Modelling of human skin diseases using in vitro 3D reconstructed skin

Elucidation of disease aetiology and pathogenesis is often approached using
animal models or traditional cell-based in vitro systems. In experimental
dermatology, the use of three-dimensional (3D) reconstructed skin models has
proven to be a powerful tool to investigate the biology of normal skin and skin
diseases. This has prompted us to initiate a research project to develop and
apply these models, aiming to resolve the biology underlying genetic risk
factors of psoriasis and atopic dermatitis (AD) and to investigate the
biological function of genes known to be mutated in rare Mendelian skin
disorders (genodermatoses). The outcome of this project will contribute to
understanding the effect of genetic variation on disease and to knowledge on
gene function, and it may contribute to personalized therapeutic approaches.

In this project we will establish primary keratinocyte cell lines of patient
skin biopsies from selected skin diseases, to generate 3D skin constructs. In
addition, patient DNA will be genotyped for genetic risk factors (psoriasis and
AD) or causative mutations (genodermatoses) to analyze in vitro biological
responses in relation to genetic background.

Patients participating in the study are treated in regular care. Inclusion of
patients and collection of samples will be performed at regular outpatient
visits as much as possible. If not enough patients can be included, we have a
list of volunteers who have registered for participation in research and have
agreed to be approached. Patients will be provided with oral and written
information about participation in this study and informed consent will be
obtained.

* Saliva from each patient will be collected for DNA analysis (to determine
disease specific candidate genes, for example the FLG genotype in AD and the
HLA-Cw6 and LCE3BC_del genotype in psoriasis). In the case of genodermatosis
patients, the causative mutation may already be known, but will be confirmed.
* Four skin biopsies of 4 mm diameter will be collected from patients treated
for a chronic inflammatory skin disease (psoriasis and AD) and from patients
with a genodermatosis. Primary keratinocytes will be isolated from three
biopsies for generation of 3D skin equivalents as previously described. One
biopsy will be used for immunohistochemical and morphological analyses. Primary
human keratinocytes from healthy controls have already been collected
previously. These in vitro skin models will be used to determine the
pathogenesis and therapy response in relation to the genetic characteristics of
the host. The skin constructs will be analyzed in vitro for gene expression
after stimulation with inflammatory, disease-specific cytokines (in case of
psoriasis and AD). Analysis by quantitative PCR (qPCR) will be performed on a
large panel of genes. The qPCR data will be analyzed with multivariate
statistical analysis. Furthermore, the skin constructs will also be analyzed
with immunohistochemical stains for protein expression and morphology.
Epidermal extracts may be used for biochemical assays to investigate the
biological pathways that are affected e.g. in constructs of genodermatosis
patients.

Skin biopsies pose a very small risk of post biopsy bleeding, infection of the
biopsy wound and scar formation. However, despite being performed at a large
scale in daily dermatology practice, complications of biopsy-taking are hardly
ever encountered. Patients with a tendency to develop hypertrophic scars will
be excluded from the studies. Genetic analyses will focus on known genetic risk
factors for psoriasis and AD, and on mutations identified to be causative for
genodermatoses. Whole exome or whole genome sequencing will not be performed,
so there will be no risk of incidental findings.