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The Underlying Dermal Scaffold Affects Molecular Differentiation Markers in Three-Dimensional Human Epidermal Equivalents

ABSTRACT: We undertook a study to compare two commonly used dermal scaffolds for three-dimensional human epidermal equivalents (HEEs): contracted collagen (CC) and acellular dermis (ACD). Using the same population of primary keratinocytes grown at an air-liquid interface in each model, we demonstrated improved basal keratinocyte morphology and compartmentalization of basal and suprabasal keratin markers in the ACD model. Using whole-transcriptome sequencing, we found that proliferation markers were more highly expressed in the ACD model at day seven and this may be explained by increased expression of key transcription factors which govern epidermal self-renewal, DNMT1, TP63, and CEBPD.

ORGANISM(S): Homo sapiens

PROVIDER: GSE310114 | GEO | 2025/11/18

REPOSITORIES: GEO

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Project description:Epidermis, a continuously self-renewing and differentiating organ, produces a protective stratum corneum that shields us from external chemical, physical and microbial threats. Epidermal differentiation is a multi-step process regulated by influences, some unknown, others insufficiently explored. Detachment of keratinocytes from the basement membrane is one such pro-differentiation stimulus. Here, we define the transcriptional changes during differentiation, especially those caused by detachment from the substratum. Using comprehensive transcriptional profiling, we revisited the effects of detachment as a differentiation signal to keratinocytes. We identified the genes regulated by detachment, the corresponding ontological categories and, using metaanalysis, compared the genes and categories to those regulated by other pro-differentiating stimuli. We identified 762 genes overexpressed in suspended keratinocyte, including known and novel differentiation markers, and 1427 in attached cells, including basal layer markers. Detachment induced epidermis development, cornification and desmosomal genes, but also innate immunity, proliferation inhibitors, transcription regulators and MAPKs; conversely the attached cells overexpressed cell cycle, anchoring, motility, splicing and mitochondrial genes, and both positive and negative regulators of apoptosis. Metaanalysis identified which detachment-regulated categories overlap with those induced by suprabasal location in vivo, by reaching confluency in vitro, and by inhibition of JUN kinases. Attached and in vivo basal cells shared overexpression of mitochondrial components. Interestingly, melanosome trafficking components were also overexpressed in the attached and in vivo basal keratinocytes. Reaching confluency did not affect adhesion and ECM proteins. Lipid metabolism and steroid metabolism were induced by confluency and by JNK inhibition, respectively. These results suggest that specific pro-differentiation signals induce specific features of the keratinization process, which are in vivo orchestrated into harmonious epidermal homeostasis. Human epidermal keratinocytes are grown in standard growth medium either attached in tissue culture plates, or suspended, in the same medium, in bacteriological plates. After 48 hrs, attached and suspended cultures were harvested and their transcriptomes compared.

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The Underlying Dermal Scaffold Affects Molecular Differentiation Markers in Three-Dimensional Human Epidermal Equivalents

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