Figure 2.

Pluripotency validation of the naïve iPSCs derived from β-thalassemia fibroblasts. (A): Quantitative PCR analysis of genes associated with ground-state self-renewal and pluripotency in naïve iPSCs and primed iPSCs and ESCs. Data are shown as the mean ± SEM; t test; ∗, p < .05; ∗∗, p < .01; n = 3 individual experiments. (B): Western blot analysis of ground-state pluripotency-associated transcription factors such as NANOG, KLF4, and REX1 in naïve iPSC lines and primed iPSC lines. β-Actin was used as an endogenous control. (C): Immunostaining images of pluripotency-associated markers OCT4, SOX2, NANOG, SSEA3/4, and TRA-1-60. Scale bars = 20 μm. (D): Representative images of n2-iPSC morphologies after withdrawal of individual inhibitors and growth factors from 5i/L/FA culture system. Scale bars = 100 μm. (E): Quantitative PCR analysis of pluripotency-associated gene expressions in naïve iPSCs after withdrawal of individual inhibitors from 5i/L/FA culture system. Data are shown as mean ± SEM; n = 3 individual experiments. (F): Quantitative PCR analysis of pluripotency-associated gene expression in naïve iPSCs after withdrawal of growth factors from 5i/L/FA culture system. Data are shown as mean ± SEM; n = 3 individual experiments. (G): Representative images of morphological changes from naïve state to primed state as the culture system changed. Scale bar = 100 μm. Abbreviations: bFGF, basic fibroblast growth factor; Ctrl, control; ESCs, embryonal stem cells; DAPI, 4′,6-diamidino-2-phenylindole; GSK3βi, GSK3β inhibitor; hESM, human embryonic stem cell medium; iPSCs, induced pluripotent stem cells; LIF, leukemia inhibitory factor; MEKi, MEK inhibitor; PCR, polymerase chain reaction; RAFi, BRAF inhibitor; SRCi, SRC inhibitor; SSEA3, stage-specific embryonic antigen 3; SSEA4, stage-specific embryonic antigen 4; ROCKi, ROCK inhibitor.