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. 2018 Nov 29;9:1703. doi: 10.3389/fphys.2018.01703

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Copyright © 2018 Degen, Wiederkehr, La Scala, Carmann, Schnyder and Katsaros.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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(A) Schematic representation of the differentiation assay used in this study. Briefly, cells at passage 2 were thawed in regular 0.4 mM Ca²⁺ KSFM. Thereafter, cells were pushed into their basal state by culturing them for 4 days in 0.1 mM Ca²⁺ KSFM before induction of differentiation for 3 or 5 days (see section “Materials and Methods”). (B) Schematic representation of keratinized epithelium. Shown are the four epithelial cell layers (basal, spinous, granular, stratum corneum) on the left side, and specific differentiation markers expressed in the different cell layers on the right side: markers of the basal layer: Keratin 5 (KRT5), Keratin 14 (KRT14); markers of the spinous layer: Keratin 10 (KRT10), Transglutaminase 1 (TGM1), Interferon Regulatory Factor 6 (IRF6), Grainyhead-like factor 3 (GRHL3), Krüppel-like factor 4 (KLF4); markers of the granular layer: Involucrin (IVL), Loricrin (LOR), Filaggrin (FLG). BM, basement membrane. (C) Light microscopy images of control keratinocytes (Cx-Ep) and CLP keratinocytes (H7-Ep) after 5 days in differentiation media. Note that both keratinocyte cultures alter their morphology upon Ca²⁺/FCS stimuli. Asterisks: signs of stratification; Arrows: elongated cells. Scale bar: 500 μm. (D) qPCR analysis of various differentiation-related genes in ∼60% confluent cultures of control keratinocytes (Cx-Ep, black bars) and CLP-keratinocytes (H7-Ep, white bars) 3 and 5 days after induction of differentiation. Genes studied were: KRT5, KRT10, TGM1, IVL, LOR, FLG, and CDKN1B (p27, marker of cell cycle inhibition). Fold induction of mRNA levels is shown compared to the reference basal levels, which has been set to 1. Data are expressed as mean ± SEM. n = 3. Significance was reached when p ≤ 0.05 (^∗) compared to basal level. (E) Immunofluorescent microscopy of H7-Ep CLP keratinocytes 5 days after differentiation initiation by various stimuli shows induction of the granular compartment marker Involucrin. Merged images are shown of F-actin (red), IVL (green), and DAPI (blue). Note that keratinocytes in basal medium (0.1 mM Ca²⁺) are only very loosely attached to one another. Scale bar: 250 μm.