Wnt3a

Recombinant Wnt3a (R&D Systems) was reconstituted with 0.1% BSA in PBS to 200 ng/μL (human Wnt3a) or to 40 ng/μL (mouse Wnt3a), snap-frozen and stored at −80 °C. Cycloheximide (Sigma), BA (Cell Signaling Technology), MG132 (Selleck Chemicals), lenalidomide (Sigma), the DUB inhibitor PR-619 (Selleck Chemicals), MLN4924 (Selleck Chemicals), and the ROCK inhibitor Y27632 (Stemcell Technologies) were dissolved in 100% DMSO at 3 to 50 mM and stored at −20 °C. Plasmids expressing human Flag-tagged CRBN (NP_001166953.1) and HA-tagged human CK1α (NP_001883.4) were synthesized in pcDNA3.1 (Addgene) and point mutations introduced by Genscript. Smart-pool siRNA targeting specific genes (AXIN1, APC, and CRBN) were purchased (Dharmacon). Individual siRNA targeting CRBN were purchased from Dharmacon (#1: J-021086-09-0005; #2: J-021086-10-0005; #3: J-021086-11-0005). Lentiviral expressing shRNA targeting human CRBN (#1: TL305228V #B; #2: TL205228V #D), mouse Scramble control (TR30021V), or mouse Crbn (#A-D: TL503372V #A, B, C, D) were purchased (Origene).

Rescue experiments were carried out at 16–18°C in the same way as in the siRNA and caspase inhibitor experiments. Animals were pre-incubated with 150 ng/ml Wnt3a (R&D Systems) in MFSW for 1 h prior to amputation, amputated, then incubated with fresh 150 ng/ml Wnt3a along with an siRNA or a caspase inhibitor at 18°C as described above. As controls, amputated animals were treated with 150 ng/ml Bovine Serum Albumin (BSA; Thermo Fisher Scientific), human recombinant BMP-2 (Advent Bio, Elk Grove Village, IL, USA) or human recombinant FGF-10 (Advent Bio) using the same regime as described for Wnt3a.

hOPCs maintained in PDGF-AA and NT-3 were infected with either SULF2 KD or control scrambled lentivirus (1 multiplicity of infection). After 48 h, cells were seeded onto the upper surface of laminin-coasted transwell membranes (5 × 10³/insert; 8-µm pore diameter, VWR). Recombinant human WNT3a (R&D Systems), PDGF-AA (Peprotech), or the combination of PDGF-AA and CXCL1 (R&D Systems) were added to the lower chamber (WNT3a, 5 ng/ml; PDGF-AA, 10 ng/ml; CXCL1, 5 ng/ml). At 16 h, cultures were fixed with 4% PFA and stained with DAPI (4′,6-diamidino-2-phenylindole). Cells on the upper surface of the transwell were removed and the remaining migrant cells were imaged (four ×10 fields/well; Olympus IX83) and counted in an unbiased manner using a Python OpenCV-based approach. Two-way ANOVA with Tukey’s HSD post hoc statistics were performed in R.

hESCs (H9 line) were cultured in mTeSR1 basal medium (Stem Cell Technologies), in 6-well plates coated with Matrigel (Corning Matrigel Matrix hESC-qualified). Once hESCs reached 75% confluency, the differentiation was initiated, based on the method by Iyer et al. (2016), with certain modifications. Specifically, for the CDM-PVA (chemically defined medium-polyvinyl alcohol) basal cardiac differentiation medium, human insulin was purchased from Sigma-Aldrich and was used at a final concentration of 7 μg/ml, and WNT3A (R&D Systems) was used at a concentration of 50 ng/ml. Furthermore, on day 5 of differentiation, when the lateral plate mesoderm cells were passaged to generate epicardium, the cells were seeded at a 75×10⁴/cm² density with additional supplementation of 10 μM Rock inhibitor (Y-27632, Abcam). The next day, Rock inhibitor was removed by replenishing with cardiac differentiation medium, CDM-PVA supplemented with WNT3A (50 ng/ml), BMP4 (50 ng/ml, R&D Systems) and all-trans retinoic acid (4 μM, Sigma-Aldrich). The differentiation continued until day 14, with the media being replenished once more at day 11, and topped up with fresh solution as required when the color of the cell media turned orange.

For WNT ligands treatment during chondrogenesis, pellets were treated with either 10 ng ml⁻¹ TGF-β3 (control group) or a combination of 10 ng ml⁻¹ TGF-β3 and 100 ng ml⁻¹ individual WNT ligand (WNT2B, WNT3A, WNT4, WNT5B, or WNT7B, all from R&D system) in chondrogenic medium from d0 to d42 as appropriate. For WNT ligands treatment during the Cp stage, cells were supplemented with either 20 ng ml⁻¹ BMP4 (R&D Systems, #314-BP-010) alone (control group), a combination of 20 ng ml⁻¹ BMP4 and 1 μM C59, or a combination of 20 ng ml⁻¹ BMP4 and 100 ng ml⁻¹ WNT3A (R&D Systems, #5036-WN-010) in mesodermal differentiation medium from d7 to d12.

Tfr1 cells, the R1 subclone carrying the TOPflash luciferase reporter plasmid, were cultured and differentiated in the same manner as the maternal R1 ES cell line. For cell stimulation, recombinant WNT3A (R&D Systems), WNT3A conditioned media or control conditioned media^{20 (link)}, TPA, and CHIR99021 (Sigma-Aldrich) were added 12 h before harvesting. Luciferase assay kit (Promega, USA) was used according to the manufacturer's instructions for the evaluation of luciferase activity. Relative luciferase units were measured on a Chameleon V luminometer (Hidex, Finland) and normalized to the cell mass.