Skinly: A novel handheld IoT device for validating biophysical skin characteristics.

BACKGROUND: Recent advancements in artificial intelligence have revolutionized dermatological diagnostics. These technologies, particularly machine learning (ML), including deep learning (DL), have shown accuracy equivalent or even superior to human experts in diagnosing skin conditions like melanoma. With the integration of ML, including DL, the development of at home skin analysis devices has become feasible. To this end, we introduced the Skinly system, a handheld device capable of evaluating various personal skin characteristics noninvasively. MATERIALS AND METHODS: Equipped with a moisture sensor and a multi-light-source camera, Skinly can assess age-related skin parameters and specific skin properties. Utilizing state-of-the-art DL, Skinly processed vast amounts of images efficiently. The Skinly system's efficacy was validated both in the lab and at home, comparing its results to established "gold standard" methods. RESULTS: Our findings revealed that the Skinly device can accurately measure age-associated parameters, that is, facial age, skin evenness, and wrinkles. Furthermore, Skinly produced data consistent with established devices for parameters like glossiness, skin tone, redness, and porphyrin levels. A separate study was conducted to evaluate the effects of two moisturizing formulations on skin hydration in laboratory studies with standard instrumentation and at home with Skinly. CONCLUSION: Thanks to its capability for multi-parameter measurements, the Skinly device, combined with its smartphone application, holds the potential to replace more expensive, time-consuming diagnostic tools. Collectively, the Skinly device opens new avenues in dermatological research, offering a reliable, versatile tool for comprehensive skin analysis.

Development of an epigenetic clock to predict visual age progression of human skin.

Aging is a complex process characterized by the gradual decline of physiological functions, leading to increased vulnerability to age-related diseases and reduced quality of life. Alterations in DNA methylation (DNAm) patterns have emerged as a fundamental characteristic of aged human skin, closely linked to the development of the well-known skin aging phenotype. These changes have been correlated with dysregulated gene expression and impaired tissue functionality. In particular, the skin, with its visible manifestations of aging, provides a unique model to study the aging process. Despite the importance of epigenetic age clocks in estimating biological age based on the correlation between methylation patterns and chronological age, a second-generation epigenetic age clock, which correlates DNAm patterns with a particular phenotype, specifically tailored to skin tissue is still lacking. In light of this gap, we aimed to develop a novel second-generation epigenetic age clock explicitly designed for skin tissue to facilitate a deeper understanding of the factors contributing to individual variations in age progression. To achieve this, we used methylation patterns from more than 370 female volunteers and developed the first skin-specific second-generation epigenetic age clock that accurately predicts the skin aging phenotype represented by wrinkle grade, visual facial age, and visual age progression, respectively. We then validated the performance of our clocks on independent datasets and demonstrated their broad applicability. In addition, we integrated gene expression and methylation data from independent studies to identify potential pathways contributing to skin age progression. Our results demonstrate that our epigenetic age clock, VisAgeX, specifically predicting visual age progression, not only captures known biological pathways associated with skin aging, but also adds novel pathways associated with skin aging.

Multi-omics network analysis reveals distinct stages in the human aging progression in epidermal tissue.

In recent years, reports of non-linear regulations in age- and longevity-associated biological processes have been accumulating. Inspired by methodological advances in precision medicine involving the integrative analysis of multi-omics data, we sought to investigate the potential of multi-omics integration to identify distinct stages in the aging progression from ex vivo human skin tissue. For this we generated transcriptome and methylome profiling data from suction blister lesions of female subjects between 21 and 76 years, which were integrated using a network fusion approach. Unsupervised cluster analysis on the combined network identified four distinct subgroupings exhibiting a significant age-association. As indicated by DNAm age analysis and Hallmark of Aging enrichment signals, the stages captured the biological aging state more clearly than a mere grouping by chronological age and could further be recovered in a longitudinal validation cohort with high stability. Characterization of the biological processes driving the phases using machine learning enabled a data-driven reconstruction of the order of Hallmark of Aging manifestation. Finally, we investigated non-linearities in the mid-life aging progression captured by the aging phases and identified a far-reaching non-linear increase in transcriptional noise in the pathway landscape in the transition from mid- to late-life.

