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

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. Overall design: Induced pluripotent stem cells (iPSC) were differentiated into keratinocytes in keratinocyte serum free medium (KSFM) and CELLnTEC (CnT)-30 medium with retinoic acid (RA) and bone morphogenetic protein 4 (BMP-4) for the first two weeks, followed by two weeks without RA and BMP-4 and bulk RNA-sequencing was performed on the resulting induced keratinocytes (iKC), primary keratinocytes (pKC) and initial iPSC before differentiation