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. 2024 Jan 11;19(2):174–186. doi: 10.1016/j.stemcr.2023.12.002

Figure 2.

Figure 2

GATA6 and SOX2 DP ESCs have ICM-like bipotency for extra-embryonic endoderm and epiblast differentiation

(A) Representative drawing of the PrE and Epi-like differentiations performed after sorting KOSR cells into SOX2+ and DP cells by FACS.

(B) Representative images of the sorted SOX2+ (green frame) and DP (yellow frame) after 5 days (5d) in PrE differentiation media (PrE diff.). Scale bar, 50 μm.

(C) Relative mRNA levels of different PrE genes after 5 days of differentiation from sorted SOX2+ (green bar) and from DP cells (yellow bar). nEnd control values in gray bar. Dots represent three individual differentiations from different individual clones. Columns show mean ± SEM. Statistics show unpaired t tests. Ns, non-significant. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

(D) Relative mRNA levels of different Epi genes after 3 days of Epi-like differentiation from sorted SOX2+ or DP cells. Dots represent four individual differentiations using two independent clones. Gray columns show control values of four individual Epi-like differentiations starting from a 2iLIF unsorted population. Columns show mean ± SEM. Statistics show one-way ANOVA. ns, non-significant ∗p < 0.05; ∗∗p < 0.01.

(E) Immunofluorescence images showing one plane or a zoomed section of the ICM (maximum projection) of E4.5 blastocysts showing the H2B-tagged contribution from SOX2+ or DP donor cells after morula aggregation assay. H2B contribution in magenta, CDX2 staining in yellow, and GATA6 staining in cyan. Scale bar, 20 μm. Arrows show contributing donor cells co-stained with GATA6 and not CDX2, meaning PrE contribution.

(F) Quantification of the E4.5 contribution after morula aggregation assay. n = 24 E4.5 blastocysts from SOX2+ donor cells and 19 E4.5 blastocysts from DP donor cells. Epi, epiblast contribution; Epi+PrE, epiblast and PrE contribution; NC, no contribution.

(G) Immunofluorescence images showing a maximum projection of a representative E6.5 embryo showing the H2B-tagged contribution from SOX2+ or DP donor cells after morula aggregation assay. H2B contribution in magenta. GATA6 staining in cyan. Arrows show contributing donor cells co-stained with GATA6 (extra-embryonic VE contribution). DAPI in white. Scale bar, 30 μm.

(H) Quantification of the E6.5 contribution after morula aggregation assay. n = 11 E6.5 embryos from SOX2+ donor cells and 13 E6.5 embryos from DP donor cells. Epi, epiblast contribution; Epi+VE, epiblast and VE contribution; VE, VE contribution; NC, no contribution.