Methylation profiling identifies two subclasses of squamous cell carcinoma related to distinct cells of origin.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and usually progresses from a UV-induced precancerous lesion termed actinic keratosis (AK). Despite various efforts to characterize these lesions molecularly, the etiology of AK and its progression to cSCC remain partially understood. Here, we use Infinium MethylationEPIC BeadChips to interrogate the DNA methylation status in healthy, AK and cSCC epidermis samples. Importantly, we show that AK methylation patterns already display classical features of cancer methylomes and are highly similar to cSCC profiles. Further analysis identifies typical features of stem cell methylomes, such as reduced DNA methylation age, non-CpG methylation, and stem cell-related keratin and enhancer methylation patterns. Interestingly, this signature is detected only in half of the samples, while the other half shows patterns more closely related to healthy epidermis. These findings suggest the existence of two subclasses of AK and cSCC emerging from distinct keratinocyte differentiation stages.

An integrative metabolomics and transcriptomics study to identify metabolic alterations in aged skin of humans in vivo.

BACKGROUND: Aging human skin undergoes significant morphological and functional changes such as wrinkle formation, reduced wound healing capacity, and altered epidermal barrier function. Besides known age-related alterations like DNA-methylation changes, metabolic adaptations have been recently linked to impaired skin function in elder humans. Understanding of these metabolic adaptations in aged skin is of special interest to devise topical treatments that potentially reverse or alleviate age-dependent skin deterioration and the occurrence of skin disorders. RESULTS: We investigated the global metabolic adaptions in human skin during aging with a combined transcriptomic and metabolomic approach applied to epidermal tissue samples of young and old human volunteers. Our analysis confirmed known age-dependent metabolic alterations, e.g. reduction of coenzyme Q10 levels, and also revealed novel age effects that are seemingly important for skin maintenance. Integration of donor-matched transcriptome and metabolome data highlighted transcriptionally-driven alterations of metabolism during aging such as altered activity in upper glycolysis and glycerolipid biosynthesis or decreased protein and polyamine biosynthesis. Together, we identified several age-dependent metabolic alterations that might affect cellular signaling, epidermal barrier function, and skin structure and morphology. CONCLUSIONS: Our study provides a global resource on the metabolic adaptations and its transcriptional regulation during aging of human skin. Thus, it represents a first step towards an understanding of the impact of metabolism on impaired skin function in aged humans and therefore will potentially lead to improved treatments of age related skin disorders.

Acute Activation of Oxidative Pentose Phosphate Pathway as First-Line Response to Oxidative Stress in Human Skin Cells.

Integrity of human skin is endangered by exposure to UV irradiation and chemical stressors, which can provoke a toxic production of reactive oxygen species (ROS) and oxidative damage. Since oxidation of proteins and metabolites occurs virtually instantaneously, immediate cellular countermeasures are pivotal to mitigate the negative implications of acute oxidative stress. We investigated the short-term metabolic response in human skin fibroblasts and keratinocytes to H2O2 and UV exposure. In time-resolved metabolomics experiments, we observed that within seconds after stress induction, glucose catabolism is routed to the oxidative pentose phosphate pathway (PPP) and nucleotide synthesis independent of previously postulated blocks in glycolysis (i.e., of GAPDH or PKM2). Through ultra-short (13)C labeling experiments, we provide evidence for multiple cycling of carbon backbones in the oxidative PPP, potentially maximizing NADPH reduction. The identified metabolic rerouting in oxidative and non-oxidative PPP has important physiological roles in stabilization of the redox balance and ROS clearance.

The nano-scale mechanical properties of the extracellular matrix regulate dermal fibroblast function.

