Isolation and Culture of SFZ Cells

Primary SFZ cells were isolated as previously described [21 (link)]. Briefly, the primary end of the femur and the distal end of the tibia were dissected from P5 mice, and ligaments and tendons were excised. Cartilage tissues were incubated with 0.25% trypsin (Thermo Fisher Scientific, Waltham, MA) for 1 h, followed by 1.5-h digestion with 173 U/mL of type I collagenase (Worthington Biochemical Corporation, Lakewood, NJ). DZ cells were isolated by additional digestion of residual epiphyseal cartilage tissue with 43 U/ml collagenase type I for 5 h. Dissociated cells were seeded on fibronectin-coated culture dishes. Cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS). The cells were cultured as a monolayer in all experiments. We used 2 μM 4-hydroxytamoxifen (4OHT; Sigma Aldrich, St Louis, MO, USA) to induce Cre recombination in the cultured SFZ cells. Recombinant human (rh) WNT5A (R&D Systems, Minneapolis, MN), rhWNT5B (R&D Systems), rhWNT9A (R&D Systems), CHIR99021 (Cayman, Ann Arbor, MI), FH535 (Cayman), and 666-15(Tocris Bioscience, Bristol, UK) were used at the concentrations described in the figures and figure legends. For the luciferase assay, ATDC5 cells (Riken BRC, Tsukuba, Japan) were cultured in DMEM/F-12 (Wako) containing 5% FBS.