Preclinical corrective gene transfer in xeroderma pigmentosum human skin stem cells.

Xeroderma pigmentosum (XP) is a devastating disease associated with dramatic skin cancer proneness. XP cells are deficient in nucleotide excision repair (NER) of bulky DNA adducts including ultraviolet (UV)-induced mutagenic lesions. Approaches of corrective gene transfer in NER-deficient keratinocyte stem cells hold great hope for the long-term treatment of XP patients. To face this challenge, we developed a retrovirus-based strategy to safely transduce the wild-type XPC gene into clonogenic human primary XP-C keratinocytes. De novo expression of XPC was maintained in both mass population and derived independent candidate stem cells (holoclones) after more than 130 population doublings (PD) in culture upon serial propagation (>10(40) cells). Analyses of retrovirus integration sequences in isolated keratinocyte stem cells suggested the absence of adverse effects such as oncogenic activation or clonal expansion. Furthermore, corrected XP-C keratinocytes exhibited full NER capacity as well as normal features of epidermal differentiation in both organotypic skin cultures and in a preclinical murine model of human skin regeneration in vivo. The achievement of a long-term genetic correction of XP-C epidermal stem cells constitutes the first preclinical model of ex vivo gene therapy for XP-C patients.

Maintenance effect of a once-weekly regimen of a Selenium Disulfide-based shampoo in moderate-to-severe scalp seborrheic dermatitis after initial treatment with topical corticosteroid/salicylic acid.

INTRODUCTION: Seborrheic dermatitis (SD) is a chronic, relapsing, inflammatory disorder of the head and trunk. OBJECTIVES: To explore the potential of a 1% Selenium disulphide (SeS2)-based shampoo to prevent relapses of scalp SD (SSD) following corticosteroid/salicylic acid (TCS/SA) treatment. MATERIALS & METHODS: After a 2-week treatment with TCS/SA, adult patients with moderate-to-severe SSD received either the SeS2-based shampoo or its vehicle for eight weeks in a randomized, double-blinded fashion. Visits took place at baseline, weeks 2, 6 and 10. SSD severity was assessed based on erythema, flakes and pruritus; patients assessed the severity of pruritus. Global investigator and patient satisfaction were assessed at week 10. RESULTS: Forty-eight adults were included. After four and eight weeks of post TCS/SA maintenance regimen, 8.1% and 16.7% in the SeS2, and 41.7% and 54.2% in the vehicle group relapsed, respectively. First median time-to-relapse in the vehicle group was 56 days; this was not reached for SeS2. After two weeks of TCS/SA, the prevalence of patients with no pruritus was 29.2% in the SeS2 group, and 41.7% in the vehicle group; it increased to 76.2% with SeS2 and to 57.1% with the vehicle at the end of the study. The clinical benefit of treatment with TCS/SCA was maintained in the SeS2 group only. Investigators and patients were highly satisfied with the efficacy of SeS2. Tolerance to SeS2 was excellent, with no reported adverse events. CONCLUSION: The SeS2-based shampoo significantly reduces the time-to-relapse of moderate-to-severe SSD flares. Its tolerance was excellent, with no reported adverse events.

Interactions between fibroblasts and keratinocytes in morphogenesis of dermal epidermal junction in a model of reconstructed skin.

De novo dermal epidermal junction morphogenesis was studied in a skin model including dermal fibroblasts and epidermal keratinocytes. Sequential gene expression, protein deposition, and localization of basement membrane zone components were studied during 15 days. The morphogenesis of dermal epidermal junction is characterized by an implementation of the different components and then a subsequent plateau phase occurring at day 11. Three groups of genes were identified depending on cellular origin and expression profile: 1/genes of fibroblastic origin (col I alpha1, col III alpha1, nidogen, and fibrillin 1); 2/genes expressed in fibroblasts and keratinocytes with symmetrical expression pattern between both cell types (col IV alpha1, col VII alpha1, and tenascin C); 3/laminin beta3 only expressed in keratinocytes. Use of modified organotypic models excluding one cell type revealed a tight interplay between fibroblasts and keratinocytes for synthesis and localization of the components of dermal epidermal junction. Keratinocytes downregulated mRNA and proteins of fibroblastic origin, upregulated col VII in fibroblasts and were absolutely required for dermal-epidermal junction localization of fibroblastic proteins. Fibroblasts downregulated mRNA of keratinocytes and were needed for extracellular secretion and correct localization of type VII collagen and laminin 5.

