Immortalized N/TERT keratinocytes as an alternative cell source in 3D human epidermal models

Abstract

The strong societal urge to reduce the use of experimental animals, and the biological differences between rodent and human skin, have led to the development of alternative models for healthy and diseased human skin. However, the limited availability of primary keratinocytes to generate such models hampers large-scale implementation of skin models in biomedical, toxicological, and pharmaceutical research. Immortalized cell lines may overcome these issues, however, few immortalized human keratinocyte cell lines are available and most do not form a fully stratified epithelium. In this study we compared two immortalized keratinocyte cell lines (N/TERT1, N/TERT2G) to human primary keratinocytes based on epidermal differentiation, response to inflammatory mediators, and the development of normal and inflammatory human epidermal equivalents (HEEs). Stratum corneum permeability, epidermal morphology, and expression of epidermal differentiation and host defence genes and proteins in N/TERT-HEE cultures was similar to that of primary human keratinocytes. We successfully generated N/TERT-HEEs with psoriasis or atopic dermatitis features and validated these models for drug-screening purposes. We conclude that the N/TERT keratinocyte cell lines are useful substitutes for primary human keratinocytes thereby providing a biologically relevant, unlimited cell source for in vitro studies on epidermal biology, inflammatory skin disease pathogenesis and therapeutics.

Figure 1

N/TERT keratinocytes express terminal differentiation genes. mRNA expression of epidermal differentiation genes by N/TERT keratinocyte monolayer cultures (N = 3 for N/TERT 1 and N/TERT2G) were compared to primary keratinocytes (N = 6 donors). Bars represent mean ± SEM. *p < 0.05 relative to primary keratinocyte at the same time point.

Figure 2

N/TERT1 keratinocytes are suitable for culture in HEE models. (A) Haematoxylin Eosin (HE) staining of N/TERT1 keratinocytes in the HEE model system while developing from day 2 to day 10 of air exposure. (B) Epidermal barrier properties of the HEE constructs by biotin penetration (red) and Lucifer yellow penetration (green). Scale bar = 100 µm.

Figure 3

N/TERT keratinocytes and primary keratinocytes respond similar to pro-inflammatory cytokine stimulation in conventional monolayer cultures. (A) Terminal differentiation genes and (B) psoriasis marker mRNA expression of monolayer N/TERT keratinocytes and primary keratinocytes after stimulation with Th1 cytokines (IL-1α, TNFα, and IFNγ). (C) Terminal differentiation genes and (D) AD marker mRNA expression of monolayer N/TERT keratinocytes and primary keratinocytes after stimulation with Th2 cytokines (IL-4 and IL-13). Bars represent mean ± SEM. *p < 0.05 **p < 0.01 ***p < 0.001 relative to primary keratinocytes.

Figure 4

N/TERT1 keratinocytes are suitable to generate a psoriasis-like HEE (PS-HEE) disease model. (A,B) N/TERT HEEs were harvested for mRNA expression analysis and (C) morphological analysis after stimulation with Th1 cytokines (TNFα, IL-6, and IL-1α) and rescued by addition of all trans retinoic acid (ATRA). (D,E) N/TERT HEEs were harvested for mRNA expression analysis and (F,G) morphological analysis after stimulation with Th17 cytokines (IL-17 and IL-22) and rescued by addition of all trans retinoic acid (ATRA). Images are representative of N = 3 N/TERT HEE experiments. Bars represent mean ± SEM. *p < 0.001 **p < 0.01 relative to control unstimulated keratinocytes. Scale bar = 100 µm.

Figure 5

N/TERT1 keratinocytes can be used to generate a atopic dermatitis-like HEE (AD-HEE) disease model. (A) mRNA expression of N/TERT1 HEEs after coal tar stimulation. Activation of the aryl hydrocarbon receptor (AHR) is measured via the induction of the AHR target gene, CYP1A1. (B) N/TERT HEEs were harvested for mRNA and (C) morphological analysis after stimulation with Th2 cytokines (IL-4 and IL-13) and rescued by addition of coal tar extract. (D) Protein expression was visualised by immunohistochemistry. Bars represent mean ± SEM. *p < 0.05 **p < 0.01 ***p < 0.001. Scale bar = 100 µm.