Fig. 2

Generation of anterior foregut cell cultures from DE monolayers. a Differentiation of hiPSC into the DE and AFE. b–k Expression levels of the markers of the DE and AFE during differentiation characterized with quantitative polymerase chain reaction (b, g) and immunofluorescent staining for SOX17 (c), FOXA2 (d, i), EPCAM (e), cytokeratin K8 (f, k), SOX2 (h), and p63 (j), scale bar = 200 μm. Controls in b, g are hiPSC. In b, value of 0 = 1 for hiPSC control, and in g 0 = 0 expression for controls. We used one-way ANOVA to assess statistical significance between gene expression levels. Except for cytokeratin K13 with p-value = 0.06, p-values for other tested genes were below the threshold p-value < 0.05 indicating that expression levels of these genes significantly differed during differentiation. Error bars represent ± standard error of the mean, n = 3 per group. l, m Gating strategy for confirmation of DE and AFE cell populations by flow cytometry. Cell populations were sorted based on GFP expression and CXCR4 and EPCAM for DE (l) and CD56 and NGFR + for AFE (m). ACTA activin A, AFE anterior foregut endoderm, DE definitive endoderm, hiPSC human induced pluripotent stem cells, VBP vocal fold basal progenitors. Source data provided as a Source Data file