'Speed-ageing' of human skin in serum-free organ culture ex vivo: An instructive novel assay for preclinical human skin ageing research demonstrates senolytic effects of caffeine and 2,5-dimethylpyrazine.

Preclinical human skin ageing research has been limited by the paucity of instructive and clinically relevant models. In this pilot study, we report that healthy human skin of different age groups undergoes extremely accelerated ageing within only 3 days, if organ-cultured in a defined serum-free medium. Quantitative (immuno-)histomorphometry documented this unexpected ex vivo phenotype on the basis of ageing-associated biomarkers: the epidermis showed significantly reduced rete ridges and keratinocyte proliferation, sirtuin-1, MTCO1 and collagen 17a1 protein levels; this contrasted with significantly increased expression of the DNA-damage marker, γH2A.X. In the dermis, collagen 1 and 3 and hyaluronic acid content were significantly reduced compared to Day 0 skin. qRT-PCR of whole skin RNA extracts also showed up-regulated mRNA levels of several (inflamm-) ageing biomarkers (MMP-1, -2, -3, -9; IL6, IL8, CXCL10 and CDKN1). Caffeine, a methylxanthine with recognized anti-ageing properties, counteracted the dermal collagen 1 and 3 reduction, the epidermal accumulation of γH2A.X, and the up-regulation of CXCL10, IL6, IL8, MMP2 and CDKN1. Finally, we present novel anti-ageing effects of topical 2,5-dimethylpyrazine, a natural pheromone TRPM5 ion channel activator. Thus, this instructive, clinically relevant "speed-ageing" assay provides a simple, but powerful new research tool for dissecting skin ageing and rejuvenation, and is well-suited to identify novel anti-ageing actives directly in the human target organ.

Spatial Distribution and Functional Impact of Human Scalp Hair Follicle Microbiota.

Human hair follicles (HFs) constitute a unique microbiota habitat that differs substantially from the skin surface. Traditional HF sampling methods fail to eliminate skin microbiota contaminants or assess the HF microbiota incompletely, and microbiota functions in human HF physiology remain ill explored. Therefore, we used laser-capture microdissection, metagenomic shotgun sequencing, and FISH to characterize the human scalp HF microbiota in defined anatomical compartments. This revealed significant compartment-, tissue lineage-, and donor age-dependent variations in microbiota composition. Greatest abundance variations between HF compartments were observed for viruses, archaea, Staphylococcus epidermidis, Cutibacterium acnes, and Malassezia restricta, with the latter 2 being the most abundant viable HF colonizers (as tested by propidium monoazide assay) and, surprisingly, most abundant in the HF mesenchyme. Transfection of organ-cultured human scalp HFs with S. epidermidis-specific lytic bacteriophages ex vivo downregulated transcription of genes known to regulate HF growth and development, metabolism, and melanogenesis, suggesting that selected microbial products may modulate HF functions. Indeed, HF treatment with butyrate, a metabolite of S. epidermidis and other HF microbiota, delayed catagen and promoted autophagy, mitochondrial activity, and gp100 and dermcidin expression ex vivo. Thus, human HF microbiota show spatial variations in abundance and modulate the physiology of their host, which invites therapeutic targeting.

Management of the human hair follicle microbiome by a synthetic odorant.

