Figure 2.

Long-term culture of NMPs in the presence of WNT/FGF and inhibited RA signaling generates epithelial SOX2⁺/CDX2⁺ cell colonies

(A) Tissue culture scheme for generating NMPs and maintaining neural progenitors in vitro.

(B) Representative immunostaining of CDX2 (gray), Brachyury (magenta), SOX2 (red), and DAPI (blue) at 36 h, passage (P) 1, P3, P5, and P10. Scale bars, 100 μm.

(C) qRT-PCR analysis of NMP markers at each passage up to P10. Expression levels are normalized to the reference gene PBDG. Error bars show SD (n = 3 technical replicates, independent differentiations provided in Figures S3A and S3B).

(D) Representative immunostaining of TBX6 (green), CDX2 (gray), and DAPI (blue) at 36 h to P4. Scale bars, 100 μm.

(E) Representative bright-field images of cells at the indicated stages. Dashed lines in P5 and P10 outline examples of compact epithelial colonies, which are surrounded by flat mesenchymal cells. Scale bars, 200 μm.

(F) Representative immunostaining of CDX2 (gray), SNAI1 (green), and DAPI (blue) at P5. Scale bars, 100 μm.

(G) Representative immunostaining of CDX2 (gray), GFP (ZO1-mEGFP iPSC, green), and DAPI (blue) at P5. Scale bars, 100 μm.