Stem cells were isolated from human scalp hair follicles and immortalized by transduction with human papilloma virus 16 E6/E7 (Tel-E6E7). Tel-E6E7 cells, similar to early passage primary hair follicle stem cells (HFSC), form tight colonies, are relatively stationary and have high adherence to collagen IV. Cells also express the stem cell markers KERATIN 15 (KRT15), β1 INTEGRIN and TENASCIN-C and can differentiate into epidermal, hair follicle, and sebaceous lineages.
Bulb hair fragments dissected from human scalp skin, were digested for 10 minutes in trypsin (0.05%) containing ethylene diamine tetraacetic acid (EDTA). Versene (1.33:1) was added and cells were incubated for an additional 20 minutes. Cells were plated on mitomycin C (15 mg/mL), treated J2-3T3 fibroblasts and grown in keratinocyte medium (KCM). Cells were fed every 2 days. Primary cells were immortalized by transduction with a retroviral vector (LXSN- 16E6E7). Briefly, primary cells were co-cultured with mitomycin C-treated virus-producing fibroblasts (PA317/L(E6E7)) in KCM for 6 days. Virus-producing cells were then removed and tranduced cells were plated on a feeder layer of mitomycin-treated 3T3-J2 NHP cells and selected by treatment with G418 (0.2 mg/ml) for 6 days. The resulting Tel-E6E7 cells were maintained on a feeder layer in KCM. KCM is comprised of Dulbecco's modified Eagles medium/Nutrient Mixture F-12 (DMEM/F12, 4:1), fetal bovine serum (FBS, 10%), cholera toxin (0.1 nM), penicillin (100 U/ml), streptomycin (100 mg/ml), hydrocortisone (0.4 mg/ml), transferrin (5 µg/ml), triiodo-l-thyronine (T3, 2 nM), insulin (5 mg/ml), epidermal growth factor (EGF, 10 ng/ml), pH 7.2.