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aNSCs differentiation assay After 24 h of incubation in the proliferation medium, half of the medium was replaced with differentiation medium containing Neural Stem Cell Basal Medium, B27 without vitamin A, GlutaMAX, antibiotics (100 IU/mL penicillin and 100 g/mL streptomycin), 1 mg/mL epidermal growth factor, 1 mg/mL fibroblast growth factor 2, 1 M retinoic acid (R2625, Sigma-Aldrich), and 1 M forskolin (F6886, Sigma-Aldrich)

186,187,188,189 In addition, the transcription factor Twist1 upregulates the expression of downstream target genes of TCF4, such as HGF, VEGF, and IGF-1, by forming complexes with TCF4 and -catenin, promoting the proliferation of dermal papilla cells and inducing hair follicle regeneration

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The protein was further purified by size-exclusion chromatography on a Superdex 75 pg 26/60 column (Cytiva, Freiburg, Germany) pre-equilibrated with phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 2 mM KHPO, pH 7.4) for polishing