Clone 5e10

The cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. After washing once with PBS, the cells were permeabilized with permeabilization buffer (0.05% SDS and 0.1% Triton X-100 in PBS, subjected to 0.22-μm syringe filtration) for 15 min. Then, 3% BSA (wt/vol) was used for blocking and hybridization after washing once with PBS. The cells were incubated with monoclonal antibodies targeting Nanog (ReproCELL, Beltsville, MD; 09-0020; 1:300) and CD90 (BD Pharmingen; Clone 5E10; 550402; 1:150) overnight at 4 °C. After washing three times with PBS, the cells were incubated with a tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibody (Thermo Fisher Scientific; A11012; 1:100; goat anti-rabbit IgG (H+L) conjugate) and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Thermo Fisher Scientific; A11001; 1:100; goat anti-mouse IgG (H+L) conjugate) for 2 h at room temperature. The nuclei were counterstained with Hoechst 33342 dye (Sigma; 14533; 5 µg/mL) for 15 min at room temperature after washing once with PBS. The stained cells were examined under an Axiovert 200 microscope (Carl Zeiss, Göttingen, Germany) or a confocal laser scanning microscope (C1 si, Nikon, Japan) with MetaXpress (Molecular Devices, Sunnyvale, CA, USA).

For cell characterization, 1 x 10⁵ primary hiPSCs or hiPSC-derived cells were seeded in 12-well plates, grown to 50 - 70% confluence and fixed with 4% formaldehyde for 10 minutes at room temperature. Cells were washed in PBS and permeabilized in citrate buffer and blocked with 1x Dako wash buffer/10% FBS (S300685-2, Agilent). Anti-human CD90-PE (40 ng/µl, clone 5E10, BD Biosciences), anti-human CD31-PE (12.5 ng/µl, clone WM59, BD Biosciences), anti-human Cytokeratin 14 (4 ng/µl, clone LL001, Santa Cruz), anti-human Cytokeratin 5 (20 ng/µl, clone 2C2, Thermo Fischer) primary antibodies and appropriately titrated isotype controls were applied overnight at 4°C. As secondary antibody, a goat anti-mouse PE (40 ng/µl, BD Biosciences) was applied for 1 hour at room temperature. Cell nuclei were stained with 4′,6-Diamidin-2-phenylindol (DAPI, 1:1000, D1306, Molecular Probes) at room temperature for 10 minutes. In situ reporter staining during endothelial cell differentiation was performed by adding 125 ng/ml of anti-human CD31-PE (clone WM59, BD Biosciences) antibody in basal medium and incubating for 30 min at 37°C. After washing the cells with basal medium, EGM-2 / 10% hPL was added back before reporter staining was analyzed by fluorescence imaging.