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. 2022 Apr 25;10:879024. doi: 10.3389/fbioe.2022.879024

FIGURE 2.

FIGURE 2

Differentiation towards definitive endoderm followed by hindgut specification. (A) H9-derived EBs were treated with 100 ng/ml Activin and the resulting spheroids were stained with the DE markers: SOX17 (red) and FOXA2 (green) and counterstained with DAPI (blue). Scale bar: 100 μm. (B) Quantification of the fluorescent images showed that about 92% of the cells in Activin-treated EBs are co-expressing SOX17 and FOXA2. (C) qRT-PCR showed significantly increased expression of the DE genes SOX17 and FOXA2 and the mesoderm marker TBXT but in lower amounts. (D) DE spheroids were further treated with FGF4 and CHIR99021 to induce hindgut specification. After 4 days of treatment, the spheroids were stained for the hindgut marker CDX2. Scale bar: 100 μm. (E) Quantification of the fluorescent images showed that about 90% of the cells were CDX2+. (F) qRT-PCR confirmed the robust expression of CDX2, whereas there was no significant expression of the foregut marker ALB. Low levels of the mesenchymal marker VIM were also detected. Error bars indicate mean ± S.E.M. (n = 3).