Foxa2

The iPSC lines were differentiated to three germ layers with STEMdiff Trilineage Differentiation Kit (StemCell Technologies, 05230) (Additional file 2). The iPSCs were maintained in StemFlex medium (Thermo Fisher, A3349401) on matrigel plates. Upon confluency, iPSCs were dissociated with Versene (Thermo Fisher, 15,040,066) and plated at the following seeding densities (300,000 cells/well for both endoderm and ectoderm, 100,000 cells/well for mesoderm). Cells were fixed with 4% paraformaldehyde and stained for the following lineage-specific markers: endoderm: FoxA2 (Cell Signaling, 8186S, 1:400), Sox17 (Cell Signaling, 81778S, 1:1600); mesoderm: Brachyury (Cell Signaling, 81694S, 1:1600), NCAM (Cell Signaling, 3576S, 1:250); ectoderm: Nestin (Cell Signaling, 33475S, 1:1600), Pax6 (BioLegend, 901,301, 1:250). Undifferentiated iPSCs were assessed for pluripotency with the markers Nanog (Cell Signaling, 4893S, 1:2000) and Oct-4A (Cell Signaling, 2890S, 1:1600). Details on the antibodies used are listed in Additional file 1: Table S2.

Tissues were either fixed overnight and embedded in paraffin. Sections were cut 4–5 mm thick. Paraffin sections were deparaffinized, dehydrated, and we performed antigen retrieval by steaming slides in sodium citrate buffer for 30 min. Sections were blocked in the blocking serum buffer (5% serum in PBS + 0.5% Triton X-100) for 30 min. Primary antibodies were diluted in blocking buffer and incubated on tissue sections overnight at 4 °C. Slides were washed and incubated in secondary antibody in blocking buffer for 2 h at room temperature. Slides were washed and mounted using Fluormount-G and observed under a microscope. Antibody information: FOXA2 (Cell Signaling, #3143); DLK1 (Abcam, ab21682); HNF4A (LSBio, LS-C413074); LZTS1 (Bioss, bs-5705R).

Cells were grown on ibidi µ-bottom plates and fixed with 4% paraformaldehyde. Cells were permeabilized with ice-cold 100% methanol, blocked with 5% donkey serum, incubated with primary antibody, washed, and incubated with DAPI and secondary antibody coupled to Alexa488 Alexa568, or Alexa647. Images were acquired with a Zeiss 40× plan apo objective (NA 1.3) with the appropriate filter sets. Data was analyzed using custom written code in MATLAB. Antibodies and dilutions used in this study: Klf4 (Abcam ab129473, 1:400); Nanog (eBiosciences 14–5761, 1:800); Oct4 (Santa Cruz sc-8628, 1:800; Cell Signaling 2840, 1:400); Sox2 (eBiosciences 14–9811, 1:800); Otx2 (Neuromics GT15095, 1:400); T (Brachyury) (Santa Cruz sc-17745, 1:200); FoxA2 (Cell Signaling 8186, 1:400); Gata4 (eBiosciences 14–9980, 1:400); Sox1 (Cell Signaling 4194, 1:200); Pax6 (DSHB Pax6, 1:200); Msx1 +2 (DSHB 4G1, 1:200); Slug (Cell Signaling 9585, 1:200), Snai1 (Cell Signaling 2879, 1:200).