Extended Data Fig. 3: Screen for regulators of CDX2 expression in TS cells.

a, Calibration of the cell lines. The assay is performed using CDX2–eGFP⁺ TS cells and as described in the schematic and the Methods. For the initial calibration of the assay, gating was set so that wild-type TS cells do not appear in the gate (non-specific fluorescence, left FACS plot). In that condition, after 48 h, the following appeared in the gate: 50% of the CDX2–eGFP⁺ cells cultured in blank TX medium; 88% of the CDX2–eGFP⁺ TS cells cultured in TX medium including FGF4 (25 ng ml⁻¹) and TGFβ1 (2 ng ml⁻¹); 10% of the CDX2–eGFP⁺ TS cells cultured in TX medium including FGF4 (25 ng ml⁻¹), TGFβ1 (2 ng ml⁻¹) and PD0325901. Biological triplicates show very minor variability (n = 3 independent biological samples, right). Error bars are s.d. Scale bar, 100 µm. b, Calibration of the assay and primary screen. For the primary screen of proteins and small molecules, the condition with TX medium including FGF4 and TGFβ1 was used as a positive control. The gating was set up such that 50% of these cells appear in the gate. The condition with TX medium without FGF4 and TGFβ1 was used as a negative control, and 20% of these cells appear in the gate. 8Br-cAMP (0.04 to 5 mM), IL-11 (4 to 500 ng ml⁻¹), LIF (3 to 375 ng ml⁻¹), BMP4 (1 to 125 ng ml⁻¹), and IGF2 (1 to 125 ng ml⁻¹) were added to the medium. The value is the measurement of a single sample. The typical s.d. for this assay is shown in a. c, Secondary screen: combinations of hits. 8Br-cAMP (1 mM), IL-11 (30 ng ml⁻¹), or 8Br-cAMP (1 mM) + IL-11 (30 ng ml⁻¹) were added to TX medium including FGF4 and TGF. The full blastoid medium was also tested (see Methods). d, e, Markers of multipotency, differentiation and epithelization in stimulated TS cells. TS cells were grown for 48 h in blank medium (see Methods), TX medium (including FGF4 and TGFβ1) or TX medium supplemented with 8Br-cAMP and IL-11. Representative bright-field images of TS cells grown for 48 h in TX medium or in blastoid medium are shown (d), along with gene expression as characterized using bulk RNA sequencing (e, n = 3). Scale bars, 1,000 µm.