Reconstructed Human Skin with Hypodermis Shows Essential Role of Adipose Tissue in Skin Metabolism

Abstract

Background: Dysregulation of skin metabolism is associated with a plethora of diseases such as psoriasis and dermatitis. Until now, reconstructed human skin (RhS) models lack the metabolic potential of native human skin, thereby limiting their relevance to study human healthy and diseased skin. We aimed to determine whether incorporation of an adipocyte-containing hypodermis into RhS improves its metabolic potential and to identify major metabolic pathways up-regulated in adipose-RhS.

Methods: Primary human keratinocytes, fibroblasts and differentiated adipose-derived stromal cells were co-cultured in a collagen/fibrin scaffold to create an adipose-RhS. The model was extensively characterized structurally in two- and three-dimensions, by cytokine secretion and RNA-sequencing for metabolic enzyme expression.

Results: Adipose-RhS showed increased secretion of adipokines. Both RhS and adipose-RhS expressed 29 of 35 metabolic genes expressed in ex vivo native human skin. Addition of the adipose layer resulted in up-regulation of 286 genes in the dermal-adipose fraction of which 7 were involved in phase I (CYP19A1, CYP4F22, CYP3A5, ALDH3B2, EPHX3) and phase II (SULT2B1, GPX3) metabolism. Vitamin A, D and carotenoid metabolic pathways were enriched. Additionally, pro-inflammatory (IL-1β, IL-18, IL-23, IL-33, IFN-α2, TNF-α) and anti-inflammatory cytokine (IL-10, IL-12p70) secretion was reduced in adipose-RhS.

Conclusions: Adipose-RhS mimics healthy native human skin more closely than traditional RhS since it has a less inflamed phenotype and a higher metabolic activity, indicating the contribution of adipocytes to tissue homeostasis. Therefore it is better suited to study onset of skin diseases and the effect of xenobiotics.

Keywords: Adipocytes; Metabolism; Organotypic models; Skin substitutes; Subcutaneous adipose tissue.

Fig. 1

Characterization of adipose-derived stromal cell differentiation to adipocytes in monolayer. A Lipid droplet formation in bright field (BF) and with AdipoRed staining on day 7. B Gene expression of key adipogenic genes throughout differentiation, relative to housekeeping genes (day 0 was set to 1). C, D Secretion of adipokines and pro-inflammatory cytokines into supernatant. ADIPOQ, Adiponectin; FABP4, Fatty-acid-binding protein 4; KLF15, Krüppel-like factor 15; PLIN1, Perilipin-1; RBP4, Retinol binding protein 4; IL, Interleukin; MCP-1, Monocyte chemoattractant protein 1. Scale bar = 200 µm. *p < 0.05. Unconditioned control medium levels as well as gray data points were below detection limit. Shapes represent different donors and bars mean ± SEM; n = 3 independent experiments performed in duplicates

Fig. 2

Adipose-RhS characterization by histology and adipokine secretion. A H&E staining of the full thickness of adipose-RhS. B Comparison of native skin, RhS and adipose-RhS for marker expression of K15, K10 (epidermis) and vimentin (dermis). C 3D representation of the adipose layer within adipose-RhS stained for lipids with AdipoRed and nuclei with DAPI. D Adipokine secretion into the supernatant of 14-day-old RhS and adipose-RhS. H&E, Hematoxylin & eosin; K, Cytokeratin; RBP4, Retinol binding protein 4. Scale bar = 100 µm in both overviews of A and C, 50 µm in B and magnifications of A and 5 µm in magnification of C. *p < 0.05, **p < 0.01. Representative images from n ≤ 4 independent repeats

Fig. 3

RNA-seq reveals metabolic gene expression in RhS/adipose-RhS and differentially expressed genes and pathways after addition of the adipose layer. A Metabolic genes reported to be present (on mRNA, protein or activity level) in ex vivo native human skin but not yet described to be present in RhS. B Separation of epidermal and dermal samples on PC1 and RhS/adipose-RhS dermis on PC2. C Significant differentially expressed genes in red: 295 DEGs were identified, 9 down-regulated and 286 up-regulated. D GO analysis with input of 286 up-regulated genes identified in C. Shown are the top 10 pathways, ranked by p-value, enriched in the WikiPathway 2021 Human library. Bars contain pathway terms, WikiPathway (WP) identifiers and p-values. PC, principal component. n = 4 independent experiments, RNA pooled from intra-experimental triplicates

Fig. 4

Cytokine secretome of RhS and adipose-RhS. A, B Pro-inflammatory and anti-inflammatory cytokines secreted into the medium of both models. Cultures were kept at the air–liquid interface for 14 days. IL, Interleukin; IFN, Interferon; TNF, Tumor necrosis factor. Unconditioned control medium levels were below detection limit. **p < 0.01, ***p < 0.001, ****p < 0.0001. Shapes represent different donors as mean ± SEM; n = 4 independent experiments performed in triplicates. Cytokine secretion from adipose-RhS was normalized to mean secretion of RhS of each donor, which was set to 1