Changes in the mechanical properties of dermis occur during skin aging or tissue remodeling and affect the activity of resident fibroblasts. With the aim to establish elastic culture substrates that reproduce the variable softness of dermis, we determined Young's elastic modulus E of human dermis at the cell perception level using atomic force microscopy. The E of dermis ranged from 0.1 to 10 kPa, varied depending on body area and dermal layer, and tended to increase with age in 26-55-year-old donors. The activation state of human dermal fibroblasts cultured on "skin-soft" E (5 kPa) silicone culture substrates was compared with stiff plastic culture (GPa), collagen gel cultures (0.1-9 kPa), and fresh human dermal tissue. Fibroblasts cultured on skin-soft silicones displayed low mRNA levels of fibrosis-associated genes and increased expression of the matrix metalloproteinases (MMPs) MMP-1 and MMP-3 as compared with collagen gel and plastic cultures. The activation profile exhibited by fibroblasts on "skin-soft" silicone culture substrates was most comparable with that of human dermis than any other tested culture condition. Hence, providing biomimetic mechanical conditions generates fibroblasts that are more suitable to investigate physiologically relevant cell processes than fibroblasts spontaneously activated by stiff conventional culture surfaces.

A tissue-engineered human dermal construct utilizing fibroblasts and transforming growth factor ß1 to promote elastogenesis.

Numerous studies have shown that extracellular matrix (ECM)-based scaffolds are suitable for dermal constructs for the differentiation of various cell types in vitro and for constructive tissue remodeling after implantation in vivo. However, a shortcoming of these ECM materials is its limited elastogenesis. Elastic fibers constitute an essential component of mammalian connective tissue and the presence of elastic fibers is crucial for the proper function of the cardiovascular, pulmonary, and intestinal systems. Since it is still largely unknown how cells coordinate the molecular events of elastic-fiber assembly, understanding the ability to regenerate elastic fibers in tissues remains a significant challenge. For this reason, human neonatal dermal fibroblasts (HDFneo) were analyzed for their potential to serve as a cell culture model for elastic fiber assembly. Using optical technologies such as multiphoton laser-scanning microscopy (MPSLM) we demonstrate that HDFneo stimulated with transforming growth factor ß1 (TGF-ß1) are able to produce a distinct and complex elastic fiber system in vitro. As shown by the desmosine and isodesmosine content, crosslinked elastic fibers were formed within the 3D ECM-based scaffold. This tissue-engineered dermal construct may prove to be an effective template for the development of medicinal approaches in regenerative soft skin tissue reconstruction through TGF-ß1 induction.

UV-mediated downregulation of the endocytic collagen receptor, Endo180, contributes to accumulation of extracellular collagen fragments in photoaged skin.

BACKGROUND: Collagen is the most abundant protein in human skin and is responsible for its resilience. In particular during photoaging, collagen homeostasis is out of balance leading to a continuous loss of intact collagen and to the observed signs of aged skin such as diminished tensile strength and wrinkle development. The process of collagen turnover is very slow and the relevance of cellular uptake of damaged collagen, most likely mediated via Endo180 or integrin α2ß1, still remains a matter of investigation. OBJECTIVE: We investigated the role of different collagen receptors on dermal fibroblasts for collagen internalization and their impact on collagen homeostasis during photoaging. METHODS: TaqMan Real-Time PCR, flow cytometry, UV irradiation, knockdown experiments and immunostaining. RESULTS: We show that Endo180 and integrin α2 are regulated in photoaged skin and after acute UV stress in vivo and in vitro. Knockdown experiments revealed that Endo180 is essential for cellular uptake of collagen fragments by dermal fibroblasts, whereas integrin α2 is important for initial binding of collagen. UV irradiation decreases collagen endocytosis. This correlates with reduced Endo180 expression and pericellular accumulation of collagen fragments during photoaging. CONCLUSION: Our findings correlate for the first time impaired collagen uptake via Endo180 with the pericellular accumulation of collagen fragments during photoaging. We assume an altered pericellular niche of fibroblasts in photoaged skin that has an impact on collagen homeostasis.

MicroRNA profiling during human keratinocyte differentiation using a quantitative real-time PCR method.

