Dispase 2

Liver tissues were homogenized in 200 U mg⁻¹ type 1 collagenase (Worthington Biochemical), 1 mg ml⁻¹ dispase 2 (Sigma) and 0.5 mg ml⁻¹ DNase 1 (Sigma) using a GentleMacs Tissue Dissociator (Miltenyi Biotec). The single-cell suspension was filtered through a 70-μM filter. The washed cells were incubated with biotinylated anti-F4/80 antibody (1:25 dilution; clone BM8; BioLegend) followed by magnetically coupled streptavidin microbeads (Miltenyi Biotec). F4/80⁺ cells were positively selected by magnetic separation (Miltenyi Biotec) to >97% purity. The flow-through fraction containing the non-macrophages was saved for analysis.

Cell extraction from tissues was modified according to the described protocol60 (link). For lymphocyte extraction, organs were digested for 30–60 minutes at 37 °C, and shaken at 240 rpm in HBSS containing 1 mg/ml Collagenase Type I (Life Technologies). Digested slurry was filtered and washed through a 70 μM mesh filter and resuspended in Percoll (GE Healthcare) diluted to 90% using HBSS. Layers of 60% and 37% Percoll were then sequentially overlayed on the 90% cell-Percoll mix. Cell separation was accomplished by centrifugation at 4 °C, 500 × g for 18 minutes with the brakes off. Cells were washed and resuspended in staining buffer containing 5% FBS in PBS in preparation for flow cytometry. Neural cells extraction was performed using previously described methods60 (link). Briefly, cells were digested using 0.8 mg/ml Dispase 2 (Sigma), 0.2 mg/ml collagenase P (Sigma), and 0.1 mg/ml DNase I (Sigma). Density separation of cells was done in one step using only 40% Percoll, and subsequent pelleted cells were filtered and washed through a 100 μM filter before preparation for flow cytometry.

NSCs were isolated from the young adult 8–10 weeks old DG, mechanically dissociated and enzymatically digested for 30 min at 37 °C in DMEM/F‐12 (#C11330500BT, Gibco, Grand Island, USA) media containing 10 mg mL⁻¹ Papain (#LK003178, Worthington Biochemicals, Lakewood, USA), 20 mg mL⁻¹ Dispase II (#04942078001, Sigma‐Aldrich, St. Louis, MO, USA), and 1 mg mL⁻¹ DNase I (#DN25, Sigma‐Aldrich, St. Louis, MO, USA) based on published methods. Cells were then plated in neurobasal media containing neurobasal (#21103‐049, Gibco, Grand Island, USA), supplemented with B27 (#17504‐044, Gibco, Grand Island, USA), l‐glutamic acid (#G3126, Sigma‐Aldrich, St. Louis, MO, USA), and penicillin streptomycin (#03‐031‐1BCS, Biological Industries, Kibbutz Beit‐Haemek, Israel). Media was changed every 2 days. About 4 weeks later, neurospheres would be formed and then passaged for further study. The PDGF‐BB (100 ng mL⁻¹) was added in culture for 36 h. Characterization of neural stem/progenitor cell cultures was evaluated by immunofluorescent staining with the specific neural stem cell markers neuroepithelial stem cell protein (Nestin).

The experimental procedures were approved by the Animal Committee of Tokyo Medical and Dental University. Primary osteoblast-like cells were collected from the cranial bones of neonatal littermates of C57BL/6 J mice (Sankyo Labo Service Co. Tokyo, Japan) and cultured as previously described [12 (link)]. In brief, the cranial bone was carefully cut up and digested with 0.1% collagenase X (Wako Pure Chemical Industries Ltd., Osaka, Japan), 0.1% dispase II (Sigma-Aldrich, St. Louis, MO, USA), and 0.05% trypsin–EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at 37 °C. The suspension was then centrifuged to remove the supernatant, and the precipitate was resuspended and seeded in cell culture flasks (Corning, NY, USA) with alpha-Modified Eagle’s Medium (Sigma-Aldrich, MO, USA) medium containing 10% fetal bovine serum (Sigma-Aldrich, MO, USA) in an incubator with 5% CO₂ at 37 °C.