Af1924

Cells cultured in 2D were fixed with 4% paraformaldehyde (Santa Cruz, SC 281692, Dallas, TX, USA) blocked with a blocking solution comprised of 10% donkey serum and 0.1% Triton X-100 in PBS −/−. The cells were incubated with primary antibodies followed by secondary antibody incubation and 4′,6-diamidino-2-phenylindole (DAPI) staining. Immunofluorescence was observed using an Olympus IX73 microscope. The following primary antibodies were used to detect hPSC-associated markers: OCT4/POU5F1 (Abcam, ab19857, Cambridge, UK), NANOG (R&D systems, AF1997, Minneapolis, MN, USA), TRA-1-81 (ReproCELL USA, Inc., 09-001, Beltsville, MD, USA), TRA-1-60 (Millipore, MAB4360, Burlington, MA, USA) and SSEA-4 (Millipore, MAB4304, Burlington, MA, USA). The following primary antibodies were used to detect expression of germ-layer specific markers: SOX17 (R&D systems, AF1924, Minneapolis, MN, USA), FOXA2 (Abcam, Ab108422, Cambridge, UK), NESTIN (R&D systems, MAB1259, Minneapolis, MN, USA), PAX6 (Biolegend, #901301), α-actinin (Sigma, A7811, St. Louis, MO, USA) and SMA (Millipore, CBL171, Burlington, MA, USA).

Human iPSCs were differentiated into definitive endoderm (DE) as previously described [39 (link)]. Briefly, 0.25 × 10⁶ single cells were seeded onto L7™ hPSC Matrix-coated 24-well plates on day 0 with L7™ TFO2 media containing L7™ hPSC medium supplement and 10 µM Y27632 (ReproCELL USA, Inc., 04-0012, Beltsville, MD, USA). On day 1, the STEMdiff™ Definitive Endoderm Kit (Stem Cell Technologies, 05110, Vancouver, Canada) was used to induce DE differentiation according to manufacturer’s protocol.
The cells were washed, fixed on day 5 and stained for DE-specific markers SOX17 (R&D systems, AF1924, Minneapolis, MN, USA) and FOXA2 (Abcam, Ab108422, Cambridge, UK).

For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 20 min at 4 °C. After washing with PBS, the cells were treated with 0.3% Triton X-100 in PBS for 10 min and blocked with PBS containing 3% bovine serum albumin (Bovogen, BSAS0.1) for 1 h at 25 °C. The cells were then treated with primary antibodies at the following concentrations: OCT4 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, SC-5279), NANOG (1:500, Abcam, Cambridge, UK, ab80892), EOMES (1:500, Abcam, ab23345), GATA4 (1:200, Abcam, ab84593), tubulin beta III isoform (TUJ1; 1:500, Millipore, Burlington, MA, USA, MAB1637), smooth muscle actin (SMA; 1:500, Abcam, ab7817), SOX17 (1:500, R&D Systems, AF1924), and CDX2 (1:1250, Abcam, ab76541). The following day, the primary antibodies were removed and the cells washed thrice with PBS for 10 min. Finally, the cells were labeled with fluorescence-conjugated secondary antibodies to detect the primary antibodies at the following concentrations: Alexa Fluor 488 (1:500) and Alexa Fluor 568 (1:500). Lastly, they were treated with DAPI or Hoechst in 0.3% Triton X-100 in PBS for 3 min at room temperature and washed.