Cnt 07 media

For validation of pBi-L-Dek expression, the plasmid was transfected into previously isolated and cultured K5-tTA expressing murine keratinocytes [91 ]. Cells were collected for Dek protein expression by western blot analysis. K5-tTA keratinocytes were grown in E-media supplemented with 0.05 mM Ca2 and 15% serum as previously published [92 ].
Keratinocytes were isolated from Bi-L-Dek_K5-tTA mice and single transgenic littermate controls using a previously published protocol with modifications [93 ]. Briefly, pups were euthanized within 48 hours of birth, rinsed in 70% ethanol, and placed in PBS. Flank skin was removed, and placed dermis side down in 1 mL of Dispase (Dispase Gibco/Invitrogen, Calsbad, CA, USA, product# 17105–041) and 1 mL of DMEM (1:1 mixture) in a 35mm plate, and incubated overnight at 4° Celsius. The epidermis was removed and placed in 1 mL of accutase (Sigma, St. Louis, MO, USA, product # A6964) for 20 minutes with agitation to release the keratinocytes. Cells were collected and centrifuged, then plated on irradiated MEFs and overlaid with CnT07 media (CellnTec, Bern, Switzerland). Cells were used for experiments in passage 0 or 1.

Normal epidermal keratinocytes were isolated and cultured in CnT-07 media (CELLnTEC). Normal dermal fibroblasts were isolated in 0.3% collagenase and cultured in fibroblast medium (DMEM culture media (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 1% penicillin-streptomycin (Life Technologies)). Cells were incubated at 37 °C in a 5% CO₂ atmosphere and media were replenished every second or third day.

On day 25 of differentiation, hiPSC-bKs were collected with Accumax (Gibco, Life Technologies, Carlsbad, CA, USA) and plated onto collagen I-coated dishes (100 µg/mL, Advanced Biomatrix) in CnT-07 media (CellnTec, Bern, Switzerland) supplemented with 10 µM Y-27632 (Sigma-Aldrich, Saint Louis, MO, USA). A rapid attachment step of approx. 20 min was performed to allow attachment of only hiPSC-bKs. Cells were fed every other day until confluent when they were used for downstream analysis. For assessing terminal differentiation ability of hiPSC-bKs and control hKs, cells were changed to CnT-PR-D media (CellnTec, Bern, Switzerland) without Y-27632 24h before confluency and, the next day, 1.2 mM CaCl₂ (Sigma-Aldrich, Saint Louis, MO, USA) was added to the differentiation media. Samples were taken for analysis after 4 and 8 days.