Epidermal Progenitor Cell Derivation

Primary epidermal progenitor cells were established from postnatal day (P) 0 pups and maintained according to published protocols (Nowak and Fuchs, 2009 (link)) using 3T3-J2 feeders and E-Low media [75% DMEM (Gibco, 21068028), 25% F12 (Gibco,21700026), supplemented with 15% chelated fetal bovine serum (FBS) (Hyclone, #SH30071.03), 0.45 µg/ml hydrocortisone (Merck, 386698-25MG), 0.1125 nM Cholera toxin (Sigma-Aldrich, D0564-1MG), 50 µg/mL transferrin (Sigma-Aldrich, T-2252), 50 µg/ml insulin (Sigma-Aldrich, I-5500), 0.02 nM 3T (3,3′,5-triiodo-L-thyronine, Sigma-Aldrich, T-2752), 1× penicillin-streptomycin (Gibco, 15140122), 8 mM L-glutamine (Gibco, 25030081) and 0.03% sodium bicarbonate (Gibco, 25080094)]. E-low media contained 63.53 µM calcium and in vitro differentiation media contained 1.5 mM calcium. Epidermal progenitor cells expressing shId1, shCebpa, shTcf3/4/12, control shScr or protein coding sequences for ID1 or CEBPA were generated by lentiviral infection followed by puromycin (1 µg/ml) selection. ID1 and CEBPA expression was induced by doxycycline (1 µg/ml) for 3 days using the pCW75.1 backbone (Addgene plasmid #38240). Primary epidermal progenitors were within 20 passages.