Figure 2.

Schematic representation of the experimental design. Schematic representation of the experimental regime, in which hESC line H9 and two hiPSC lines (AD3 and CaSR) were cultured for an extended period under the same culture conditions. Stock cultures of all three lines were propagated manually by separately selecting cultures with good and bad colony morphology, and phase-contrast images were recorded for colonies of different morphological phenotype from 24 to 120 h in the early, middle, and late passages. Each colony of good or bad phenotype was then recloned manually to generate clonal subcultures. Parent cultures propagated via bulk expansion were further cultured under bulk conditions to generate nonclonal cohorts. At the end of the expansion period, cultures were propagated again manually or bulk-expanded to obtain cultures of good and bad morphological phenotypes from which morphological parameters (phase-contrast/ImageJ) and genetic material were extracted for gene expression via qRT-PCR analyses.