Abstract
Skin aging is driven by intrinsic and extrinsic factors impacting on skin functionality with progressive age. One factor of this multifaceted process is cellular senescence, as it has recently been identified to contribute to a declining tissue functionality in old age. In the skin, senescent cells have been found to markedly accumulate with age, and thus might impact directly on skin characteristics. Especially the switch from young, extracellular matrix-building fibroblasts to a senescence-associated secretory phenotype (SASP) could alter the microenvironment in the skin drastically and therefore promote skin aging. In order to study the influence of senescence in human skin, 3D organotypic cultures are a well-suited model system. However, only few “aged” skin- equivalent (SE) models are available, requiring complex and long-term experimental setups. Here, we adapted a previously published full-thickness SE model by seeding increasing ratios of stress-induced premature senescent versus normal fibroblasts into the collagen matrix, terming these SE “senoskin”. Immunohistochemistry stainings revealed a shift in the balance between proliferation (Ki67) and differentiation (Keratin 10 and Filaggrin) of keratinocytes within our senoskin equivalents, as well as partial impairment of skin barrier function and changed surface properties. Monitoring of cytokine levels of known SASP factors confirmedly showed an upregulation in 2D cultures of senescent cells and at the time of seeding into the skin equivalent. Surprisingly, we find a blunted response of cytokines in the senoskin equivalent over time during 3D differentiation.
Highlights
The human skin is exposed daily to internal and external stressors, which leads to an “aged“ phenotype with wrinkling, sagging, thinning of the epidermis, dry skin, and reduced barrier integrity
The slides for fluorescence analysis were counterstained with DAPI (1:5000, ThermoFisher Scientific, MA, USA), mounted with Fluoprep, and analyzed by fluorescence microscopy on a Leica DMI-6000 microscope
The first derivative was calculated, and after unit vector normalization, spectra were used for principal component analysis (PCA)
Summary
The human skin is exposed daily to internal and external stressors, which leads to an “aged“ phenotype with wrinkling, sagging, thinning of the epidermis, dry skin, and reduced barrier integrity. For senescence induction with H2O2, the cells were treated nine times with 100 μM H2O2 supplemented to the media for 1 h followed by a media change. For senescence induction with doxorubicin, cells were treated two times with 200 nM doxorubicin. RNA isolation and RT-qPCR Cells were lysed in TRI Reagent (Sigma, St. Louis, MO, USA), and RNA was isolated following the manufacturer’s protocol.
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