Galardo, Francesco (2024) Bioengineered human skin model to study multiple aging-related phenomena in vitro. [Tesi di dottorato]

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Abstract

Skin aging has always raised a lot of interest from a research point of view, as skin represents one of the most important organ barriers in the human body. Aging leads to a change in the morphology of the skin as well as decay in its mechanical and structural properties. Ultimately, this results in a loss of functionality, leading to impaired repairing capability and thus making people prone to different pathological conditions. Photoaging refers to the cumulative damage affecting many skin components as a consequence of prolonged exposure to both UVA and UVB rays. There is a growing interest in elucidating the sequence of events eventually resulting in reduced strength and elasticity of dermal compartment, as well as an increasing thickening of the outermost epidermal layer. However, most of the readily available skin constructs fail in replicating relevant physiological structures, therefore limiting both the descriptive and predictive ability of in vitro constructs. To address this challenge, the aim of this PhD project is to leverage the potential of a bioengineered human skin equivalent characterized by the presence of an endogenously produced extracellular matrix (ECM). As UV irradiation deeply affects ECM constituents and their organization, a skin model which is able to recapitulate relevant in vivo structures can effectively provide reliable characterizations and readouts. By exploiting a previously described bottom-up approach, a human dermis equivalent (HDE) can be obtained starting with micro-tissue precursors (μTPs). The rationale behind this procedure is to let fibroblasts expand and produce their own ECM, being at the same time the main drivers for its organization. This would resemble what is actually observed during in vivo tissue morphogenesis, therefore representing an ideal setup to recapitulate physiological structures. After the establishment of a dermis equivalent, human keratinocytes are seeded on top of each construct, allowing the formation of a stratified epithelial layer after two weeks of air-liquid interface (ALI) culture. In the first phase of the project, the full-thickness models thus obtained, referred to as human skin equivalents (HSEs), are subjected to intermittent UV exposure for 14 days through a UV lamp, with experimental time accurately selected to mimic the daily exposure to sunlight. The experimental setup includes two time points: the first one after 7 days of UV exposure, and the second one at the end of the experiment. Histological and immunofluorescent analyses are then conducted to qualitatively and quantitatively assess morphological changes occurring within the irradiated ECM. Progressive loss in the organization of collagen fibers as well as an accelerated development of the epithelial layer are among the main indicators of an aged phenotype. Furthermore, immunostaining allows the assessment of keratinization state. Subsequently, mRNA sequencing analyses are performed to elucidate the biological pathways involved in the response to UV-induced stresses. Assessing gene expression changes upon induction of a certain stimulus is a widely used procedure favored by the rapid advancements in sequencing technologies in recent years. Through annotation and enrichment analyses, relevant genes and gene sets can be retrieved, thus enabling a more comprehensive understanding of the molecular activity of cells. If coupled with morphological analyses, a complete description of the effects of repeated UV exposures can be achieved. Furthermore, the experimental setup allows the evaluation of both time and exposure as sources of gene expression changes, replicating the slow and steady accumulation of damage experienced during a lifetime. Additionally, comparison with transcriptomic profiles retrieved from open-source databases can help evaluate the accuracy of the realized skin constructs in correctly describing pathophysiological conditions. In parallel, epigenetics, which studies mechanisms that regulate gene expression without altering the DNA sequence offers another layer of understanding. Some examples of epigenetic modifications include DNA methylation, post-translational modification of histones as well as histone ubiquitination. DNA methylation describes the addition of a methyl group to the fifth carbon of the cytosine residues by DNA methyltransferases (DNMts). Changes in methylation patterns have been linked to aging and age-related diseases, making it a valuable tool for understanding the molecular mechanisms driving aging. Specifically, DNA methylation analysis on UV-irradiated skin samples provides valuable insights into the epigenetic mechanisms of aging. UV radiation, a key factor in skin aging, deeply contributes to epigenetic changes by generating reactive oxygen species (ROS) that damage DNA and alter the activity of DNMTs. Reduced representation bisulfite sequencing (RRBS) is performed on DNA extracted from both control and treated samples.

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