Fig. 1. Schematic representation of the experimental design.

Schematic representation of the experimental regime, in which MShef4 and MShef11 human ES cell lines were cultured for an extended period under different growth-conditions. Stock cultures of MShef4 and MShef11 were cloned by single-cell deposition to ensure a homogeneous starting point. A clone of each was then recloned to generate Parent Clones that were then cultured under the different growth-conditions for a period exceeding three months—MShef4 clone B8, standard conditions, 109 days (19 passages); MShef11 clones E4 & E7, standard conditions, 111 days (25 passages); MShef11 clones F1 & F4, standard conditions + Y27632, 115 days, (25 passages); MShef11 clone G8 low oxygen, 111 days (25 passages) to give subclone cohort N, and 111 days + 28 days in standard oxygen (25 + 6 passages) to give subclone cohort Q. At the end of the expansion period cultures were recloned again into standard conditions to obtain subclone cohorts (J, O, P, K, L, N and Q, respectively, as shown), from which genetic material was extracted as early as possible (5 passages) for sequencing analysis.