Challenges and Opportunities for the Translation of Single-Cell RNA Sequencing Technologies to Dermatology

Skin is a complex and heterogeneous organ at the cellular level. This complexity is beginning to be understood through the application of single-cell genomics and computational tools. A large number of datasets that shed light on how the different human skin cell types interact in homeostasis—and what ceases to work in diverse dermatological diseases—have been generated and are publicly available. However, translation of these novel aspects to the clinic is lacking. This review aims to summarize the state-of-the-art of skin biology using single-cell technologies, with a special focus on skin pathologies and the translation of mechanistic findings to the clinic. The main implications of this review are to summarize the benefits and limitations of single-cell analysis and thus help translate the emerging insights from these novel techniques to the bedside.

Reporter System Controlled by the Involucrin Promoter as a Tool to Follow the Epidermal Differentiation

Different approaches have been explored to study skin biology, including the use of stem cells. Mesenchymal stem cells (MSC) from umbilical cord can be safely and easily obtained, however a simple strategy to monitor their differentiation is essential. Involucrin is a marker of keratinocyte terminal differentiation, and its promoter (pINV) directs stratum-specific expression of this protein. We designed a reporter system containing EGFP under control of pINV to assess MSC differentiation into keratinocytes. The functional sequence of pINV was inserted into a lentiviral vector, originating LeGO-GpINV. MSC were transduced with the LeGO-GpINV and induced to differentiate into keratinocytes upon cultivation with Keratinocyte Serum Free Medium supplemented. MSC differentiation was confirmed by morphological changes and by the expression of epidermal markers, by flow cytometry, quantitative PCR and western blot. The activity of kallikreins 5, 6 and 7 was detected using fluorogenic substrates. After 14 days of differentiation, MSC transduced with LeGO-GpINV showed an increase in EGFP fluorescence and expressed CK10, CK14, involucrin and filaggrin. There was also an increase in the kallikrein activity. This reporter system allowed to temporally assess the epidermal differentiation, simultaneously with involucrin expression, opening perspectives for the in vivo study of skin biology and in regenerative medicine.

Identification of Chicken Transglutaminase 1 and In Situ Localization of Transglutaminase Activity in Avian Skin and Esophagus

Transglutaminase 1 (TGM1) is a membrane-anchored enzyme that cross-links proteins during terminal differentiation of epidermal and esophageal keratinocytes in mammals. The current genome assembly of the chicken, which is a major model for avian skin biology, does not include an annotated region corresponding to TGM1. To close this gap of knowledge about the genetic control of avian cornification, we analyzed RNA-sequencing reads from organotypic chicken skin and identified TGM1 mRNA. By RT-PCR, we demonstrated that TGM1 is expressed in the skin and esophagus of chickens. The cysteine-rich sequence motif required for palmitoylation and membrane anchorage is conserved in the chicken TGM1 protein, and differentiated chicken keratinocytes display membrane-associated transglutaminase activity. Expression of TGM1 and prominent transglutaminase activity in the esophageal epithelium was also demonstrated in the zebra finch. Altogether, the results of this study indicate that TGM1 is conserved among birds and suggest that chicken keratinocytes may be a useful model for the study of TGM1 in non-mammalian cornification.

Ex Vivo Human Skin: An Alternative Test System for Skin Irritation and Corrosion Assays

Native human skin has been reported in the literature as being an important experimental model for studying skin biology. Studies performed by our group have shown that ex vivo skin, from elective plastic surgery, maintains the biological characteristics of native skin under specific culture conditions. As such, it might be a feasible model for the safety and efficacy testing of topical substances. While Brazil is at the forefront of global regulation implementation, Brazilian researchers are not always able to transfer certain widely used protocols to their laboratories, particularly protocols that involve the use of reconstructed tissues with limited viability, such as those for skin corrosion (OECD TG 431) and irritation testing (OECD TG 439). In this study, we investigated the applicability of the ex vivo skin model to the evaluation of irritation and corrosion potential of a number of proficiency substances described in TG 431 and TG 439. The skin fragments were standardised in size and diameter, and placed into cell culture inserts. The experimental protocol was conducted according to TG 431 and TG 439. The results obtained show that ex vivo skin could represent a promising tool for the evaluation of irritation and corrosion potential of substances (subject to inclusion and exclusion criteria), as recommended by OECD guidelines. While this is a proof-of-concept study, the use of ex vivo skin should be considered for such testing.

