Activin a

Human ESCs were passaged with Accutase (Sigma) and plated at a density of 100,000 cells/cm² in mTeSR with 10 μM Y27632 (Selleck) on Matrigel (BD), Laminin (BD), and collagen IV (BD) (3:1:1) mixed gel coated-plate (Corning). In the restriction of definitive endoderm (DEs) stage (S1), cells were cultured for 24 hrs in RPMI with B27 supplement (1:50, Gibco), 100 ng/ml Activin A (R&D) and 3 μM CHIR99021 (Selleck), and then treated with 100 ng/ml Activin A for 2 days. In the hepatic specification stage to get hepatic progenitor cells (S2), the culture medium was replaced with RPMI (Gibco) supplemented with B27 supplement (1:50), 20 ng/ml BMP4 and 10 ng/ml FGF2 for 5 days. And in the stage of hepatic maturation (S3), cells were cultured in Hepatocyte Culture Medium (HCM, Lonza) with 20 ng/ml HGF, 10 ng/ml OSM and 1 μM dexamethasone for 10–15 days. During stages 2 and 3, cells are fed every 48 hr. The final stage was also carried out with 10 μM SB203580 (Selleck), 50 μM Vitamin K2 (Sigma), 50 μM 2-APB (Tocris), or 0.5 μM Anisomycin (Selleck), and 0.1% dimethyl sulfoxide (DSMO) was used as control, as described in the text. Cells were photographed during differentiation using a Nikon phase contrast microscope (Nikon Microscopes).

Bone marrow cells derived from WT mice or CX₃CR1-EGFP/TRAP-tdTomato mice were cultured with 10 ng/mL M-CSF (R&D Systems) in α-MEM containing 10% fetal calf serum for 3 days. Then bone marrow macrophages were cultured for 3 days in the presence of 50 ng/mL RANKL (PeproTech) and 10 ng/mL M-CSF to induce differentiation into osteoclasts^{58 (link)}. Human/mouse recombinant activin A (R&D Systems), mouse recombinant SFRP2 (R&D Systems), and mouse recombinant IGF2 (R&D Systems) were added at the concentrations indicated in Figure legends. Nuclei were stained with DAPI (D523; Dojindo Molecular Technologies, Kumamoto, Japan). To evaluate the inhibition of activin A-mediated osteoclast differentiation, the ALK4 inhibitor SB505124 (#S2186; Selleck Chemicals, Houston, TX, USA) was added at a concentration of 10 μM; 0.005% DMSO was used as vehicle control.

Chondrogenic induction was performed following the description in our previous report (Hino et al, 2015 (link); Matsumoto et al, 2015 (link)) with some modifications (Ruhl & Beier, 2019 (link)). For 2D chondrogenic induction, cells (6 × 10⁵/well) were seeded into a fibronectin-precoated 12-well plate, and the chondrogenic induction was initiated after cells were adhered uniformly. For 3D chondrogenic induction, cells (2.5 × 10⁵/well) were suspended and transferred to a PrimeSurface 96U plate and centrifuged to form a cell pellet. Chondrogenic induction was carried out using the chondrogenic medium with Activin A (100 ng/ml), with or without IACS-010759 (Selleck), and the medium was changed every 2 d until day 9 or day 21 for 2D and 3D chondrogenic induction, respectively, unless stated otherwise. All cultures were maintained at 37°C under 5% (vol/vol) CO₂ and 100% humidity.