Extended Data Fig. 8: Differentiation of MTSCs in vitro and in vivo.

(a) Immunostaining of TFAP2C and CK7 in ESCs cultured in the MTSC medium for 10 or 20 days. (b) Immunostaining of CDX2 and GATA6 in ESCs cultured in the MTSC medium for 10 days. Stages 1, 2, and 3 represent three distinct phases during the ESC to MTSC transition. Emerging CDX2 positive cells (white arrowheads). (c) Chimeric blastocysts formed after injection of 4-6 MTSCs (GFP positive) into 32-cell embryos. Bottom: green fluorescent images; Top: merged light field and green fluorescent images. (d) Deciduas of E8.5 embryos from the MTSC chimeric blastocysts; progenies of MTSCs (GFP positive) contributed to the placenta formation of E14.5. (e) Flow cytometry analysis of percentage of GFP positive cells in E16.5 mouse conceptuses (related to Fig. 7k). (f) Immunostaining of CK7, TPBPA, and SYNA proteins after MTSCs were cultured in the differentiation medium on day zero (related to Fig. 7h). (g) Immunostaining for TPBPA, SYNA, and CK7 in TSCs after removal of FGF4 for 2 or 4 days. BF, bright field. (h) Immunostaining for TPBPA, SYNA, and CK7 of the E16.5 chimeric placenta (progenies of MTSCs, GFP labeled). (i) Proliferation of trophoblast organoids derived from MTSCs cultured in vitro for 6 days. Experiments were repeated three times (a-i) with similar results. Scale bars: 20 μm in b and f; 50 μm in a, c, and g-i; 1 mm in d.