Figure 1.

Activation of Wnt pathway to different levels leads to distinct cell fates in hPSCs

On day 0, SOX17-mCherry knockin H9 cells were treated with CH at different concentrations in RPMI medium for 24 h and then cultured in RPMI plus B-27 minus insulin supplement for another 48 h without CH. On day 3, cells were treated with 2 μM Wnt inhibitor C59 for 48 h and then the medium was changed to the RPMI plus B-27 supplement.

(A) Representative bright-field images were taken on day 0 and day 1. Scale bar, 100 μm.

(B) Representative mCherry images of cells treated with CH at 3, 4, or 5 μM were taken from day 2 to day 5. Scale bar, 100 μm.

(C) Flow cytometry analysis of mCherry expression and number of cells in SOX17-mCherry H9 cells from day 1 to day 4. Error bars represent SEM of three independent replicates.

(D and E) SOX17-mCherry knockin H9 cells treated with CH at 3 or 5 μM on day 0 were analyzed on day 13 for cTNT expression by immunostaining (D) or flow cytometry (E). Scale bar, 100 μm. Error bars represent the SD of three or four independent experiments. ^∗∗∗∗p < 0.0001, 3 μM CH versus 5 μM CH; Student's t test.

(F) Diagram summarizing that SOX17-mCherry knockin H9 cells treated with 3 μM CH had high percentage of SOX17-expressing cells and became non-cardiomyocytes (CMs), while 5 μM CH induced low SOX17 expression and CM cell fate.

(G and H) Unmodified H9 cells (G) or H1 cells (H) were treated with CH at different concentrations similar to the cells in (D). On day 4, the cells were collected for flow cytometry against SOX17.

(I) RUES2_GLR cells (SOX17-tdTomato) were treated with 3 μM CH in RPMI for 24 h and then cultured in RPMI with B-27 minus insulin supplement. Time-lapse imaging was performed from day 2 to day 3 of differentiation. Representative images were shown at indicated time points. Scale bar, 100 μm.

See also Figures S1 and S2 and Videos S1 and S2.