Journal: Stem Cell Reports
Article Title: Development of a high-efficiency induction system for embryonic oral epithelium from human pluripotent stem cells
doi: 10.1016/j.stemcr.2025.102781
Figure Lengend Snippet: 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.
Article Snippet: From day 2 onward, various signaling molecules were individually added daily at the time of medium change, including 10 ng/mL BMP-4 (PeproTech, #120-05), 100 nM LDN (STEMCELL Technologies, #72147), 5 μM CHIR (Reprocell, #04-0004), 500 nM IWP2 (Reprocell, #04-0034), 5 μM SB-431542 (Stemgent, #04-0010-05), 400 nM SAG (Selleckchem, #S6384), 10 ng/mL EGF (PeproTech, #AF-100-15), 5 ng/mL FGF2 (R&D Systems, #233-FB), 100 ng/mL FGF7 (R&D Systems, #251-KG), and 200 ng/mL FGF10 (Qkine, #QK003-0500).
Techniques: Knock-In, CRISPR, Cell Culture, Fluorescence, Expressing, Quantitative RT-PCR, FACS, Control, Activation Assay
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![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 <t>SAG-treated</t> 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 <t>6</t> <t>[IWP2],</t> from three independent experiments); statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5381/pmc12985381/pmc12985381__gr1.jpg)