Fig. 1. Effect of O2 tension on maintenance of the undifferentiated state of hESCs. hESCs (passage 44) were cultured on MEF feeder cells under different O2 tensions. (A-F) Phase contrast images of hESC colonies cultured under different O2 tensions. (G-L) Immunostaining to detect expression of the undifferentiated markers Oct4 (red) and Nanog (green). DAPI (blue) was used as a nuclear counterstain. (M) Quantitative analysis of Oct4 and Nanog expression in colonies. Randomly selected fields (n = 5) from a minimum of three independent experiments were analyzed. *P < 0.05. Results for each O2 condition were present as a percentage of the expression of undifferentiated-cell markers in colonies containing 1,000-2,000 DAPI-positive cells. (N) Expression of HIF2α in undifferentiated hESCs was analyzed by Western blotting. (O) Immunostaining to detect the expression of HIF2α (green) and DAPI (blue) in hESC colonies. Magnification, 100×.
