2.1 Cell culture2.1.1 Fibroblasts and keratinocytes

Dermal fibroblasts and keratinocytes (KCs) were isolated from human foreskin as described previously [20, 21]. KCs were amplified in DermaLife K medium (Lifeline Cell Technology, Frederick, MD) with 1% penicillin/streptomycin (Life Technologies Corporation, Grand Island, NY) on 0.5 µg/cm2 collagen IV (Sigma-Aldrich, St. Louis, MO) coated plates. Medium was switched to keratinocyte medium I (KCI) and DermaLife K medium (1:1) one day before using the cells for construction of the epidermis. KCI consists of Dulbecco's modified Eagle's medium (DMEM; Lonza, Basel, Switzerland) and Ham’s F12 (Corning, Corning, NY) in a ratio of 3:1, supplemented with 1% penicillin/streptomycin, 5% FetalClone III (HyClone, Logan, UT), 1 µM hydrocortisone (Sigma-Aldrich), 1 µM isoproterenol hydrochloride (Sigma-Aldrich) and 0.1 µM insulin (Sigma-Aldrich). Fibroblasts were amplified in fibroblast medium: DMEM supplemented with 1% penicillin/streptomycin and 5% FetalClone III. Medium for both cell types was exchanged every 3–4 days, KCs were cultured at 37 °C, 7.5% CO2 and fibroblasts at 37 °C, 5% CO2.

2.1.2 Adipose-derived stromal cells

The ASC fraction was isolated from fat attached to skin as described previously and grown in fibroblast medium [20]. Five days before construction of the adipose layer, adipocyte differentiation was induced by ASC differentiation medium [22] consisting of DMEM:Ham’s F12 1:1, supplemented with 1% penicillin/streptomycin, 33 µM biotin (Sigma-Aldrich), 17 µM d-pantothenic acid hemicalcium salt (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), 100 nM humulin R (Lilly, Indianapolis, IN), 1 µM rosiglitazone (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 2 nM 3,3′,5-triiodo-l-thyronine sodium salt (Sigma-Aldrich) and 10 µg/ml human transferrin (Sigma-Aldrich). Three days after differentiation induction, medium was changed to ASC maintenance medium (ASC-MM) consisting of DMEM:Ham’s F12 1:1, supplemented with 1% penicillin/streptomycin, 33 µM biotin, 17 µM D-pantothenic acid hemicalcium salt, 10 nM dexamethasone and 10 nM humulin R. Cells were cultured at 37 °C, 5% CO2.

2.2 Reconstructed human skin

RhS and adipose-RhS were constructed in transwells (0.4 µm, 24 mm; Corning). For the adipose layer, 6 mg/ml collagen, isolated from rat tails and dissolved in 0.1% acetic acid (VWR, Radnor, PA), was mixed with 5 mg/ml fibrinogen (Diagnostica Stago, Paris, France) in a ratio of 1:1. Then, 3.2 × 106 adipocytes were added to 800 µl hydrogel per construct. For fibrin formation and polymerization, 0.5 U/ml thrombin (Merck, Darmstadt, Germany) was added and constructs were incubated at 37 °C, 5% CO2 for 90 min. The polymerized adipose layer was submerged in ASC-MM and incubated overnight. Next, the dermis was constructed with 1.4 × 105 dermal fibroblasts in 2 ml hydrogel, composed of 3 mg/ml collagen, 1 mg/ml fibrinogen and 0.5 U/ml thrombin, and incubated overnight. Constructs with adipose layer were submerged in ASC-MM and KCI (1:1) and constructs without adipose layer in KCI. The next day, 5 × 105 KCs were seeded on top and culture medium was supplemented with 2 ng/ml keratinocyte growth factor (Sigma-Aldrich). Skin constructs were cultured submerged for 3 days, at 37 °C, 7.5% CO2 from now on, and subsequently cultured at the air–liquid interface in keratinocyte medium II (KCII: DMEM:Ham's F12 3:1, supplemented with 1% penicillin/streptomycin, 1% FetalClone III, 1 µM hydrocortisone, 1 µM isoproterenol hydrochloride, 0.1 µM insulin, 10 µM l-carnitine (Sigma-Aldrich), 10 mM l-serine (Sigma-Aldrich) and 50 μg/ml ascorbic acid (Sigma-Aldrich) or KCII:ASC-MM (1:1), respectively. Cultures were kept at the air–liquid interface for 14 days and incubated in KCII without hydrocortisone 24 h before harvesting. Supernatants were stored at -20 °C until further analysis. Samples were taken for RNA-seq and histology. For RNA-seq, epidermis and dermis were separated, tissue samples were snap frozen in liquid N2 and stored at -80 °C. For histology, tissue samples were fixed overnight in 4% formaldehyde (VWR) before preparation for histological analysis.