BACKGROUND: Human scalp hair follicles (HFs) engage in olfactory receptor (OR)-dependent chemosensation. Activation of olfactory receptor family 2 subfamily AT member 4 (OR2AT4) by the synthetic, sandalwood-like odorant Sandalore® up-regulated HF antimicrobial peptide expression of dermcidin (DCD), which had previously been thought to be produced exclusively by sweat and sebaceous glands. OBJECTIVES: To understand if intrafollicular DCD production can be stimulated by a commonly used cosmetic odorant, thus altering human HF microbiome composition in a clinically beneficial manner. METHODS: DCD expression was compared between fresh-frozen scalp biopsies and microdissected, full-length scalp HFs, organ-cultured in the presence/absence of the OR2AT4 agonist, Sandalore® and/or antibiotics and/or the competitive OR2AT4 antagonist, Phenirat®. Amplicon-based sequencing and microbial growth assays were performed to assess how this treatment affected the HF microbiome. RESULTS: Synthetic odorant treatment upregulated epithelial DCD expression and exerted antimicrobial activity in human HFs ex vivo. Combined antibiotic and odorant treatment, during an ex vivo dysbiosis event, prevented HF tissue damage and favoured a more physiological microbiome composition. Sandalore®-conditioned medium, containing higher DCD content, favoured Staphylococcus epidermidis and Malassezia restricta over S. aureus and M. globosa, while exhibiting antimicrobial activity against Cutibacterium acnes. These effects were reversed by co-administration of Phenirat®. CONCLUSIONS: We provide the first proof-of-principle that a cosmetic odorant impacts the human HF microbiome by up-regulating antimicrobial peptide production in an olfactory receptor-dependent manner. Specifically, a synthetic sandalwood-like odorant stimulates intrafollicular DCD production, likely via OR2AT4, and thereby controls microbial overgrowth. Thus, deserving further exploration as an adjuvant therapeutic principle in the management of folliculitis and dysbiosis-associated hair diseases.

Application of Topical Sandalore® Increases Epidermal Dermcidin Synthesis in Organ-Cultured Human Skin ex vivo.

INTRODUCTION: Several olfactory receptors (ORs) are expressed in human skin, where they regulate skin pigmentation, barrier function, wound healing, and hair growth. Previously, we found that the selective activation of OR family 2 subfamily AT member 4 (OR2AT4) by the synthetic, sandalwood-like odorant Sandalore® differentially stimulates the expression of antimicrobial peptides (AMPs) in human scalp hair follicle epithelium ex vivo. As OR2AT4 is also expressed by epidermal keratinocytes, we hypothesized that it may modulate intraepidermal AMP synthesis, thereby contributing to skin microbiome management. METHODS: We investigated this hypothesis in organ-cultured human skin in the presence of Sandalore® and antibiotics and evaluated epidermal production of two AMPs, LL37 (cathelicidin) and dermcidin (DCD), as well as OR2AT4, by quantitative immunohistomorphometry. Moreover, we quantified DCD secretion into the culture medium by ELISA and studied the effect of culture medium on selected bacterial and fungal strains. RESULTS: Topical application of Sandalore®to organ-cultured human skin increased OR2AT4 protein expression, the number of DCD-positive intraepidermal cells, and DCD secretion into culture media, without significantly affecting epidermal LL37 expression. In line with the significantly increased secretion of DCD into the culture medium, we demonstrated, in a spectrophotometric assay, that application of conditioned media from Sandalore®-treated skin promotes Staphylococcus epidermidis, Malassezia restricta, and, minimally, Cutibacterium acnes and inhibits Staphylococcus aureus growth. CONCLUSION: In addition to demonstrating for the first time that DCD can be expressed by epidermal keratinocytes, our pilot study suggests that topical treatment of human skin with a cosmetic odorant (Sandalore®) has the potential to alter the composition of the human skin microbiome through the selective upregulation of DCD. If confirmed, Sandalore® could become an attractive adjuvant, nondrug treatment for dermatoses characterized by dysbiosis due to overgrowth of S. aureus and Malassezia, such as atopic dermatitis and seborrheic dermatitis.

Intermediate Hair Follicles from Patients with Female Pattern Hair Loss Are Associated with Nutrient Insufficiency and a Quiescent Metabolic Phenotype.

Female pattern hair loss (FPHL) is a non-scarring alopecia resulting from the progressive conversion of the terminal (t) scalp hair follicles (HFs) into intermediate/miniaturized (i/m) HFs. Although data supporting nutrient deficiency in FPHL HFs are lacking, therapeutic strategies are often associated with nutritional supplementation. Here, we show by metabolic analysis that selected nutrients important for hair growth such as essential amino acids and vitamins are indeed decreased in affected iHFs compared to tHFs in FPHL scalp skin, confirming nutrient insufficiency. iHFs also displayed a more quiescent metabolic phenotype, as indicated by altered metabolite abundance in freshly collected HFs and release/consumption during organ culture of products/substrates of TCA cycle, aerobic glycolysis, and glutaminolysis. Yet, as assessed by exogenous nutrient supplementation ex vivo, nutrient uptake mechanisms are not impaired in affected FPHL iHFs. Moreover, blood vessel density is not diminished in iHFs versus tHFs, despite differences in tHFs from different FPHL scalp locations or versus healthy scalp or changes in the expression of angiogenesis-associated growth factors. Thus, our data reveal that affected iHFs in FPHL display a relative nutrient insufficiency and dormant metabolism, but are still capable of absorbing nutrients, supporting the potential of nutritional supplementation as an adjunct therapy for FPHL.

