Fig 1: Generation of a PITX2::EGFP reporter system for monitoring oral epithelial differentiation from hiPSCs(A) Schematic of PITX2::EGFP knockin strategy. An HA tag and EGFP were inserted in-frame downstream of PITX2 open reading frame using CRISPR-Cas9. LHA, left homology arm; RHA, right homology arm; PGK, phosphoglycerate kinase promoter; NeoR, neomycin resistance gene.(B) Protocol for oral epithelial induction. hiPSCs were seeded in StemFit medium on laminin-511-coated plates. From day 2, cells were cultured in oral epithelial induction medium.(C) Bright-field and fluorescence images of PITX2::EGFP reporter cells cultured in CnT medium. EGFP signals were barely detectable on day 5 but were observed by day 10. Scale bars, 100 μm.(D) Flow cytometric analysis of PITX2::EGFP expression before (day 0) and after (day 10) differentiation in CnT medium.(E) RT-qPCR analysis in induced pluripotent stem cells (iPSCs), unsorted cells (unsort), EGFP-negative (GFP_nega), and EGFP-positive (GFP_posi) populations at day 10. Data are presented as relative fold change; error bars represent means ± SD (n = 6 wells from three independent experiments). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test.(F) Representative fluorescence-activated cell sorting (FACS) plots showing PITX2::EGFP and EPCAM expression in control and SAG-treated cells at day 10. SHH pathway activation via SAG enhanced the proportion of PITX2::EGFP- and EPCAM-double-positive cells.(G) Quantification of PITX2::EGFP- and EPCAM-double-positive cell populations following treatment with various signaling molecules from day 2. SAG significantly increased the proportion of double-positive cells compared to control and treatment conditions. Data represent means ± SD (n = 8 wells [Ctrl, SAG], 7 wells [BMP4, EGF, FGF2], 10 wells [SB], 9 wells [LDN, CHIR, FGF7, FGF10], and 6 [IWP2], from three independent experiments); statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test.