The terminal differentiation of epidermal keratinocytes requires transcriptional and posttranscriptional regulatory mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that play key roles during differentiation processes by regulating protein expression at the posttranscriptional level. Several studies have investigated miRNA expression in murine or human skin using northern blotting, microarrays, deep sequencing, or real-time PCR (Andl et al., Curr Biol 16:1041-1049, 2006; Hildebrand et al., J Invest Dermatol 131:20-29, 2011; Sonkoly et al., PLoS One 2:e610, 2007; Yi et al., Nat Genet 38:356-362, 2006; Yi et al., Proc Natl Acad Sci U S A 106:498-502, 2009). Conventional techniques such as northern blotting and microarrays often fail to detect miRNAs of low abundance, while the per-sample cost of deep sequencing approaches is still prohibitive in many cases. In contrast, stem loop primer-based real-time PCR methods for simultaneous detection of up to 380 miRNAs allow fast, specific, and reliable miRNA profiling. These methods are suitable for in vitro material, but also for samples which are of limited availability, such as epidermal stem cells isolated from human skin biopsies. Here, we describe a general real-time PCR method for miRNA profiling using isolated epidermal stem cells, transiently amplifying cells and terminally differentiated keratinocytes of human skin.

A novel niche for skin derived precursors in non-follicular skin.

BACKGROUND: Skin derived precursors (SKP) comprise a subset of specialized dermal cells that can be distinguished from fibroblast by their capacity for spheroidal growth. Recent investigations have shown that hair follicles constitute a niche for this cell type, but their localization and their definite function in non-follicular skin remains largely unknown. OBJECTIVE: To identify the dermal niche of non-follicular SKPs and to analyze whether functional aspects correlate with this localization. METHODS: SKPs were isolated from separate anatomical regions of human abdominal skin. Fluorescence activated cell sorting then was used to obtain a pure population of non-follicular SKPs. Functional characterization of these cells was performed applying differentiation and proliferation assays. Information on specific in vivo functions was derived from histological evaluation of quantity and localization patterns. RESULTS: Sphere forming capacity and differentiation assays show that SKPs reside in the papillary part of the dermis. Further delineation revealed that the dermal capillaries represent a niche for these cells which subsequently could be isolated by FACS utilizing a perivascular marker. Whereas functional properties described for follicular SKPs could also be detected in the perivascular SKP population, histological analyses additionally point to a cross-talk with epidermal stem cells and a reduction during chronological aging. CONCLUSION: Our data show that SKPs isolated from non-follicular skin originate from a perivascular niche. Compared to their follicular counterparts, no functional differences could be observed upon cultivation, but ex vivo analyses also point to unique functions and a contribution to the phenotype of aged skin.

Approach to quantify human dermal skin aging using multiphoton laser scanning microscopy.

Extracellular skin structures in human skin are impaired during intrinsic and extrinsic aging. Assessment of these dermal changes is conducted by subjective clinical evaluation and histological and molecular analysis. We aimed to develop a new parameter for the noninvasive quantitative determination of dermal skin alterations utilizing the high-resolution three-dimensional multiphoton laser scanning microscopy (MPLSM) technique. To quantify structural differences between chronically sun-exposed and sun-protected human skin, the respective collagen-specific second harmonic generation and the elastin-specific autofluorescence signals were recorded in young and elderly volunteers using the MPLSM technique. After image processing, the elastin-to-collagen ratio (ELCOR) was calculated. Results show that the ELCOR parameter of volar forearm skin significantly increases with age. For elderly volunteers, the ELCOR value calculated for the chronically sun-exposed temple area is significantly augmented compared to the sun-protected upper arm area. Based on the MPLSM technology, we introduce the ELCOR parameter as a new means to quantify accurately age-associated alterations in the extracellular matrix.

Impact of collagen crosslinking on the second harmonic generation signal and the fluorescence lifetime of collagen autofluorescence.

