony forming units (CFU) were performed as described²⁵. Briefly, MSCs were serially diluted in cell culture medium to obtain 100 cells/10 mL of media and plated in triplicate on a 100 mm tissue culture dish (Falcon). After 10 days, the colonies were fixed with methanol and stained with 0.5% crystal violet solution (Sigma). The dishes were placed on a grid and visible colonies were counted by a blinded observer.