AZA treatment of primary MSCs does not alter their viability, morphology, or immunophenotype. Healthy and MDS MSCs were grown to 80% confluency and treated once on day 0 with 10 µM AZA. After 48 hours, stromal layers were washed and fresh medium was added. Untreated MSCs were used as controls. (A) MSC viability was assessed by flow cytometry using Annexin V/PI on day 6. Shown are mean frequencies ± SEM of Annexin V−/PI− cells measured in triplicate for n = 3 healthy and n = 4 MDS MSCs. (B) Cell counts of MSCs were determined by trypan-blue staining at indicated time points. Values represent mean ± SEM of n = 3 healthy and n = 4 MDS MSC cell counts normalized to the cell count before treatment on day 0. (*P = .045 for MDS MSC AZA vs untreated on day 4, all other n.s.) (C) Representative light microscopy images showing MSC morphology at indicated conditions (magnification ×10). (D) Expression of MSC-defining markers was assessed by flow cytometry on day 6.