Fig 2: Chemicals altered Lgr5 expression in OE colonies. (A-H) Confocal images of Lgr5-EGFP+ cells in OE colonies cultured in NWR-based medium (A), supplemented with CHIR99021 (B), VPA (C), IGF-1 (D), pVc (E), 616452 (F), bFGF (G) and CVIA6F cocktail (H). (I) Statistical analysis on the ratio of Lgr5-EGFP+ cells per colony in NWR-based medium with different chemical treatments. (J) Quantitative PCR showed differential Lgr5-mRNA levels in OE colonies under stimulations of various single chemicals and cocktails CVIA6F and VIAFSNT. (K) Quantitative PCR analysis on Lgr5-mRNA level in OE colonies cultured in the absence of Wnt3a, Noggin, R-Spondin 1 or of NWR. (L) The Lgr5-mRNA level in OE colonies cultured in EFI-based medium supplemented with single chemicals and cocktails CVIA6F and VIAFSNT. (M) The ratio of Lgr5-mRNA level between colonies cultured in EFI- and NWR-based media, supplemented with single chemicals and cocktails CVIA6F and VIAFSNT. The statistical difference was determined by unpaired t test. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars were 20 µm. Abbreviations of chemicals were as follows: CHIR99021—C, VPA -V, IGF-1—I, pVc—A, 616452—6, bFGF—F, SB431542—S, Nicotinamide—N, N-Acetyl cysteine—T.

Fig 3: Expansion of colonies from human olfactory mucosa. (A) Diagram showing the human olfactory mucosa, from which colonies were derived. (B) Presence of biomarkers in human olfactory mucosa and derived colonies by RT-PCR. (C) Images of colonies in NWR-based medium with and without treatment of A83-01/SB431542 on Day 7, 10 and 14 post in vitro culture. (D, E) Immunostaining against OMP, PGP9.5, Ki67, Sox2 and Tuj1 in human cystic colonies cultured in NWR-based medium, supplemented with 616452 and 616452/A83-01/SB431542. (F) Statistical analysis on the ratios of Ki67+, PGP9.5+ and Tuj1+ cells in human cystic colonies with treatments of 616452, A83-01/SB431542 and 616452/A83-01/SB431542. (G) Quantitative PCR analysis showed Lgr5-, OMP- and Krt5-mRNA levels in human colonies treated with IGF-1, 616452, pVc, VPA, bFGF and CHIR99021. The statistical difference was determined by two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05, ***p < 0.001. Scale bars, 100 µm in (C), 25 µm in (D) and (E), 10 µm in enlarged images from boxed areas.

Fig 4: bFGF promoted SHEDs proliferation. Colony formation was stained with Coomassie blue at day 14 (A and B). In some conditions, cells were pretreated with an FGFR or PI3K inhibitor for 30 min prior to bFGF exposure. Further, the cells were maintained with or without the inhibitor and bFGF for 14 days. Black bars in (B) indicate 200 μm. Cells were treated with bFGF for 72 h and subsequently stained with PI/RNase. Cell cycle analysis was performed using flow cytometry. DNA histograms (C). The percentage of cell cycle subpopulations (D). Asterisks indicate a significant difference compared with the same subpopulation group in the control.

Fig 5: Cell fate commitment along the developmental trajectory toward epiblast fate(A and B) (A) Assessing cellular proliferation and differentiation during in vitro conversion of naive mouse ESCs into EpiLCs. (B) Flow cytometry analysis of CellTrace Violet-labeled Rex1-GFP reporter cells following 48 h of EpiLC stimulation under hourly ACCP-mediated medium exchange cycles and conventional batch culture. CellTrace Violet-stained Rex1-GFP reporter ESCs (t = 0) are shown as a reference. Graphs represent averages from two independent biological experiments. Error bars denote ±SE. Representative flow cytometry profiles are shown.(C–F) Investigating cell fate commitment during the ESC-to-EpiLC transition through timed medium switch experiments.(D) Culture schemes and fluidic routines employed.(E and F) Flow cytometry-based quantification of the fraction of Rex1-GFP positive (GFP+) cells after different durations of EpiLC induction. bFGF, basic fibroblast growth factor; ActA, activin A.