Figure 2.

Immunocytochemistry, FC and RT-qPCR confirmed both protein and genic expression of pluripotency markers in hiPSC clones PC4.3, PC4.5, PC4.6 and ACP5. (a) Expression of pluripotency markers NANOG, OCT4, SOX2, DNMT3B and LIN28 in hiPSC clones at high passages, determined by RT-qPCR. The gene expression of the hiPSC was normalized to that of BR1 (hESC). PC4 clones were obtained by reprogramming fibroblast cells (PC fibro), which were used as negative control for pluripotency. Data is presented as mean ± SD, n = 3. *p < 0.05 by ANOVA with Tukey’s post hoc test. (b) Immunostaining of ACP5 clone at passage 20 showing expression of pluripotency markers OCT4, NANOG (both nuclear) and TRA-1-60 (membrane). Experiments were performed for all hiPSC clones. (c)–(e) Cytometry data of clones at passages 10, 30 and 50 using NANOG, OCT4 and SOX2 markers. In (c), light and dark gray indicate negative control (fibroblasts) and stained cells (iPSC), respectively. Statistical analysis revealed percentage positivity was not different for any marker when comparing clones in (d) (n = 3) or passages (n = 4) in (e).