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.2018 Sep 7;8(1):13434.
doi: 10.1038/s41598-018-31829-0.

Establishment of keratinocyte cell lines from human hair follicles

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Establishment of keratinocyte cell lines from human hair follicles

Tanja Wagner et al. Sci Rep..

Abstract

The advent of organotypic skin models advanced the understanding of complex mechanisms of keratinocyte differentiation. However, these models are limited by both availability of primary keratinocytes and donor variability. Keratinocytes derived from cultured hair follicles and interfollicular epidermis were immortalized by ectopic expression of SV40 and hTERT. The generated keratinocyte cell lines differentiated into stratified epidermis with well-defined stratum granulosum and stratum corneum in organotypic human skin models. They behaved comparable to primary keratinocytes regarding the expression of differentiation-associated proteins, cell junction components and proteins associated with cornification and formed a barrier against biotin diffusion. Mechanistically, we found that SV40 large T-antigen expression, accompanied by a strong p53 accumulation, was only detectable in the basal layer of the in vitro reconstructed epidermis. Inhibition of DNA-methylation resulted in expression of SV40 large T-antigen also in the suprabasal epidermal layers and led to incomplete differentiation of keratinocyte cell lines. Our study demonstrates the generation of keratinocyte cell lines which are able to fully differentiate in an organotypic skin model. Since hair follicles, as source for keratinocytes, can be obtained by minimally invasive procedures, our approach enables the generation of cell lines also from individuals not available for skin biopsies.

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Conflict of interest statement

J.G. and R.G.V. are co-funders of Evercyte GmbH. A.S. is an employee of Evercyte GmbH.

Figures

Figure 1
Figure 1
Establishment and growth characteristics of human KC cell lines. For KC isolation plucked hair roots were placed on growth inactivated fibroblasts (a), round KC colonies that formed after 2–3 weeks (b) were transferred to cell culture flasks and the cells were subjected to different immortalization protocols (a). Growth curves show the extension of KC life span after SV40 transfection (SV; blue lines) and the establishment of stable KC cell lines after SV40 transfection and subsequent hTERT transduction (SVTERT; green and red lines) (c). Scale bar = 480 µm.
Figure 2
Figure 2
Primary and SVTERT KC differentiate in monolayer culture. Primary and SVTERT KC derived from epidermis were cultured post-confluent for six days and mRNA expression levels were analyzed by real-time PCR at the indicated time points. Primary and SVTERT epidermal KC showed no induction of the basal marker keratin 5 and showed upregulation of differentiation-associated proteins keratin 10, filaggrin and loricrin (a). Similarly, the cell junction components tight junction protein 1, claudin 1, occludin and desmocollin 1 (b) and the cornification and desquamation associated fators transglutaminase 1, small proline rich protein 1 A and 2 G and serine protease inhibitor Kazal-type 5 SPINK5 (c) were induced during differentiation in primary and SVTERT KC.
Figure 3
Figure 3
Primary and SVTERT epidermal KC form fully differentiated skin equivalents. Normal abdominal skin and human skin equivalents (HSE) at day seven were analyzed by hematoxylin and eosin (H&E) and immunfluorescence staining. HSE with primary and SVTERT KC developed a multilayered epidermis with intact stratum granulosum and stratum corneum resembling human skin. The differentiation associated proteins keratin 10, keratin 2, involcurin and filaggrin were detected by immunofluorescence staining, both in normal skin and HSE cultures. One representative experiment out of three is shown; Scale bar = 120 µm.
Figure 4
Figure 4
Primary and SVTERT hair KC form completely differentiated skin equivalents. Normal scalp skin and HSE at day seven were analyzed by hematoxylin and eosin (H&E) and immunfluorescence staining. HSE developed a multilayered epidermis with intact stratum granulosum and stratum corneum resembling human skin. The differentiation associated proteins keratin 10, involcurin and filaggrin were detected by immunofluorescence staining, both in scalp skin and HSE cultures. keratin 2 was detected in scalp skin, but not in SE cultures. One representative experiment out of three is shown; Scale bar = 120 µm, for scalp 240 µm.
Figure 5
Figure 5
Biotin does not diffuse into the stratum corneum of skin equivalents with primary and SVTERT KC. Biotin was added to the surface of fully developed human skin equivalents (HSE). In all HSE biotin could not penetrate the stratum corneum into the deeper layers within 60 min. Scale bar = 120 µm.
Figure 6
Figure 6
Skin equivalent cultures with SVTERT KC show p53 expression limited to the basal layer. HSE cultures from primary and SVTERT skin and hair KC were established and analyzed at day seven by immunfluorescence staining (a) and Western Blot (b). In primary KC no p53 and SV40 large T-antigen expression was detected. In SVTERT KC the expression of SV40 large T-antigen was restricted to the basal and granular cell layer; p53 was only detected in the basal cell layer. One representative experiment out of three is shown and Ponceau S stainings was used as loading control in Western Blots; scale bar = 120 µm.
Figure 7
Figure 7
Inhibition of DNA methylation results in loss of normal epidermal architecture in skin equivalents with SVTERT KC. HSE cultures with primary and SVTERT skin KC were incubated with 10 µM of DNA methylation inhibitor 5-Aza-2′-Deoxycytidine and analyzed at day seven. Hematoxylin and eosin (H&E) staining showed no effect on primary KC, but completely disturbed epidermal morphology for SVTERT KC (a). Immunfluorescence staining showed that SV40 large T-antigen and p53 was not confined to the basal layer, but was detected in all layers (b). Bar = 120 µm.
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