Nuria Illera

Development of a Bioengineered Skin-Humanized Mouse Model for Psoriasis: Dissecting Epidermal-Lymphocyte Interacting Pathways

Over the past few years, whole skin xenotransplantation models that mimic different aspects of psoriasis have become available. However, these models are strongly constrained by the lack of skin donor availability and homogeneity. We present in this study a bioengineering-based skin-humanized mouse model for psoriasis, either in an autologous version using samples derived from psoriatic patients or, more importantly, in an allogeneic context, starting from skin biopsies and blood samples from unrelated healthy donors. After engraftment, the regenerated human skin presents the typical architecture of normal human skin but, in both cases, immunological reconstitution through intradermal injection in the regenerated skin using in vitro-differentiated T1 subpopulations as well as recombinant IL-17 and IL-22 Th17 cytokines, together with removal of the stratum corneum barrier by a mild abrasive treatment, leads to the rapid conversion of the skin into a bona fide psoriatic phenotype. Major hallmarks of psoriasis were confirmed by the evaluation of specific epidermal differentiation and proliferation markers as well as the mesenchymal milieu, including angiogenesis and infiltrate. Our bioengineered skin-based system represents a robust platform to reliably assess the molecular and cellular mechanisms underlying the complex interdependence between epidermal cells and the immune system. The system may also prove suitable to assess preclinical studies that test the efficacy of novel therapeutic treatments and to predict individual patient response to therapy.

The first COL7A1 mutation survey in a large Spanish dystrophic epidermolysis bullosa cohort: c.6527insC disclosed as an unusually recurrent mutation

Background  Dystrophic epidermolysis bullosa (DEB) is a genodermatosis caused by mutations in COL7A1. The clinical manifestations are highly variable from nail dystrophy to life‐threatening blistering, making early molecular diagnosis and prognosis of utmost importance for the affected families. Mutation identification is mandatory for prenatal testing.

In Vivo Assessment of Acute UVB Responses in Normal and Xeroderma Pigmentosum (XP-C) Skin-Humanized Mouse Models

In vivo studies of UVB effects on human skin are precluded by ethical and technical arguments on volunteers and inconceivable in cancer-prone patients such as those affected with Xeroderma Pigmentosum (XP). Establishing reliable models to address mechanistic and therapeutic matters thus remains a challenge. Here we have used the skin-humanized mouse system that circumvents most current model constraints. We assessed the UVB radiation effects including the sequential changes after acute exposure with respect to timing, dosage, and the relationship between dose and degree-sort of epidermal alteration. On Caucasian-derived regenerated skins, UVB irradiation (800 J/m(2)) induced DNA damage (cyclobutane pyrimidine dimers) and p53 expression in exposed keratinocytes. Epidermal disorganization was observed at higher doses. In contrast, in African descent-derived regenerated skins, physiological hyperpigmentation prevented tissue alterations and DNA photolesions. The acute UVB effects seen in Caucasian-derived engrafted skins were also blocked by a physical sunscreen, demonstrating the suitability of the system for photoprotection studies. We also report the establishment of a photosensitive model through the transplantation of XP-C patient cells as part of a bioengineered skin. The inability of XP-C engrafted skin to remove DNA damaged cells was confirmed in vivo. Both the normal and XP-C versions of the skin-humanized mice proved proficient models to assess UVB-mediated DNA repair responses and provide a strong platform to test novel therapeutic strategies.

Targeted silencing of DEFB4 in a bioengineered skin‐humanized mouse model for psoriasis: development of siRNA SECosome‐based novel therapies

Psoriasis is a complex inflammatory skin disease that presents a wide variety of clinical manifestations. Human β defensin‐2 (hBD‐2) is highly up‐regulated in psoriatic lesions and has been defined as a biomarker for disease activity. We explored the potential benefits of targeting hBD‐2 by topical application of DEFB4‐siRNA‐containing SECosomes in a bioengineered skin‐humanized mouse model for psoriasis. A significant improvement in the psoriatic phenotype was observed by histological examination, with a normalization of the skin architecture and a reduction in the number and size of blood vessels in the dermal compartment. Treatment leads to the recovery of transglutaminase activity, filaggrin expression and stratum corneum appearance to the levels similar to those found in normal regenerated human skin. The availability of a reliable skin‐humanized mouse model for psoriasis in conjunction with the use of the SECosome technology may provide a valuable preclinical tool for identifying potential therapeutic targets for this disease.

