Ssea4

Cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (RT), followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min at RT. Cells were blocked for 30 min in 3% BSA in PBS and incubated with primary antibodies overnight at 4 °C. Cells were treated with fluorescently coupled secondary antibodies and were incubated for 1 h at RT. The nuclei were stained with DAPI (Sigma/merck, Billerica, MA, USA) for 5 min at RT. All images were captured using an inverted fluorescence microscope. The antibodies used in this study are summarized as follows: Oct4 (Cell Signaling Technology, Danvers, MA, USA, Cat. No. 2840, 1 : 400), Nanog (Cell Signaling Technology, Cat. No. 3580, 1 : 800), Sox2 (Epitomics, Burlingame, CA, USA, Cat. No. 2683, 1 : 500), SSEA4 (Cell Signaling Technology, Cat. No. 4755, 1 : 500), TRA-1-60 (Cell Signaling Technology, Cat. No. 4746, 1 : 1000), TRA-1-81 (Cell Signaling Technology, Cat. No. 4745, 1 : 1000), CDH1 (Cell Signaling Technology, Cat. No. 3195, 1 : 200), CDH2 (Cell Signaling Technology, Cat. No. 14215, 1 : 200), cTnT (Abcam, Cambridge, MA, USA, Cat. No. ab8295, 1 : 200), Nestin (Millipore, Cat. No. MAB5326, 1 : 1000), sox17(Abcam, Cat. No. ab155402, 1 : 200), anti-rabbit IgG, Alexa Fluor 488 (Invitrogen, Cat. No. A-11008, 1 : 1000), anti-mouse IgG, Alexa Fluor 488 (Invitrogen, Cat. No. A-11005, 1 : 1000).

The PBMC-originated, iPSCs were characterized by flow cytometry. Approximately 2 × 10⁵ cells were labeled with SSEA4 and TRA-1-60 (Cell Signaling Technology, Danvers, MA, USA) primary antibodies for 1 hour at 4°C followed by treatment with FITC-conjugated goat anti-mouse IgG antibody (against SSEA4 and TRA-1-60; MilliporeSigma) for 40 minutes at 4°C. A total of 10,000 events were acquired for each sample using a flow cytometer (Guava easyCyte; MilliporeSigma).

The hESCs and PPs were stained by immunocytochemistry (ICC/IF). For improved staining quality, hESCs were cultured and differentiated on Matrigel-coated 24-well ibiTreat µ-plates (IBIDI, Gräfelfing, Germany). For fixation, cells were washed with PBS and incubated in 4% PFA+100 mM sucrose solution for 20 min at RT. After three rounds of PBS washing, 50 mM NH₄Cl (Sigma-Aldrich) was added for quenching for 10 min again followed by three washing steps. Cells were then permeabilized using PBS-T, blocked for 45 min in 5% normal goat serum (Jackson ImmunoResearch) or 5% DS in PBS-T and incubated o/n at 4 °C with primary antibodies diluted in blocking solution. Next, cells were washed three times with PBS before secondary antibody solution was added for 1 h at RT in the dark. After PBS wash, cells were incubated with 500 ng/mL DAPI in PBS for 10 min and finally kept in PBS for imaging. As primary antibodies OCT4 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-5279, 1:200), NANOG (Cell Signaling, 3580, 1:500), SSEA4 (Cell Signaling, 4755, 1:500), PDX1 (R&D, AF2419, 1:500), NKX6-1 (DSHB, F55A12, 1:150), and as secondary antibodies Alexa-Fluor conjugated antibodies (1:500) were used at specified concentrations. SSEA4 staining was performed without permeabilization step and plates were imaged on a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan).

T-MPCs were harvested and fixed as described above and incubated with corresponding antibodies: SSEA4 (1:100), TRA-1-81 (1:250), and TRA-1-60 (1:250) (Cell Signaling) in blocking solution (10% FBS, 1% BSA in DPBS) on ice for 30 min. Mouse embryonic stem cells were used as positive controls. Negative control MPCs were incubated with mouse IgG or rat IgG with the corresponding secondary antibodies. Following washes with blocking solution, cells were mixed with the corresponding secondary antibody in blocking solution and incubated for 20 min on ice. Cells were washed with DPBS and analyzed by flow cytometer.

Cells were fixed in 4% paraformaldehyde at room temperature for 20 min, washed three times with PBS, and then permeabilized with 0.5% PBS-Tween (PBST) overnight. Cells were then blocked with blocking solution (10% fetal bovine serum in 0.1% PBST) for 1 h at room temperature and then incubated with the primary antibody diluted in blocking buffer overnight at 4 degrees Celsius. Primary antibodies used here include the following: OCT4 (Cell Signaling Technology, Cat. 2,840) 1/400, SSEA4 (Cell Signaling Technology, Cat. 4,755) 1/500, MAP2 (Cell Signaling Technology, Cat. 4,542) 1/400, NESTIN, (BioLegend, Cat. 656,802) 1/500, SOX2 (Cell Signaling Technology, Cat. 3,579) 1/400, PAX6 (BioLegend, Cat. 901,301) 1/300. The next day, the cells were washed three times with 0.1% PBST and then incubated with the secondary antibody for 1 h at room temperature followed by DAPI staining.