Extended Data Fig. 1: Optimization of extra embryonic lineage induction using transient overexpression of GATA4 and GATA6.
From: Complete human day 14 post-implantation embryo models from naive ES cells
a, representative Flow Cytometry (FACS) plots of Pdgfra-PE/Cy7 marking primitive endoderm (PrE)-like cells priming from mouse embryonic stem cells (ESC) using iGata4 with DOX for 48 h (right) versus the control condition without DOX (left). b, representative FACS plots of PDGFRa-APC for putative PrE/ExEM-like priming from human naïve ESCs (nESCs) using iGATA6 with DOX in different media (N2B27 and HENSM) as indicated. c, quantification of the PDGFRa+ population by FACS analysis among the PrE/ExEM-like cell optimization conditions presented in this study (number of biological replicates is indicated for each condition). Average values and s.d. error bars are shown. Two-sided Student t-test p values are indicated where relevant. >40% PDGFRa+ set as a threshold for follow-up, followed by extended characterization and validation. d, representative FACS plots of PDGFRa-APC for putative PrE/ExEM-like priming/induction from human nESCs after three days in RCL medium (conventional 2D conditions) followed by three days of RCL (left) or basal N2B27 (right) in aggregation setting. e, representative RT-qPCR gene expression (normalized by GAPDH and ACTIN) of the endodermal marker genes, in RCL (yellow), RACL (blue), NACL (dark blue), and basal N2B27 (grey) media conditions versus naïve PCSs used as a control (white). PrE/ExEM and definitive endoderm (DE)-specific genes are separately underlined. Bar plot based panel showing the average value of each sample (which represents average value of 3 technical replicates), error bars indicate s.d. A single representative experiment out of N = 3 biological replicates performed is shown. f, integration of day 3 RCL starting cell population with Pham et al. 2022 ExEM conversion dataset10. UMAPs of RCL starting cell population integrated with the published reference dataset of day 6 RACL conversion protocol from naïve ESCs containing PrE cells, ExEM, and intermediate epiblast. Selected cell type annotations are shown.
