Human skin equivalents cultured under hypoxia display enhanced epidermal morphogenesis and lipid barrier formation

Arnout Mieremet,Gert Gooris,Adela Vázquez García,Joke A Bouwstra,Rianne Van Dijk,Abdoelwaheb El Ghalbzouri,Walter Boiten

doi:10.1038/s41598-019-44204-4

Arnout Mieremet, Gert Gooris + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-44204-4

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Abstract

Human skin equivalents (HSEs) are three-dimensional cell models mimicking characteristics of native human skin (NHS) in many aspects. However, a limitation of HSEs is the altered in vitro morphogenesis and barrier formation. Differences between in vitro and in vivo skin could have been induced by suboptimal cell culture conditions, of which the level of oxygen in vitro (20%) is much higher than in vivo (0.5–8%). Our aim is to study how external oxygen levels affect epidermal morphogenesis and barrier formation in HSEs. In the present study, fibroblast and keratinocyte monocultures, and HSEs were generated under 20% (normoxia) and 3% (hypoxia) oxygen level. In all cultures under hypoxia, expression of hypoxia-inducible factor target genes was increased. Characterization of HSEs generated under hypoxia using immunohistochemical analyses of morphogenesis biomarkers revealed a reduction in epidermal thickness, reduced proliferation, similar early differentiation, and an attenuated terminal differentiation program compared to normoxia, better mimicking NHS. The stratum corneum ceramide composition was studied with liquid chromatography coupled to mass spectrometry. Under hypoxia, HSEs exhibited a ceramide composition that more closely resembles that of NHS. Consequently, the lipid organization was improved. In conclusion, epidermal morphogenesis and barrier formation in HSEs reconstructed under hypoxia better mimics that of NHS.

Highlights

Three-dimensional (3D) human skin equivalents (HSEs) are mainly used for toxicology screenings and for research purposes to increase the knowledge on skin biology, or the skin barrier formation
The main objective of this study is to investigate the influence of oxygen level on dermal and epidermal morphogenesis and barrier formation in Human skin equivalents (HSEs)
Our results demonstrate that a reduced oxygen level (i) activates Hypoxia-Inducible Factors (HIFs) signaling in vitro leading to metabolic reprogramming of primary human fibroblasts and keratinocytes in mono and 3D cultures, (ii) decreases epidermal thickness in full thickness skin models (FTMs), (iii) affects epidermal morphogenesis, and (iv) alters the stratum corneum (SC) lipid composition and organization

Summary

Introduction

Three-dimensional (3D) human skin equivalents (HSEs) are mainly used for toxicology screenings and for research purposes to increase the knowledge on skin biology, or the skin barrier formation. As reviewed recently by Niehues et al.[8], while all of these models have advantages and drawbacks, a known limitation of the in vitro developed skin models is the altered barrier formation and resulting reduced functionality when compared to native human skin (NHS)[9,10,11] This could lead to inadequate in vitro – in vivo correlations regarding pharmacokinetics and permeability testing of compounds or misinterpretation of adverse outcome pathways[12]. The alterations in the lipid composition of HSEs as compared to NHS include an altered CERstotal subclass profile, reduction of CER carbon chain length, and a higher level in unsaturation These alterations directly affect the lipid organization, including the reduction in lamellar phase repeat distance and the conversion in lateral packing from a predominant orthorhombic to a hexagonal packing[22,24]. Previous in vivo murine studies have already revealed that the HIF pathway is a crucial determinant of skin homeostasis[32,33]

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