Key regulatory role of dermal fibroblasts in pigmentation as demonstrated using a reconstructed skin model: impact of photo-aging.

To study cutaneous pigmentation in a physiological context, we have previously developed a functional pigmented reconstructed skin model composed of a melanocyte-containing epidermis grown on a dermal equivalent comprising living fibroblasts. The present studies, using the same model, aimed to demonstrate that dermal fibroblasts influence skin pigmentation up to the macroscopic level. The proof of principle was performed with pigmented skins differing only in the fibroblast component. First, the in vitro system was reconstructed with or without fibroblasts in order to test the global influence of the presence of this cell type. We then assessed the impact of the origin of the fibroblast strain on the degree of pigmentation using fetal versus adult fibroblasts. In both experiments, impressive variation in skin pigmentation at the macroscopic level was observed and confirmed by quantitative parameters related to skin color, melanin content and melanocyte numbers. These data confirmed the responsiveness of the model and demonstrated that dermal fibroblasts do indeed impact the degree of skin pigmentation. We then hypothesized that a physiological state associated with pigmentary alterations such as photo-aging could be linked to dermal fibroblasts modifications that accumulate over time. Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3) was compared to that of tissues containing natural photo-aged fibroblasts (n = 3) which express a senescent phenotype. A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance) and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis. Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the hyperpigmentation that is associated with photo-aging.

Human skin model containing melanocytes: essential role of keratinocyte growth factor for constitutive pigmentation-functional response to α-melanocyte stimulating hormone and forskolin.

To study human skin pigmentation in a physiological in vitro model, we developed a pigmented reconstructed skin reproducing the three-dimensional architecture of the melanocyte environment and the interactions of melanocyte with its cellular partners, keratinocytes, and fibroblasts. Co-seeding melanocytes and keratinocytes onto a fibroblast-populated collagen matrix led to a correct integration of melanocytes within the epidermal basal layer, but melanocytes remained amelanotic even after supplementation with promelanogenic factors. Interestingly, normalization of keratinocyte differentiation using keratinocyte growth factor instead of epidermal growth factor finally allowed an active pigmentary system to develop, as shown by the expression of key melanogenic markers, the production, and transfer of melanosome-containing melanin into keratinocytes. Various degrees of constitutive pigmentation were reproduced using melanocytes from different skin phenotypes. Furthermore, induction of pigmentation was achieved by treatment with known propigmenting molecules, αMSH and forskolin, thus demonstrating the functionality of the pigmentary system. This pigmented full-thickness skin model therefore represents a highly relevant tool to study the role of cell-cell, cell-matrix, and mesenchymal-epithelial interactions in the control of skin pigmentation.

Morphogenesis of dermal-epidermal junction in a model of reconstructed skin: beneficial effects of vitamin C.

In skin, cohesion between the dermis and the epidermis is ensured by the dermal-epidermal junction which is also required for control of epidermal growth and differentiation. Here we showed that addition of vitamin C optimized the formation of the dermal-epidermal junction in an in vitro human reconstructed skin model leading to a structure closer to that of normal human skin. Compared with controls, vitamin C treatment led to a better organization of basal keratinocytes, an increase in fibroblast number and a faster formation of the dermal-epidermal junction. Vitamin C also accelerated deposition of several basement membrane proteins, like type IV and VII collagens, nidogen, laminin 10/11, procollagens I and III, tenascin C and fibrillin-1 at the dermal-epidermal junction. The mechanism of action of vitamin C was investigated by quantitative polymerase chain reaction in fibroblasts and keratinocytes respectively. Vitamin C effects passed in part through an increase in col I alpha1, col III alpha1 and fibrillin-1 mRNA levels. Effects on the other markers appeared to happen at the translational and/or post-translational level, as illustrated for tenascin C, col IV alpha2 and col VII alpha1 mRNA levels which were reduced by vitamin C in both cell types.