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IPSC-derived keratinocytes to study epidermal defects in atopic dermatitis [bulk RNA-seq]

ABSTRACT: Human induced pluripotent stem cells (iPSC) can be derived from patients or be genome-edited to introduce or correct disease-related genetic variants, and subsequently be differentiated into any skin cell type. Yet, the potential of induced keratinocytes (iKC) derived from iPSC has not been explored to model the chronic inflammatory skin disease atopic dermatitis (AD), partially because iKC are often immature and heterogeneous as compared to primary keratinocytes (pKC). We aimed to optimize iPSC to keratinocyte and subsequent epidermal differentiation protocols, and compared the response of iKC to inflammatory cytokines involved in the AD pathophysiology. We found that CELLnTEC (CnT)-30 medium greatly increased the iKC culture homogeneity based on morphology and transcriptome (e.g., 130-fold higher KRT5) as compared to commonly used keratinocyte serum free medium (KSFM). Single cell RNA sequencing of iKC revealed different cell populations in the iKC culture, and indicated cell surface markers to enrich for keratinocyte-like cells. In addition, iKC passaging enhanced overall keratinocyte marker gene expression like KRT14 to similar levels as primary keratinocytes (pKC) while maintaining the cobblestone keratinocyte morphology. Upon 2D calcium or fetal bovine serum (FBS) stimulation iKC showed flattened morphology and increased KRT1 and IVL expression, typical for epidermal differentiation. Epidermal differentiation markers were reduced again by AD cytokines IL-4, IL-13 and IL-22, mimicking the epidermis defects in AD. Finally, air exposure also induced 3D epidermal differentiation, including presence of all epidermal layers, and KRT10, IVL and FLG expression, while maintaining the proliferative capacity of keratinocytes in the basal layer based on Ki67 expression. Altogether, we show the potential of iPSC-derived keratinocytes to study epidermal defects in AD, which could facilitate patient-derived skin tissue modeling in translational research.

ORGANISM(S): Homo sapiens

PROVIDER: GSE287810 | GEO | 2025/12/19

REPOSITORIES: GEO

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Project description:In atopic dermatitis (AD), epidermal disease hallmarks are driven by a complex cutaneous inflammatory milieu that varies between patients. How these variable inflammatory signatures translate to patient-specific disease endotypes is poorly understood, which hampers the personalized use of targeted drugs. We here employed primary and immortalized keratinocyte-derived human epidermal equivalents stimulated with T helper (Th)2, -17 and -22 cytokines, to unravel which AD-associated cytokines drive specific epidermal disease hallmarks. Th2 cytokines IL-4 and IL-13 were main inducers of a pro-inflammatory and hyperproliferative response. The presence of IL-17A or IL-22 in the Th2 milieu caused spongiosis, impaired keratinocyte differentiation (loss of FLG, IVL, KRT2) and epidermal barrier defects (altered CLDN1, CLDN4, transepithelial impedance). Transcriptomic analyses revealed upregulation of proliferation genes by Th2 cytokines, and activation of different immune pathways upon Th2 cytokines alone and combination of Th2 and Th17/22 cytokines. Downregulation of epidermis development was most apparent upon combined exposure to Th2 cytokines and IL-22. Single-cell spatial transcriptomics showed expansion of keratinocytes expressing high level of proliferation genes in all epidermal layers, and downregulation of terminal differentiation genes in the upper epidermal layers. Our in vitro AD model using Th2 + IL-22 showed the highest transcriptome resemblance with in vivo AD lesional epidermis and we identified IL-22 specific alteration of epidermis development by lipid mediators like ACER1 and AKR1C3. Finally, we revealed that Aryl Hydrocarbon Receptor (AHR) ligands partially restored the barrier defects caused by Th2 and IL-22, whereas combination of AHR ligand Tapinarof with Janus Kinase (JAK) inhibitor I had an even greater restoring effect. Thus, specific combinations of cytokines upregulated in AD skin drive differential disease hallmarks highlighting the importance of personalized medicine and the need for prediction models in the targeted treatment of AD patients.

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IPSC-derived keratinocytes to study epidermal defects in atopic dermatitis [bulk RNA-seq]

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