Shh protein

Bilateral CN crush was performed and MPG tissues (n = 4) were isolated after two days, and were embedded in sterile culture plates containing 150 μl of reduced growth factor Matrigel (Corning Life Sciences). Affi-Gel beads (100–200 mesh, Bio Rad), incubated overnight at 4 °C with 1XPBS (n = 2) or SHH protein (25 μl of a 1 μg/μl solution, R&D Systems, n = 2), were embedded in the Matrigel near the MPG. Matrigel was gelled at 37 °C for 5 minutes prior to adding RPMI media (Sigma) and an antibiotic cocktail (100X Penicillin-Streptomycin-Glutamine, Thermo Fisher). Culture plates were placed in an atmosphere controlled incubator (5% CO₂) at 37 °C for three days.

Cervical loops were dissected under a Leica M80 stereomicroscope. Explants were incubated in 1% type I Collagenase (Sigma‐Aldrich) in HBSS with 1% penicillin–streptomycin and 1% Fungizone^® Antimycotic (all from Thermo Fisher Scientific) for 60 min and dissociated by pipetting 20 times every 15 min. Collagenase was deactivated with equal amount of DMEM containing 10% fetal bovine serum (Sigma‐Aldrich) and 1% penicillin–streptomycin. The cell suspension was then centrifuged at 1,000 × g for 5 min, and the resulting pellet was re‐suspended in DMEM/F12 (Thermo Fisher Scientific) containing B27 supplements (Minus vitamin A version, Thermo Fisher Scientific), recombinant mouse EGF (20 ng/ml final concentration; carrier‐free form, R&D Systems), recombinant mouse basic FGF (25 ng/ml final concentration; carrier‐free form, R&D Systems), and 1% PS. From the second passage, cells started to express typical keratinocyte‐like morphology. The exclusion of contamination of fibroblasts was confirmed by real‐time RT–PCR on Cytokeratin 14 and Vimentin (Appendix Fig S2E). All experiments were performed with cells between passages 4 and 10. For SHH stimulation experiments, cells were treated with indicated concentration of SHH protein (carrier‐free form, R&D Systems) for 24 h before further analysis.