Mab1435

Cells were fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.2% TritonX-100 PBS and incubated with 1xBlock Ace (DS PHARMA BIOMEDICAL, Osaka, Japan) containing 0.2% Tween-20 to prevent non-specific binding before overnight incubation with primary antibodies at 4 °C. The following day, secondary antibody incubations were performed for 1 h with the appropriate species-specific antiserum coupled to either FITC or Cy3 (Jackson ImmunoResearch #711-095-152, #711-165-152, #715-095-151, #715-165-151, 1/100, West Grove, PA, USA). After staining the nuclei with DAPI (Sigma-Aldrich, 1/1,000, St. Louis, MO, USA), the cells were mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA) and imaged using BZ-X710 (KEYENCE). All antibodies were diluted in 1xBlock Ace. The following primary antibodies were used at the indicated dilution rates: PAX6 (BD Biosciences #561462, 1/100, Franklin Lakes, NJ, USA), NESTIN (MERCK #AB5922, 1/1,000, Darmstadt, Germany), SSEA-4 (R&D SYSTEMS #MAB1435, 1/100, Minneapolis, MN, USA), OCT-3/4 (R&D SYSTEMS #AF1759, 1/100), PERICENTRIN (Abcam #ab4448, 1/1,000, Cambridge, UK), and α-Tubulin (Sigma-Aldrich # T9026, 1/1,000) N-CADHERIN (Abcam #18203, 1/1000). For immunostaining of a definitive endodermal marker, Alexa Fluor 488-conjugated SOX17 (BD Biosciences #562205, 1/100) was used without secondary antibody.

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).

An immunofluorescent assay was performed using human SSEA-4 antibody (mab1435, R&D Systems, Inc., Minneapolis, MN, USA) on Day 3. The BMSCs were fixated, permeabilized, blocked, then reared in the incubator with SSEA-4 primary antibody. The cultures were incubated with secondary antibody conjugated with fluorescein isothiocyanate (F2761, Abcam, Cambridge, UK), followed by staining with 4’,6-diamidino-2-phenylindole. A fluorescence microscope (Axiovert 200) was used for the analysis.