Mesencult expansion kit

For quantitative coverage of all cell transplants, we used a total of two 10-week-old B6 eGFP donor mice (Jackson Laboratory, Maine, ME, USA) from the same birth to isolate murine bone marrow-derived mononuclear cells. Mice were killed by dislocating the cervical spine. After the tibiae and femora had been dissected out, they were transferred to a Petri dish with 5 mL MesenCult™ expansion kit (supplemented with MesenCult 1:10 and MesenPure, 1:1000; STEMCELL Technologies, Vancouver, BC, Canada). After removal of the epiphyses, 2.5 mL of MesenCult medium were injected into the open medullary canal using an 18 gauge injection syringe (B. Braun, Melsungen, Germany). Cells were filtered with a 70 µm EASYstrainer (Greiner Bio-One, Frickenhausen, Germany), centrifuged at 300× g and room temperature for 10 min and cultured with a density of ~6 × 10³ per cm² at 37 °C, 5% CO₂ and 1% O₂ for 5 days. When ~90% confluence was reached, cells were passaged using 1% Trypsin/EDTA (PAN Biotech, Aidenbach, Germany) for 2 min at 37 °C and 5% CO₂. After reaching 90% of confluency, cells at passage 0, 1 and 2 were used for the follow-up experiments.

After bone marrow punctures of NCs and OP patients, density gradient centrifugation at 12 000 r·min⁻¹ for 30 min (Invitrogen) was used to extract MSCs from the bone marrow. The extracted MSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Hangzhou Sijiqing Biological Engineering Material Company, Limited).
For mouse MSC isolation, femurs and tibias were collected and cut into pieces. After filtration through a 40 µm cell strainer (BD, Cat. No. 352340), the filtrate was resuspended using the MesenCult Expansion Kit (Stemcell, Cat. No. 05513). The MSCs adhered to the flask, and nonadherent cells were removed after five days.
For osteogenic induction, MSCs were cultured in osteogenic medium consisting of 10% FBS DMEM containing 100 IU·mL⁻¹ penicillin, 100 IU·mL⁻¹ streptomycin, 0.1 μmol·L⁻¹ dexamethasone, 10 mmol·L⁻¹ β-glycerol phosphate, and 50 μmol·L⁻¹ ascorbic acid (Sigma‒Aldrich). The osteogenic medium was changed every three days. JQ1 (ApexBio, Cat. No. A1910) was used to treat MSCs.

AMSCs were obtained from inguinal adipose tissues of C57BL/6 mice, as we previously described [19 (link)]. After being digested with 0.075% collagenase type I (Sigma, St. Louis, MO, USA) and washed with PBS, cells were expanded in the murine MesenCult™ Expansion Kit (Stemcell, Vancouver, BC, Canada). AML-12 cell line was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in DMEM/F12 containing 10% FBS, 10 µg/mL insulin, 5.5 µg/mL transferrin, 5 ng/mL selenium, 40 ng/mL dexamethasone and 1% penicillin-streptomycin at 37 °C in a 5% CO₂ atmosphere. The JNK inhibitor SP600125 (HY-12041) and ATM inhibitor KU-55933 (HY-12016) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). The cells were preincubated with SP600125 (10 µM) or KU-55933 (10 µM) for 1 h to inhibit JNK or ATM before being treated with 10 mM APAP or 4 µM Anisomycin for 24 h.