Figure 1. Generation of three-dimensional posterior foregut spheroids.
a, Sox2 marks foregut endoderm and Cdx2 marks mid/hindgut endoderm in E8.5 (14 somite stage) mouse embryo. b–c, qPCR analysis (b) and wholemount immunostaining (c) for patterning markers in hPSC-DE cultures exposed to three days in media alone (control) or with the indicated growth factors/antagonists. WNT3A and FGF4 induced CDX2 expression whereas the BMP antagonist noggin repressed CDX2 and induced high levels of the foregut marker SOX2. Results are normalized to expression in Control (stage-matched, no growth factor-treated) endoderm. *, p<0.05 compared to control. **, p<0.005 compared to WNT/FGF; two-tailed student’s t-test; n=3 biological replicates per condition, data representative of 6 independent experiments. d, Quantitation of SOX2- and CDX2-expressing cells in day 6 spheroids generated in hindgut (WNT/FGF4) and foregut (WNT/FGF4/Noggin) patterning conditions. Data are expressed as the percentage of cells expressing indicated markers, normalized to the total number of cells in the spheroids. *, p<1.0×10−6; two-tailed student’s t-test; n=5 biological replicates per condition, data representative of 3 independent experiments. e, The posterior foregut in the E8.5 mouse embryo expressed both Sox2 and Hnf1β. f–g, Exposing cultures to RA on the final day of the spheroid generation step induced expression of HNF1β in SOX2-expressing epithelium, measured by both qPCR (f) and wholemount immunofluorescent staining (g), indicating the formation of posterior foregut spheroids. *, p<0.005; two-tailed student’s t-test; n=3 biological replicates per condition, data representative of 3 independent experiments. Scale bars, 100 μm in a and e, 50 μm in c and g. Error bars represent standard deviation.