An extract of Leontopodium alpinum inhibits catagen development ex vivo and increases hair density in vivo.

OBJECTIVES: Hair loss and reduction in hair volume are hallmarks of hair disorders, such as telogen effluvium, or male or female pattern hair loss, and hair ageing, which can cause severe distress in both men and women. Common anti-hair loss drugs carry some side effects; therefore, novel, safer approaches targeting milder phenotypes are highly advocated. In this context, we investigated an extract of the alpine plant Edelweiss, Leontopodium alpinum var. Helvetia, for its ability to modulate hair follicle (HF) growth ex vivo and inhibit hair loss while increasing hair regeneration in vivo. METHODS: Human amputated HFs were microdissected from three donors, two women and one man, and cultured ex vivo for 6 days. After treatment with 0.001% Edelweiss extract (EWDE), we investigated hair shaft production and anagen/catagen conversion, and measured known parameters associated with hair growth, that is hair matrix keratinocyte proliferation and apoptosis, dermal papilla inductivity, and growth factors, by quantitative (immuno)histomorphometry. To assess the anti-hair loss potential of the alpine plant compound, we performed a randomized, placebo-controlled human study enrolling Caucasian women and men, aged 18 to 65 years, with normal hair loss. After 5 months' daily use of an extract containing leave-on serum, we analysed hair density and anagen-to-catagen/telogen ratio by the Trichogram analysis. RESULTS: Our results revealed a significant prolongation in the anagen phase in HFs treated with 0.001% Edelweiss, as indicated by an increase in HFs remaining in anagen and a significant decrease in hair cycle score. In line with this effect, EWDE significantly stimulated hair matrix (HM) keratinocyte proliferation, and dermal papilla inductivity, as shown by a significant up-regulation of versican expression and alkaline phosphatase activity, and a tendential increase in FGF7 immunoreactivity in the dermal papilla of all HFs or only anagen VI HFs. Corroborating the ex vivo results, we observed a significant increase in growing hair shaft numbers (hair density) after treatment with Edelweiss extract formulation, and a tendential up-regulation in the anagen-to-catagen/telogen ratio. CONCLUSIONS: We show here, through several lines of evidence, that the selected extract of the alpine plant Leontopodium alpinum var Helvetia (Edelweiss) inhibits premature catagen induction, possibly by stimulating dermal papilla inductivity. It is therefore worth exploiting this extract clinically as an anti-hair loss agent, both for preventing ageing-associated hair shedding and as an adjuvant therapy for hair loss disorders.