2.3 RNA sample preparation and sequencing

RNA was isolated using QIAshredder spin columns (Qiagen, Hilden, Germany) and the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. Concentrations and quality (RNA integrity numbers) were measured with a TapeStation (Agilent, Santa Clara, CA). Samples were stored at -80 °C until the library was prepared and sequenced with QuantSeq FWD by Lexogen (Vienna, Austria). Reads were aligned with the Spliced Transcripts Alignment to a Reference (STAR) aligner. For DEG analysis, summary count data was used.

2.4 RNA-sequencing analysis

R (v4.1.2) was used for programming. The R scripts used for analysis can be found at https://github.com/MolecularCellBiologyImmunology/Tri-layered_RhS. Gene counts were normalized and transformed to log count per million (lcpm), low count genes with a lcpm lower than two were removed. After quality control, sample 9 was removed because of too many unmapped reads and the log2 fold changes (FCs) were re-shrunk with the method from Zhu, Ibrahim and Love [23]. DESeq2 (v1.34.0) was used to generate principal components (PCs), run principal component analysis (PCA) and identify DEGs. Resulting p-values were adjusted according to the number of tests performed and genes were regarded as significantly differentially expressed if padj < 0.05. For GO, Enrichr [24,25,26] was used with the library WikiPathway_2021_Human.

2.5 Histology, immunohistochemistry and immunofluorescent staining

Confluent adipocyte monolayers were washed with PBS and stained with AdipoRed (Lonza) in PBS (1:33) for 15 min at room temperature (RT) according to the manufacturer’s instructions before imaging. Tissue samples were embedded in paraffin and 5 µm sections were used for morphological (haematoxylin & eosin, H&E) and immunohistochemical stainings. Antibodies for cytokeratin 15 (K15, clone EPR1614Y; Abcam, Cambridge, UK), cytokeratin 10 (K10, clone DE-K10; Progen, Heidelberg, Germany) and vimentin (clone V9; Dako, Glostrup, Denmark) were used as previously described [27, 28]. Prior to embedding in Aquatex (Merck), sections were counter-stained with hematoxylin. For confocal imaging, tissue samples were fixed in 4% formaldehyde overnight, stored in PBS with 0.02% sodium azide (Merck) and subsequently stained with AdipoRed (1:33) and DAPI (1:5000) for 15 min at RT.

2.6 Microscopy

Adipocyte monolayers were imaged using a Nikon ECLIPSE Ti2 (Nikon, Tokyo, Japan). Sections were imaged with a Vectra Polaris slide scanner (Akoya, Marlborough, MA) and analyzed with QuPath (v0.2.2). Confocal images were taken with a Leica TCS SP8 (Leica Microsystems, Wetzlar, Germany) and analyzed using Imaris (v9.9.1; Oxford Instruments, Oxfordshire, UK).

2.7 Quantitative polymerase chain reaction

Adipocyte RNA was isolated with the RNeasy Mini Kit (Qiagen) and reversely transcribed with the RT2 First Strand kit (Qiagen) according to the manufacturer’s instructions. RNA and cDNA concentrations and quality were measured with a NanoPhotometer (Implen, Munich, Germany). The following primers were used (OriGene, Rockville, MD): Krüppel-like factor 15 (KLF15, HP210627), Fatty-acid-binding protein 4 (FABP4, HP205321), Adiponectin (ADIPOQ, HP208060) and Perilipin-1 (PLIN1, HP206315). The reaction mix consisted of Fast SYBR Green Master Mix (Applied Biosystems, Waltham, MA), 300 nM of primer pairs, cDNA and DEPC-Treated Water (Invitrogen, Waltham, MA) with a total volume of 10 µl. Relative changes in mRNA levels were calculated with the 2−∆∆Ct method, using GAPDH as the housekeeping gene, and normalized to expression levels at day 0.

2.8 Enzyme-linked immunosorbent assay and cytokine bead array

Culture supernatant was used to analyze cytokines, IL-6 and IL-8 by ELISA (IL-6: R&D Systems, Minneapolis, MN; IL-8: Diaclone SAS, Besancon, France) according to the manufacturer’s instructions. Cytokine bead arrays: Human Inflammation Panel (BioLegend, San Diego, CA) and Human Adipokine Panel (BioLegend) were used on an Attune NxT flow cytometer (ThermoFisher, Waltham, MA). Data was analyzed with the online available LEGENDplex Data Analysis Software Suite.

2.9 Statistical analysis

Data is presented as mean ± standard error of the mean (SEM). For Fig. 1, three independent repeats, each with different donors of ASCs, were used with two intra-experimental replicates. For RhS experiments (Figs. 2, 3, 4), four independent experiments, each with different donors, were performed with three intra-experimental replicates. KCs, fibroblasts and ASCs all derived from different donors resulting in a total of 12 skin donors were used in the experiments (to minimize donor variation). In graphs, the average from intra-experimental replicates is shown as one data point. GraphPad Prism (v9.1.0; GraphPad Software Inc., La Jolla, CA) was used for statistical analysis. For normality testing, a Shapiro–Wilk test was used and a one-way ANOVA with a Friedman test and a two-way ANOVA. Differences were considered as significant when p < 0.05.

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