FIG. 1.

(A) Schematic diagram depicting conditions under which human embryonic stem cells (hESCs) were maintained and passaged prior to RNA extraction. H1 and H9 ESCs were maintained under 4% O₂ condition from p26 and p23, respectively. The H1 and H9 cells were propagated under 4% O₂ (represented by upper open box) for seven passages on MEF feeder layer (represented by solid arrow lines); thereafter the both cells were switched to Matrigel coated culture wells for at least three passages (represented by broken arrow lines) prior to RNA collection. RNA extraction was performed within 3 min following removal of the culture plates from respective O₂ conditions and well before dissolved O₂ in the culture medium showed any detectable changes. Total six RNA samples were collected from H1 cells at two different passage numbers (p37 and p50) and H9 cells at passage 32 (p32) under 4% and 20% O₂ (represented by lower gray box) conditions, respectively. The cells maintained under 4% O₂ were split into both 4% and 20% O₂ conditions and cultured for 7 days before of RNA collection. (B) Phase contrast images of H1p50 hESC colonies under 4% (left) and 20% (right) O₂ as they appeared prior to RNA extraction. RNA was isolated at 7 days postpassage, prior to the time at which morphological differentiation is apparent in H1 hESCs maintained under 20% O₂. Bar 1 mm.