The regenerative potential of fibroblasts in a new diabetes-induced delayed humanised wound healing model.

Cutaneous diabetic wounds greatly affect the quality of life of patients, causing a substantial economic impact on the healthcare system. The limited clinical success of conventional treatments is mainly attributed to the lack of knowledge of the pathogenic mechanisms related to chronic ulceration. Therefore, management of diabetic ulcers remains a challenging clinical issue. Within this context, reliable animal models that recapitulate situations of impaired wound healing have become essential. In this study, we established a new in vivo humanised model of delayed wound healing in a diabetic context that reproduces the main features of the human disease. Diabetes was induced by multiple low doses of streptozotocin in bioengineered human‐skin‐engrafted immunodeficient mice. The significant delay in wound closure exhibited in diabetic wounds was mainly attributed to alterations in the granulation tissue formation and resolution, involving defects in wound bed maturation, vascularisation, inflammatory response and collagen deposition. In the new model, a cell‐based wound therapy consisting of the application of plasma‐derived fibrin dermal scaffolds containing fibroblasts consistently improved the healing response by triggering granulation tissue maturation and further providing a suitable matrix for migrating keratinocytes during wound re‐epithelialisation. The present preclinical wound healing model was able to shed light on the biological processes responsible for the improvement achieved, and these findings can be extended for designing new therapeutic approaches with clinical relevance.

Two novel recessive mutations in KRT14 identified in a cohort of 21 Spanish families with epidermolysis bullosa simplex

Background  Basal epidermolysis bullosa simplex (EBS) is a group of blistering genodermatoses mostly caused by mutations in the keratin genes, KRT5 and KRT14. Recessive mutations represent about 5% of all EBS mutations, being common and specific in populations with high consanguinity, where affected patients show severe phenotypes.

A prevalent mutation with founder effect in Spanish Recessive Dystrophic Epidermolysis Bullosa families.

BackgroundRecessive Dystrophic Epidermolysis Bullosa (RDEB) is a genodermatosis caused by more than 500 different mutations in the COL7A1 gene and characterized by blistering of the skin following a minimal friction or mechanical trauma.The identification of a cluster of RDEB pedigrees carrying the c.6527insC mutation in a specific area raises the question of the origin of this mutation from a common ancestor or as a result of a hotspot mutation. The aim of this study was to investigate the origin of the c.6527insC mutation.MethodsHaplotypes were constructed by genotyping nine single nucleotides polymorphisms (SNPs) throughout the COL7A1 gene. Haplotypes were determined in RDEB patients and control samples, both of Spanish origin.ResultsSixteen different haplotypes were identified in our study. A single haplotype cosegregated with the c.6527insC mutation.ConclusionHaplotype analysis showed that all alleles carrying the c.6527insC mutation shared the same haplotype cosegregating with this mutation (CCGCTCAAA_6527insC), thus suggesting the presence of a common ancestor.

Differential Features between Chronic Skin Inflammatory Diseases Revealed in Skin-Humanized Psoriasis and Atopic Dermatitis Mouse Models

Psoriasis and atopic dermatitis are chronic and relapsing inflammatory diseases of the skin affecting a large number of patients worldwide. Psoriasis is characterized by a T helper type 1 and/or T helper type 17 immunological response, whereas acute atopic dermatitis lesions exhibit T helper type 2-dominant inflammation. Current single gene and signaling pathways-based models of inflammatory skin diseases are incomplete. Previous work allowed us to model psoriasis in skin-humanized mice through proper combinations of inflammatory cell components and disruption of barrier function. Herein, we describe and characterize an animal model for atopic dermatitis using similar bioengineered-based approaches, by intradermal injection of human T helper type 2 lymphocytes in regenerated human skin after partial removal of stratum corneum. In this work, we have extensively compared this model with the previous and an improved version of the psoriasis model, in which T helper type 1 and/or T helper type 17 lymphocytes replace exogenous cytokines. Comparative expression analyses revealed marked differences in specific epidermal proliferation and differentiation markers and immune-related molecules, including antimicrobial peptides. Likewise, the composition of the dermal inflammatory infiltrate presented important differences. The availability of accurate and reliable animal models for these diseases will contribute to the understanding of the pathogenesis and provide valuable tools for drug development and testing.

Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells

Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented gene-editing strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patient-derived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils, and structural integrity when analyzed 12 weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutation-containing, functionally expendable COL7A1 exons in patient epidermal stem cells.