Ssea4

hMSCs and pMSCs were assessed for expression of multipotent markers (Dominici et al., 2006), performed 24 h after MP labelling (with SiMAG and ScreenMAG, respectively) and 14 days after initial labelling, with repeated passaging and relabelling every 3 days to maintain a high MP level throughout. Cells were harvested with trypsin–EDTA and pelleted by centrifugation for 5 min at 200 × g before washing in PBS. The cell pellets were then resuspended in 100 μl PBS supplemented with 5 μl antibodies against CD29 (Abcam, UK), CD105, CD34 and CD73 (AbdSerotec, UK), CD90 and SSEA4 (eBiosciences, USA) for 30 min at room temperature, before two PBS washes and flow‐cytometry analysis.

LNCaP xenografted tumors were sectioned (4 μm thick) and mounted on glass slides, then stained with Meyer’s hematoxylin and eosin (H&E), and immunostained with primary antibodies for IL-1β (Invitrogen). After glass slides were mounted, each sample was observed at a 200× magnification of the microscopic field. For human PCa immunohistochemistry evaluation, tissue sections were immunostained with primary antibodies for SSEA-4 (eBioscience, San Diego, CA, USA), 4-HNE (Abcam), and IL-1β (Invitrogen). Each sample was observed at a 400× magnification of the microscopic field in 10 randomly selected areas. The intensity and extent of staining were evaluated by score, assigning from 0 to 3 where 0 = none, 1 = weak, 2 = intermediate, and 3 = strong. Final scores were computed using a composite of intensity scores multiplied by the extent of staining score. A score of 1–4 was assessed as low expression and 6–9 as high expression.

For the immunocytochemical determination of pluripotency and differentiation markers, cells were fixed with PBS containing 4% (vol per vol) paraformaldehyde for 10 min at room temperature. The cells were permeabilized with PBS containing 0.1% Triton X-100 for 10 min at room temperature, then washed with PBS and treated with 1% BSA for blocking. For hiPSCs or reprogramming HDFs, the primary antibodies used were SSEA4 (0.5 µg per mL; eBiosciences), TRA-1-60 (0.5 µg per mL; eBiosciences), NANOG (2 µg per mL, AF1997; R&D Systems), OCT3/4 (1:200, sc-5279; Santa Cruz Biotechnology), MAP2 (1:1000, AB5622; Millipore), α-SMA (1:1000, A2547; Sigma), and AFP (2 µg per mL, MAB1368; R&D Systems). The secondary antibodies used were Alexa Fluor 488- or 555-conjugated goat anti-mouse IgG (1:200; Invitrogen), Alexa 488- or 555-conjugated goat anti-rabbit IgG (1:200; Invitrogen), and Alexa 488- or 555-conjugated donkey anti-goat IgG (1:200; Invitrogen). Nuclei were stained with the 4’,6-diamidino-2-phenylindole (DAPI) contained in the VectaShield set (Vector Laboratories). The samples were analyzed in randomly selected images with BZ-X710 (Keyence, Osaka, Japan).

Cells were harvested using 0.05% trypsin-EDTA, centrifuged at 200× g for 5 min, and incubated at room temperature with 5 µL of anti-CD29 (Abcam), -CD90 (eBioscience, Altrincham, UK), -CD105 (AbD Serotec, Altrincham, UK) or -SSEA4 (eBioscience, Altrincham, UK) fluorescently-conjugated antibodies for 30 min. Cells were washed and stored on ice in the dark until analysis using a Beckman Coulter FC500 flow cytometer (Beckman Coulter, High Wycombe, UK). Each surface marker was analysed in triplicates, and the experiment was repeated three times with 50,000 events recorded for each measurement.