BACKGROUND/PURPOSE: Collagen is the major structural protein of the skin and its crosslinks are essential for its mechanical stability. In photodamaged skin, a decrease of the mature collagen crosslink histidinohydroxylysino-norleucine was reported. In this study, we investigated the consequences and measurability of the reduced crosslinking. METHODS: In order to determine the consequences of reduced collagen crosslinking, in vitro models of reduced collagen crosslinking were established. The collagen synthesis and structure was analyzed using the signals second harmonic generation (SHG) and the fluorescence lifetime of the collagen autofluorescence by a multiphoton laser scanning microscope. RESULTS: Reduced collagen crosslinking results in a posttranscriptionally diminished collagen synthesis, a modified structure of the collagen fibers and fibrils and a higher intensity of the SHG signal. The SHG signal might be influenced by the interspaces of the collagen molecules within one collagen fibril. Because of these findings, it can be speculated that reduced collagen crosslinking changes the interspace of single collagen molecules within the collagen fibril, resulting in an enhanced SHG signal. Alternative explanations are discussed. Furthermore, the fluorescence lifetime was reduced in the in vitro models of reduced collagen crosslinking. In the crosslink sites of the collagen molecules, the main ratio of fluorescence is found. As the fluorescence lifetime is determined not only by the fluorescent molecule itself but also by its microenvironment, the change in the fluorescence lifetime might be explained by reduced crosslinking at the crosslink site. CONCLUSION: A reduction of collagen crosslinking (as seen in photodamaged skin) results in an increase of the SHG signal and a decrease of the fluorescence lifetime in vitro. In vivo measurements of the two parameters might reveal the status of collagen crosslinking and therefore help to identify the status of dermal photodamage or pathogenesis using collagen crosslinking determination.

Dermal penetration of creatine from a face-care formulation containing creatine, guarana and glycerol is linked to effective antiwrinkle and antisagging efficacy in male subjects.

BACKGROUND: The dermal extracellular matrix provides stability and structure to the skin. With increasing age, however, its major component collagen is subject to degeneration, resulting in a gradual decline in skin elasticity and progression of wrinkle formation. Previous studies suggest that the reduction in cellular energy contributes to the diminished synthesis of cutaneous collagen during aging. AIMS: To investigate the potential of topically applied creatine to improve the clinical signs of skin aging by stimulating dermal collagen synthesis in vitro and in vivo. PATIENTS/METHODS: Penetration experiments were performed with a pig skin ex vivo model. Effects of creatine on dermal collagen gene expression and procollagen synthesis were studied in vitro using cultured fibroblast-populated collagen gels. In a single-center, controlled study, 43 male Caucasians applied a face-care formulation containing creatine, guarana extract, and glycerol to determine its influence on facial topometric features. RESULTS: Cultured human dermal fibroblasts supplemented with creatine displayed a stimulation of collagen synthesis relative to untreated control cells both on the gene expression and at the protein level. In skin penetration experiments, topically applied creatine rapidly reached the dermis. In addition, topical in vivo application of a creatine-containing formulation for 6 weeks significantly reduced the sagging cheek intensity in the jowl area as compared to baseline. This result was confirmed by clinical live scoring, which also demonstrated a significant reduction in crow's feet wrinkles and wrinkles under the eyes. CONCLUSIONS: In summary, creatine represents a beneficial active ingredient for topical use in the prevention and treatment of human skin aging.

Damage at the root of cell renewal--UV sensitivity of human epidermal stem cells.

BACKGROUND: The epidermis harbors adult stem cells that reside in the basal layer and ensure the continuous maintenance of tissue homeostasis. Various studies imply that stem cells generally possess specific defense mechanisms against several forms of exogenous stress factors. As sun exposition is the most prevalent impact on human skin, this feature would be of particular importance in terms of sensitivity to UV-induced DNA damage. OBJECTIVE: To investigate whether human epidermal stem cells are susceptible to UV-induced DNA damage and subsequent functional impairment. METHODS: A method to isolate human epidermal stem cells from suction blister epidermis was established and validated. Volunteers were treated with solar-simulated irradiation on test areas of the forearm and stem cells were isolated from suction blister material of this region. DNA damage was analyzed by staining for cyclobutane thymidine dimers. The functional consequences of UV-induced damages were assessed by colony forming efficiency assays and gene expression analyses. RESULTS: Compared to an unirradiated control, stem cells isolated from areas that were exposed to solar-simulated radiation showed significantly more DNA lesions. Although the number of stem cells was not reduced by this treatment, a functional impairment of stem cells could be shown by reduced colony forming efficiency and altered gene expression of stem cell markers. CONCLUSIONS: Despite their essential role in skin maintenance, epidermal stem cells are sensitive to physiological doses of UV irradiation in vivo.

