Nanog

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).

The sorted cells were attached to glass coverslips, washed extensively with phosphate-buffered saline (PBS), and then fixed with 4 % (w/v) paraformaldehyde (PFA) in PBS for 10 min at room temperature. After washing with PBS, the cells were permeabilized with 0.1 % Triton X-100 in PBS for 10 min at room temperature. Following another wash with PBS and blocking with 10 % (v/v) normal donkey serum in PBS for 1 h, the cells were incubated overnight at 4 °C with primary antibodies against Oct3/4 (1:200, R&D Systems) and Nanog (1:200, R&D Systems). Subsequently, the cells were washed extensively with PBS and then incubated with the secondary antibody conjugated to Alexa Fluor 568 (Molecular Probes) for 40 min at room temperature. The nuclei were visualized by staining with DAPI (Sigma-Aldrich). Cells incubated with fluorescein-conjugated secondary antibody in the absence of primary antibodies were used as negative controls. Fluorescent images of the cells were captured using a fluorescence microscope (DM 2500; Leica, USA).

For immunostaining, cells were seeded onto eight-well Nunc Lab-Tek II Chambered Coverglass (ThermoFisher Scientific, Waltham, MA, USA, #155411), fixed, and permeabilized with 4% PFA (paraformaldehyde) in DPBS (Dulbecco’s modified PBS) for 15 min on RT (room temperature). Next, cells were blocked for 60 min at RT in a blocking solution (DPBS supplemented with 2 mg/mL BSA, 0.1% Triton-X 100, and 1% fish gelatin with or without 5% goat serum). Then, the samples were incubated for 60 min at RT in the blocking solution supplemented with the following primary antibodies: OCT3-4 (1:50, Santa Cruz, CA, USA, #SC-5279) and NANOG (1:100, R&D Systems, Minneapolis, MN, USA, #AF1997) as pluripotency markers; AFP (1:500, Sigma, St. Louis, MO, USA#A8452), SMA (1:500, Abcam, Cambridge, UK, #ab7817), and ß-III-tubulin (1:2000, R&D Systems, Minneapolis, MN, USA, #RD-MAB1195) as markers specific for the three lineages. After washing with DPBS, the cells were incubated for 60 min at RT with Alexa Fluor 647-conjugated goat anti-mouse and Alexa Fluor 594-conjugated donkey anti-goat IgG antibodies (1:250, ThermoFisher Scientific, Waltham, MA, USA, #A11029, #A11012). DAPI (ThermoFisher Scientific, Waltham, MA, USA, # D1306) was used for nuclear staining. The samples were then examined by a Zeiss LSM 710 confocal laser scanning microscope.