Induced pluripotent stem cell line heterozygous for p.R2447X mutation in filaggrin: KCLi002-A.

We have generated an induced pluripotent stem cell (iPSC) line KCLi002-A (iOP107) from a female donor, heterozygous for the loss-of-function mutation p.R2447X in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization of KCLi002-A line included molecular karyotyping, mutation screening using restriction enzyme digestion Sanger sequencing and next generation sequencing (NGS), whereas pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and in vivo teratoma assay.

Keratin gene mutations influence the keratinocyte response to DNA damage and cytokine induced apoptosis.

The keratin filament cytoskeleton is vital to the normal function of epithelial cells. It provides structural support and regulates different aspects of cell metabolism. Mutations in keratins 5 and 14 cause a skin fragility disorder, epidermolysis bullosa simplex (EBS). Patients with severe EBS have an increased cumulative risk for basal cell carcinoma. In this study, we tested how keratin 5 and 14 mutant EBS patient-derived keratinocytes behave in the face of two different types of stressors that are able to induce cell death: ionizing radiation and cytokines TNF-α and TRAIL. The data point out to a substantial difference between how normal and keratin mutant keratinocytes deal with such stresses. When case of DNA damage, the ATM/Chk2-pathway is one of the two main tracks that can prevent the progression of mitosis and so allow repair. This was altered in all investigated keratin mutants with a particular down-regulation of the activated form of checkpoint kinase 2 (pChk2). Keratin mutants also appear less sensitive than normal cells to treatment with TNF-α or TRAIL, and this may be linked to the up-regulation of two pro-survival proteins, Bcl-2 and FLIP. Such changes are likely to have a profound effect on mutant keratinocytes ability to survive and withstand stress, and in theory this may be also a contributing factor to cell transformation.

Breast cancer resistance protein identifies clonogenic keratinocytes in human interfollicular epidermis.

INTRODUCTION: There is a practical need for the identification of robust cell-surface markers that can be used to enrich for living keratinocyte progenitor cells. Breast cancer resistance protein (ABCG2), a member of the ATP binding cassette (ABC) transporter family, is known to be a marker for stem/progenitor cells in many tissues and organs. METHODS: We investigated the expression of ABCG2 protein in normal human epidermis to evaluate its potential as a cell surface marker for identifying and enriching for clonogenic epidermal keratinocytes outside the pilosebaceous tract. RESULTS: Immunofluorescence and immunoblotting studies of human skin showed that ABCG2 is expressed in a subset of basal layer cells in the epidermis. Flow cytometry analysis showed approximately 2-3% of keratinocytes in non-hair-bearing epidermis expressing ABCG2; this population also expresses p63, ß1 and α6 integrins and keratin 14, but not CD34, CD71, C-kit or involucrin. The ABCG2-positive keratinocytes showed significantly higher colony forming efficiency when co-cultured with mouse 3T3 feeder cells, and more extensive long-term proliferation capacity in vitro, than did ABCG2-negative keratinocytes. Upon clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and were capable of generating a stratified differentiating epidermis in organotypic culture models. CONCLUSIONS: These data indicate that in skin, expression of the ABCG2 transporter is a characteristic of interfollicular keratinocyte progentior cells and suggest that ABCG2 may be useful for enriching keratinocyte stem cells in human interfollicular epidermis.

Intestinal cell barrier function in vitro is severely compromised by keratin 8 and 18 mutations identified in patients with inflammatory bowel disease.

Keratin 8 and 18 (K8/K18) mutations have been implicated in the aetiology of certain pathogenic processes of the liver and pancreas. While some K8 mutations (K8 G62C, K8 K464N) are also presumed susceptibility factors for inflammatory bowel disease (IBD), the only K18 mutation (K18 S230T) discovered so far in an IBD patient is thought to be a polymorphism. The aim of our study was to demonstrate that these mutations might also directly affect intestinal cell barrier function. Cell monolayers of genetically engineered human colonocytes expressing these mutations were tested for permeability, growth rate and resistance to heat-stress. We also calculated the change in dissociation constant (Kd, measure of affinity) each of these mutations introduces into the keratin protein, and present the first model of a keratin dimer L12 region with in silico clues to how the K18 S230T mutation may affect keratin function. Physiologically, these mutations cause up to 30% increase in paracellular permeability in vitro. Heat-stress induces little keratin clumping but instead cell monolayers peel off the surface suggesting a problem with cell junctions. K18 S230T has pronounced pathological effects in vitro marked by high Kd, low growth rate and increased permeability. The latter may be due to the altered distribution of tight junction components claudin-4 and ZO-1. This is the first time intestinal cells have been suggested also functionally impaired by K8/K18 mutations. Although an in vitro colonocyte model system does not completely mimic the epithelial lining of the intestine, nevertheless the data suggest that K8/K18 mutations may be also able to produce a phenotype in vivo.

A rare connexin 26 mutation in a patient with a forme fruste of keratitis-ichthyosis-deafness (KID) syndrome.

BACKGROUND: Keratitis-ichthyosis-deafness (KID) syndrome is a rare ectodermal dysplasia characterized by generalized erythrokeratotic plaques, sensorineural hearing loss, and vascularizing keratitis. Cutaneous changes and hearing loss typically present in early childhood, whereas ocular symptoms present later. Mutations in the connexin (Cx) 26 gene, GJB2, are now established to underlie many of the affected cases, with the majority of patients harboring the p.D50N mutation. METHODS: A rare patient demonstrating features of incomplete KID syndrome associated with an uncommon Cx26 gene mutation is described. RESULTS: The patient presented late in adolescence with partial features of KID syndrome. There was limited cutaneous involvement and the rare association of cystic acne. Both hearing impairment and ophthalmic involvement were mild in severity. Genetic mutation analysis revealed a previously described, rare mutation in GJB2, resulting in a glycine to arginine change at codon 12 (p.G12R). CONCLUSIONS: This report describes a patient exhibiting characteristics suggestive of a late-onset, incomplete form of KID syndrome with the GJB2 mutation (p.G12R). The p.G12R mutation has only been described in one other patient with KID syndrome, whose clinical presentation was not characterized.

Intermediate filament protein expression and mesoderm formation in the rabbit embryo : A double-labelling immunofluorescence study.

This study aims to describe the regulation of vimentin and cytokeratin expression during differentiation of primary mesenchymal cells in the 7 day old rabbit embryo; unusual intermediate filament protein expression patterns have already been found in this species at later embryonic stages. Double-labelling indirect immunofluorescence assays with a panel of monoclonal intermediate filament antibodies are performed on frozen sections and compared with aldehyde-fixed plastic-embedded tissues. The histological part of the study, serving as a basis for the topographical orientation in the immunostained frozen sections, emphasises many similarities between the primitive streak embryos of the rabbit and the chick. The immunohistochemical analysis reveals cytokeratin expression to varying degrees in all germ layers. Vimentin expression, always in combination with cytokeratin expression, is found in a few cells of the ectoderm, endoderm and lateral mesoderm, but not in the primary mesenchymal cells of either the primitive node or the primitive streak. The results are discussed in relation to recent experimental findings on differentiation and morphogenetic processes in the primitive streak embryo. While these complex expression patterns make it seem unlikely that intermediate filament protein subtypes are expressed independently of cellular function during development, no indication can be found for a relation between vimentin expression and the morphogenetic changes thought to be important during mesoderm formation.