Folic acid and creatine improve the firmness of human skin in vivo.

BACKGROUND: The decrease in firmness is a hallmark of skin aging. Accelerated by chronic sun exposure, fundamental changes occur within the dermal extracellular matrix over the years, mainly impairing the collagenous network. AIMS: Based on the qualitative and quantitative assessment of skin firmness, in vitro and in vivo studies were carried out to elucidate the effects of topical folic acid and creatine to counteract this age-dependent reduction in the amount of collagen. PATIENTS/METHODS: Topical application of a commercially available formulation containing folic acid and creatine was performed to study effects on skin firmness in vivo using cutometric analysis. Imaging and quantification of collagen density were carried out using multiphoton laser scanning microscopy (MPLSM). To investigate the effects of these compounds on collagen gene expression, procollagen synthesis, and collagen fibril organization, complementary in vitro studies on cultured fibroblast-populated collagen gels were carried out. RESULTS: The underlying structural changes in the collagen network of young and aged sun-exposed facial skin in vivo were visualized by MPLSM. Topical application of a folic acid- and creatine-containing formulation significantly improved firmness of mature skin in vivo. Treatment of fibroblast-populated dermal equivalents with folic acid and creatine increased collagen gene expression and procollagen levels and improved collagen fiber density, suggesting that the in vivo effects are based on the overall improvement of the collagen metabolism. CONCLUSIONS: Employing MPLSM, dermal changes occurring in photo-aged human skin were visualized in an unprecedented manner and correlated to a loss of firmness. Treatment of aged skin with a topical formulation containing folic acid and creatine counteracted this age-dependent decline by exerting sustained effects on collagen metabolism. Our results support previous findings on the efficacy of these actives.

A comprehensive analysis of microRNA expression during human keratinocyte differentiation in vitro and in vivo.

Here, we report a comprehensive investigation of changes in microRNA (miRNA) expression profiles on human keratinocyte (HK) differentiation in vitro and in vivo. We have monitored expression patterns of 377 miRNAs during calcium-induced differentiation of primary HKs, and have compared these patterns with miRNA expression profiles of epidermal stem cells, transient amplifying cells, and terminally differentiated HKs from human skin. Apart from the previously described miR-203, we found an additional nine miRNAs (miR-23b, miR-95, miR-210, miR-224, miR-26a, miR-200a, miR-27b, miR-328, and miR-376a) that are associated with HK differentiation in vitro and in vivo. In situ hybridization experiments confirmed miR-23b as a marker of HK differentiation in vivo. Additionally, gene ontology analysis and functional validation of predicted miRNA targets using 3'-untranslated region-luciferase assays suggest that multiple miRNAs that are upregulated on HK differentiation cooperate to regulate gene expression during skin development. Our results thus provide the basis for further analysis of miRNA functions during epidermal differentiation.

In situ localization of epidermal stem cells using a novel multi epitope ligand cartography approach.

Precise knowledge of the frequency and localization of epidermal stem cells within skin tissue would further our understanding of their role in maintaining skin homeostasis. As a novel approach we used the recently developed method of multi epitope ligand cartography, applying a set of described putative epidermal stem cell markers. Bioinformatic evaluation of the data led to the identification of several discrete basal keratinocyte populations, but none of them displayed the complete stem cell marker set. The distribution of the keratinocyte populations within the tissue was remarkably heterogeneous, but determination of distance relationships revealed a population of quiescent cells highly expressing p63 and the integrins alpha(6)/beta(1) that represent origins of a gradual differentiation lineage. This population comprises about 6% of all basal cells, shows a scattered distribution pattern and could also be found in keratinocyte holoclone colonies. The data suggest that this population identifies interfollicular epidermal stem cells.