Figure 2.
GETMS-iPSCs and GETMS-iTSCs Are Fully Functional
(A) (Left) Oct4-GFP MEFs were infected with GETMS factors, with Esrrb marked by EGFP and Eomes marked by tdTomato. 6 days post-dox induction, 16,000 of Esrrb-EGFP-positive and Eomes-tdTomato-high cells or 16,000 of Esrrb-EGFP-positive and Eomes-tdTomato-low cells were sorted and plated on feeder cells. Reprogramming continued for 20 more days in ESC medium followed by 10 days of dox removal. Left: number of Oct4-GFP-positive iPSC colonies is shown. Right: FACS analysis of Oct4-GFP-positive cells is shown. (Right) Oct4-GFP MEFs were infected with GETMS factors, with Eomes marked by EGFP and Esrrb marked by tdTomato. 6 days post-dox induction, 80,000 of Eomes-EGFP-positive and Esrrb-tdTomato-high cells or 80,000 of Eomes-EGFP-positive and Esrrb-tdTomato-low cells were sorted and plated on feeder cells. Reprogramming continued for 20 more days in TSC medium followed by 10 days of dox removal and one split for colony stabilization. Left: number of iTSC colonies is shown. Right: FACS analysis for the trophoblast-specific cell surface marker, Cd40, is shown. Asterisk indicates p ≤ 0.05 using Student’s t test.
(B) tdTomato-marked ESC, OSKM-iPSC, GETMS-iPSC, and EGFP-marked bdTSC and GETMS-iTSC clones were injected into blastocysts. The resulting embryos and placentas were analyzed at 13.5 DPC. Bright field, red and green channel images of the chimeric embryos and placentas are depicted.
(C) Immunofluorescent and immunohistochemical staining images of 13.5-DPC chimeric placenta showing a clear EGFP signal in the central and marginal areas (marked by blue and orange rectangles, respectively) following GETMSEGFP-iTSCs injection.
(D) (Left) Hemorrhagic lesions 6 to 7 days following subcutaneous injection of the indicated GETMS-iTSC and bdTSC lines. (Right) H&E staining of paraffin sections of hemorrhagic lesions shows necrotic tissue with blood and scattered trophoblastic cells.
(E) PCA of global gene expression profiles from RNA-seq data of two biological replicates of 2 MEF, 2 OSKM-iPSC, 2 GETMS-iPSC, 2 ESC, 2 GETM-iTSC, 2 GETMS-iTSC, and 2 bdTSC lines.
Related to Figure S2.