Mab2155

Newly formed neurospheres and differentiated cells after 48 h of hypoxia were fixed with 4% paraformaldehyde for 10 min at 4°C, washed with PBS and blocked with 10% normal serum for 20 min in PBS that contained 0.5% Triton X-100. The cells were incubated for 24 h at 4°C with primary antibodies against SOX-2 (1:100, MAB2018, R&D Systems, USA), OCT-4 (1:100, MAB1759, R&D Systems, USA), Nanog (1:100, Human: AF1997; Mouse: AF2729, R&D Systems, USA), KLF-4 (1:100, Human: AF3640; Mouse: AF3158, R&D Systems, USA), CD133 (1:150, MBS462020, MyBiosource, USA), CD15 (1:100, MAB2155, R&D Systems, USA), NESTIN (1:100, Human: MAB1259; Mouse: MAB2736, R&D Systems, USA), ABCG2 (1:100, ab130244, Abcam, USA), VEGF (1:100, MAB293, R&D Systems, USA) and HIF1α (1:100, MAB1536, R&D Systems, USA). Neurospheres and cells were washed three times with PBS for 5 min and then incubated at 37°C for 1 h with appropriate fluorophore-labeled secondary antibodies. Images were acquired with a laser scanning confocal microscope (LSM780, ZEISS, Germany).

Established pluripotent (beyond passage 15) DF‐iPSCs and ACL‐iPSCs were seeded into 24‐well plates and grown for 4 days. Pluripotent cells and outgrown EBs were fixed with 4% paraformaldehyde in PBS for 10 min. Cells were then incubated with primary antibodies against markers of pluripotency; NANOG (1:400, cat. no. 4903; Cell Signaling Technology, London, UK), OCT‐4 (1:100, cat. no. 611202; BD Biosciences, Oxford, UK), SOX2 (1:400, cat. no. 3579; Cell Signaling Technology), SSEA‐3 (1:200, cat. no MAB1434; R&D Systems, Abingdon, UK), SSEA‐4 (1:200, cat. no MAB1435; R&D Systems), TRA‐1‐60 (1:200, cat. no. Ab16288; Abcam, Cambridge, UK), TRA‐1‐81 (1:200, cat. no. Ab16289; Abcam), marker of early differentiation; SSEA‐1 (1:200, cat. no. MAB2155; R&D Systems), marker of mesoderm; α‐smooth muscle actin (αSMA) (1:100, cat. no. MAB1420; R&D Systems), marker of endoderm; GATA6 (1:1600, cat. no. 5851; Cell Signaling Technology) and marker of ectoderm; Neurofilament (1:100, cat. no. 2837; Cell Signaling Technology), in the presence of 1% goat serum, followed by Alexa Fluor secondary antibodies (1:200; Thermo Fisher Scientific) and nuclei stained using 4′,6‐diamidino‐2‐phenylindole (DAPI) (cat no. D1306; Thermo Fisher Scientific). Images were captured using BX51 fluorescence microscope (Olympus, Southend‐on‐Sea, UK).

Cells cultured in hypoxia for 0, 12, 24, 48 and 72 h were collected, subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk and incubated with antibodies against SOX-2 (1:1000, MAB2018, R&D Systems, USA), OCT-4 (1:1000, MAB1759, R&D Systems, USA), Nanog (1:1000, Human: AF1997; Mouse: AF2729, R&D Systems, USA), KLF-4 (1:1000, Human: AF3640; Mouse: AF3158, R&D Systems, USA), CD133 (1:1000, MBS462020, MyBiosource, USA), CD15 (1:1000, MAB2155, R&D Systems, USA), NESTIN (1:1000, Human: MAB1259; Mouse: MAB2736, R&D Systems, USA), ABCG2 (1:1000, ab130244, Abcam, USA), VEGF (1:1000, MAB293, R&D Systems, USA) and HIF1α (1:1000, MAB1536, R&D Systems, USA). Enhanced chemiluminescence was conducted for visualization.