Figure 2.

hPSCs transiently gain the ability to form epithelial cavitating structures during the naive-to-primed transition

(A) A scheme of human peri-implantation development and correspondent hPSC states (PreEPI, preimplantation epiblast; PostEPI-E and PostEPI-L, early and late postimplantation epiblast, respectively; PS, primitive streak).

(B) Experimental setup. Partially primed hPSCs were treated with either an inhibitor of ALK4/5/7, MAPK, or their combination.

(C) qRT-PCR for markers after differentiation in indicated conditions; results of two independent experiments.

(D) Stitched images of the cells in 24-well plates after differentiation in indicated conditions and staining with Phalloidin.

(E) Bright-field image of 3D epithelial cavitating spheres obtained in AP condition. Note that the spheres remain attached to the surface of culture plates.

(F) Immunofluorescence for GATA3 in combination with E-cadherin and CDX2 in combination with POU5F1, of partially primed hPSCs differentiated in AP condition (“AP”) and undifferentiated control (“undiff”).

(G) Experimental setup. hPSCs on different days of the formative transition were differentiated in AP.

(H) Stitched images of scanned 24-well plates showing hPSCs after different periods of the formative transition differentiated in AP (4′,6-diamidino-2-phenylindole (DAPI) staining).

(I) Bright-field images of hPSCs after different periods of the formative transition and conventional H9 hPSCs differentiated in AP.

(J) qRT-PCR for markers during the time course of AP treatment of naive and partially primed hPSCs.

(K) Immunofluorescence of naive and partially primed hPSCs during the time course of differentiation in AP.

See also Figures S3 and S4.