IL-1 Family Antagonists in Mouse and Human Skin Inflammation

Interleukin (IL)-1 family cytokines initiate inflammatory responses, and shape innate and adaptive immunity. They play important roles in host defense, but excessive immune activation can also lead to the development of chronic inflammatory diseases. Dysregulated IL-1 family signaling is observed in a variety of skin disorders. In particular, IL-1 family cytokines have been linked to the pathogenesis of psoriasis and atopic dermatitis. The biological activity of pro-inflammatory IL-1 family agonists is controlled by the natural receptor antagonists IL-1Ra and IL-36Ra, as well as by the regulatory cytokines IL-37 and IL-38. These four anti-inflammatory IL-1 family members are constitutively and highly expressed at steady state in the epidermis, where keratinocytes are a major producing cell type. In this review, we provide an overview of the current knowledge concerning their regulatory roles in skin biology and inflammation and their therapeutic potential in human inflammatory skin diseases. We further highlight some common misunderstandings and less well-known observations, which persist in the field despite recent extensive interest for these cytokines.

Leptin System in Obese Dog Skin: A Pilot Study

Obesity predisposes to several health problems including skin diseases. However, information on the relationship between obesity and skin disorders in pets is very scarce. Leptin (LEP) is mainly produced by adipose tissue and has a prominent role in skin biology. This study evaluated the LEP system in the skin of obese dogs compared to normal-weight animals. The investigation was carried out on 10 obese (Obese group) and 10 normal-weight (Normal-weight group) dogs through Real-time PCR and immunohistochemistry. Cells of skin associated immune system were also evaluated. No differences were evidenced between the two groups as well as skin inflammation. LEP differences were no significant, while LEPR transcript appeared 10-fold higher in obesedogs than in normal-weight ones. Immunostaining for both molecules was observed in several skin structures such as the epidermis, hair follicles, and glands. No differences appeared in the skin associated immune system composition. This study is a preliminary report showing that LEP system changes in obese dog skin. The increased LEPR expression observed in the obese group suggests that the receptor plays a modulating role in the system control. However, the exact role of LEPin the skin under obesity conditions needs further elucidation.

Foxn1 Control of Skin Function

The forkhead box N1 (Foxn1) transcription factor regulates biological processes of the thymus and skin. Loss-of-function mutations in Foxn1 cause the nude phenotype in humans, mice, and rats, which is characterized by hairless skin and a lack of thymus. This review focuses on the role of Foxn1 in skin biology, including epidermal, dermal, and dermal white adipose tissue (dWAT) skin components. In particular, the role of Foxn1 in the scar-forming skin wound healing process is discussed, underscoring that Foxn1 inactivity in nude mice is permissive for scar-less cutaneous wound resolution.

Generation of human hair-bearing skin organoids from stem cells

Skin is a complex and vulnerable tissue that it is challenging to reconstitute once damaged. Here, we describe a three-dimensional organoid culture system that can generate fully stratified skin with its appendages from human pluripotent stem cells. This in vitro-based skin organoid culture system will benefit investigations into basic skin biology and disease modeling, as well as translational efforts to reconstruct or regenerate skin tissue.

Shedding Light on the Effects of Calorie Restriction and Its Mimetics on Skin Biology

During the aging process of an organism, the skin gradually loses its structural and functional characteristics. The skin becomes more fragile and vulnerable to damage, which may contribute to age-related diseases and even death. Skin aging is aggravated by the fact that the skin is in direct contact with extrinsic factors, such as ultraviolet irradiation. While calorie restriction (CR) is the most effective intervention to extend the lifespan of organisms and prevent age-related disorders, its effects on cutaneous aging and disorders are poorly understood. This review discusses the effects of CR and its alternative dietary intake on skin biology, with a focus on skin aging. CR structurally and functionally affects most of the skin and has been reported to rescue both age-related and photo-induced changes. The anti-inflammatory, anti-oxidative, stem cell maintenance, and metabolic activities of CR contribute to its beneficial effects on the skin. To the best of the author’s knowledge, the effects of fasting or a specific nutrient-restricted diet on skin aging have not been evaluated; these strategies offer benefits in wound healing and inflammatory skin diseases. In addition, well-known CR mimetics, including resveratrol, metformin, rapamycin, and peroxisome proliferator-activated receptor agonists, show CR-like prevention against skin aging. An overview of the role of CR in skin biology will provide valuable insights that would eventually lead to improvements in skin health.