Research Paper Volume 8, Issue 8 pp 1670—1689

Figure 1. Hypoxia protects hUCB-MSCs from cellular senescence and increases BMI1 expression. (A) Cumulative population doubling levels were measured to investigate the effect of hypoxic culturing on the proliferation of hUCB-MSCs. At each passage, the same numbers of cells were seeded and cultured in normoxic (20% O₂) or hypoxic (1% O₂) conditions. After 4 days, cells were harvested and counted with a hemocytometer. (n=3) (B) SA-beta galactosidase staining was conducted in early and late passages of normoxic- and hypoxic-cultured hUCB-MSCs. (C) MTT assay was conducted to assess the proliferation of normoxic- and hypoxic-cultured MSCs (n=3). (D) Cell cycle distribution was analyzed with propidium iodide using flow cytometry (n=3). (E) Immunostaining of γH2AX was performed in the passaged hUCB-MSCs in normoxia and hypoxia. Each images show the representative and the graph indicates the quantification of loci per cell. (F) Protein expression of BMI1 and p16^INK4a was determined via western blot analysis. (G) Western blotting was performed to evaluate the expression of BMI1 and p16^INK4a in hUCB-MSCs cultured for three days in normoxia or hypoxia. Average BMI1 and p16^INK4a band intensities of three independent replicate experiments were quantified and the representative immunoblots are shown. (H) Expression of BMI1 in normoxic- and hypoxic-cultured hUCB-MSCs was determined via immunocytochemistry. Representative images from at least three independent experiments are shown. Error bars represent mean±s.e.m. from three separate experiments. * P<0.05, ** P<0.01, *** P<0.005 using Student’s t-test.