Fig 1.
Epidermal morphogenesis in NHS, FTMs and CC-FTMS.
(a) Cross-sections are examined for general morphology (HE), proliferation (Ki67), early (K10) and late (LOR) differentiation and epidermal cell activation (K16) using immunohistochemistry or immunofluorescence. The expression of loricrin (LOR) is depicted in red and the nuclei in blue, whereas the yellow dotted line indicates the dermal-epidermal junction. (b) Quantification of the epidermal thickness based on the HE staining. (c) Basal cell proliferation quantified based on the Ki67 positive cells in multiple regions of the epidermis. (d) Safranin red staining was quantified by counting the amount of corneocyte layers in the SC. Scale bar represents 100μm. *indicates p<0.05. Data represents mean ±SD of four independent experiments or skin donors.
Fig 2.
The dermal-epidermal linkage and fibroblast distribution.
Cross-sections of NHS, FTMs and CC-FTMs are analysed by immunofluorescence. Collagen type IV (COL IV) is similarly expressed at the BM in both FTMs and CC-FTMs, while laminin 332 shows a delayed expression in CC-FTMs. Similar fibroblast distribution throughout the dermis is shown by vimentin (VIM). Proteins are visualized in red, nuclei are stained blue using DAPI and the yellow dotted line indicates the dermal-epidermal junction. Scale bar: 100 μm. Representative pictures are shown of four independent experiments.
Fig 3.
Lamellar organization of the extracellular lipid matrix in the Stratum Corneum (SC).
(a) Representative diffraction pattern of the lipids in the SC of FTMs and CC-FTMs. The first, second and third order of diffraction are detected, as well as diffraction peaks attributed to separate crystalline cholesterol, indicated by the asterisk (*). In FTMs, an extra peak is observed, indicated by the pound sign (#). (b) Barplot of the original repeat distance of the LPP, which is elongated in the CC-FTM. Data represents mean ±SD of four independent experiments. (c) Drawing of lamellar organization in the intercorneocyte space, where d indicates the length of the periodicity phase, adapted from van Smeden et al. [34].
Fig 4.
Lipid content and relative abundance of ceramides with specific carbon chain length.
(a) The level of lipids in the SC of the FTM and CC-FTM is equal. (b) Box and whisker plot of CERs as function of increasing number of C-atoms in the total hydrocarbon chain, with indicated benchmark values of NHS. In CC-FTMs, a decreased abundance of C34 CERs is indicated, whereas there is an increase in C44, C46 and C48 CERs. Whiskers indicate the 95% confidence interval. Data is obtained from four independent experiments.
Fig 5.
Ceramide subclass profile in FTMs and CC-FTMs.
Box and whisker plot of CER subclasses with indicated benchmark values of NHS. Significant differences in relative abundance are indicated, which are the reduction in NS and AS and an increase of NdS, NP and NH in SC of CC-FTMs compared to FTMs. Whiskers indicate the 95% confidence interval. Data is obtained from four independent experiments.
Fig 6.
Transepidermal water loss of NHS, FTMs and CC-FTMs.
TEWL was measured over 900 second time period and plotted as mean values +SEM. Data is obtained from three independent experiments and three NHS samples.
Fig 7.
Expression of lipid processing enzymes in NHS, FTM and CC-FTM.
Tissue sections of NHS, FTMs and CC-FTMs are analysed by immunofluorescence for the expression of lipid processing enzymes GBA, aSMase, CER-S3 and ELOVL1. Representative images show the localization (red) of GBA, aSMase and CER-S3 in the stratum granulosum, while the expression of ELOVL1 is more diffuse throughout the epidermis. Nuclei are stained blue using DAPI, the yellow dotted line indicates the dermal-epidermal junction. Scale bar represents 100μm.