Fig. 2: CLDN6^Low and CLDN6^High EpiSCs possess distinct germ layer differentiation bias.

a Schematics describing the differentiation propensity of CLDN6^Low and CLDN6^High EpiSCs towards anterior primitive streak derivatives (APSD). Right panel, representative IF staining for FOXA2 (green), TBX6 (red), and DAPI (blue) of CLDN6^Low and CLDN6^High EpiSCs subjected to APSD-differentiation reveals the higher potential of CLDN6^Low to differentiate towards TBX6^pos NMP cells, while CLDN6^High EpiSCs generate FOXA2^pos DE-fated cells with greater efficiency. Scale bar: 50 μm. Each differentiation experiment was performed independently more than three times, and multiple immunofluorescence images were acquired for each experiment. b Illustration of the APSD protocol to differentiate EpiSCs towards NMPs and DE. EpiSCs culture in APS medium for 24 h generated APSD containing NMP and DE progenitors, which are further differentiated toward PSM and DE by administration of PSM medium and DE medium, respectively, for 48 h. c Relative Tbx6 and Msgn1 expression measured by RT-qPCR, revealing high levels of NMP and PSM markers in CLDN6^Low EpiSCs differentiated towards APSD. d Relative expression of DE markers, Foxa2 and Sox17, measured by RT-qPCR, showing enriched expression in CLDN6^High EpiSCs differentiated towards APSD. Data are mean ± s.e.m. (n = 2, biological replicates) for (c) and (d). e Cartoon describing differentiation of CLDN6^Low and CLDN6^High EpiSCs towards neuroectoderm. Right panels, representative phase-contrast photomicrographs of the CLDN6^Low and CLDN6^High anterior neural differentiated cells, showing the formation of neurons (white arrowheads and high magnification of area framed in green boxes, panels I and II) at day (D) 4 and D7 in CLDN6^High, while CLDN6^Low EpiSCs failed to differentiate towards neuroectoderm. The images are representative of at least three independent differentiations. Scale bars: 100 μm. f Illustration depicting the prospective embryonic contribution of CLDN6^Low and CLDN6^High epiblast cells to different germ layers. CLDN6^High cells generate anterior neural tissues (neuroectoderm) (green) and DE (pink), while distal-posteriorly located CLDN6^Low cells specifically contribute to NMPs (blue), giving rise to PSM and posterior neural tissues (spinal cord).