OBJECTIFS: La perte de cheveux et la réduction du volume des cheveux sont caractéristiques des troubles capillaires, tels que l'effluvium télogène, ou la calvitie chez l'homme ou la femme, et le vieillissement des cheveux, qui peuvent causer une certaine détresse chez les hommes et les femmes. Les médicaments courants contre la chute des cheveux ont des effets secondaires, par conséquent, de nouvelles approches plus sûres ciblant des phénotypes légers sont fortement recommandées. Dans ce contexte, nous avons étudié un extrait de la plante alpine Edelweiss, Leontopodium alpinum var. Helvetia, pour sa capacité à stimuler la croissance du follicule pileux (HF) ex vivo et à inhiber la chute des cheveux tout en augmentant la régénération des fibres capillaires in vivo. MÉTHODES: Les follicules pileux (HF) humains prélevés ont été microdisséqués chez trois donneurs, deux femmes et un homme, et cultivés ex vivo pendant 6 jours. Après le traitement avec l'extrait d'Edelweiss à 0,001 % (EWDE), nous avons étudié la production de fibre capillaire et la conversion anagène/catagène, ainsi que mesuré les paramètres connus associés à la croissance des cheveux, à savoir, la prolifération des kératinocytes dans la matrice capillaire et l'apoptose, l'induction des papilles dermiques, et des facteurs de croissance, par (immuno-)histomorphométrie quantitative. Pour évaluer le potentiel des propriétés anti-chute du cheveu de l'extrait de plante alpine, nous avons réalisé une étude clinique aléatoire avec placebo, sur des femmes et des hommes de type caucasien âgés de 18 à 65 ans présentant une perte de cheveux normale. Après cinq mois d'utilisation quotidienne d'un sérum sans rinçage contenant l'extrait de plante, nous avons analysé la densité capillaire et le rapport anagène à catagène/télogène par trichogramme. RÉSULTATS: Nos résultats ont révélé une prolongation significative de la phase anagène dans les HF traités avec 0,001% d'Edelweiss, comme l'indique une augmentation des HF restant en phase anagène et une diminution significative du « hair cycle score ¼. En ligne avec cet effet, EWDE a stimulé de façon significative la matrice du cheveux (HM), la prolifération des kératinocytes, et l'induction de la papille dermique, comme le montre une augmentation significative de l'expression du versican et de l'activité de la phosphatase alcaline, et une augmentation tendancielle de l'immunoréactivité FGF7 dans la papille dermique de tous les HF ou seulement des HF anagènes VI. Corroborant les résultats ex vivo, nous avons observé une augmentation significative du nombre de fibres capillaires (densité de cheveux) après le traitement avec la formulation d'extrait d'Edelweiss, et une augmentation tendancielle dans le rapport anagène à catagène/télogène. CONCLUSIONS: Nous montrons ici, à travers plusieurs éléments de preuve, que l'extrait sélectionné de la plante alpine Leontopodium alpinum var Helvetia (Edelweiss) inhibe l'induction prématurée de la phase catagène, en stimulant la papille dermique. Il est donc possible d'utiliser cet extrait comme un agent anti-chute, à la fois pour prévenir la chute des cheveux associée au vieillissement mais aussi comme une thérapie complémentaire pour les troubles liés à la perte des cheveux.

Hydra and the hair follicle - An unconventional comparative biology approach to exploring the human holobiont.

The microbiome of human hair follicles (HFs) has emerged as an important player in different HF and skin pathologies, yet awaits in-depth exploration. This raises questions regarding the tightly linked interactions between host environment, nutrient dependency of host-associated microbes, microbial metabolism, microbe-microbe interactions and host immunity. The use of simple model systems facilitates addressing generally important questions and testing overarching, therapeutically relevant principles that likely transcend obvious interspecies differences. Here, we evaluate the potential of the freshwater polyp Hydra, to dissect fundamental principles of microbiome regulation by the host, that is the human HF. In particular, we focus on therapeutically targetable host-microbiome interactions, such as nutrient dependency, microbial interactions and host defence. Offering a new lens into the study of HF - microbiota interactions, we argue that general principles of how Hydra manages its microbiota can inform the development of novel, microbiome-targeting therapeutic interventions in human skin disease.

Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts.

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.

Methods to Study Human Hair Follicle Growth Ex Vivo: Human Microdissected Hair Follicle and Human Full Thickness Skin Organ Culture.

The culture of microdissected hair follicles (HFs) and scalp skin enriched in terminal HFs are the best currently available preclinical assays for studying hair and skin biology/pathology in the human system. While microdissected HF organ culture only allows the testing of compounds added into the culture medium, mimicking a systemic application, the scalp skin organ culture also is suitable to test topical and intradermal applications. Here, we describe different methods for isolation of human scalp HFs, the procedures for culturing the scalp skin and microdissected HFs and we also outline different delivery techniques (e.g., topical, systemic) to test active and control substances.

Human Wound Healing Ex Vivo Model with Focus on Molecular Markers.

Wound healing is a complex, multifactorial process that is divided in sequential and overlapping phases in order to restore the skin barrier. For the study of wound healing, different in vivo, in vitro, and ex vivo models have been used in the past. Here we describe in detail the methodology of the human skin punch-in-a-punch ex vivo wound healing model.