Generation and characterization of epidermolysis bullosa simplex cell lines: scratch assays show faster migration with disruptive keratin mutations

Abstract

These cell lines provide useful culture systems in which to assess aspects of EBS-induced cell changes. The faster migration after scratch wounding of the EBS keratinocytes may be a consequence of the known upregulation of stress-activated kinase pathways in these cells.

Keratinocyte cell lines derived from severe generalized recessive Epidermolysis Bullosa patients carrying a highly recurrent COL7A1 homozygous mutation: models to assess cell and gene therapies in vitro and in vivo

“…Evaluation of these approaches relies on the in vitro analysis of large number of cells and on stringent assays dealing with skin regenerative capacity of the modified cells in vivo (9)(10)(11). Although immortalized keratinocyte cell lines derived from several genodermatoses, including RDEB, have been reported (12)(13)(14)(15), detailed characterization of the immortalization process, growth performance, cytogenetic status and skin regenerative capacity has not been thoroughly addressed.…”

Functional improvement of mutant keratin cells on addition of desmin: an alternative approach to gene therapy for dominant diseases

“…KEB-7 cells, 25 which express a highly disruptive keratin mutation (K14 R125P), typical of the severe Dowling-Meara type EBS, were stably transfected with full-length human desmin cDNA, using a retroviral vector (pKDblast) ( Figure 1a …”

“…Cells expressing EBS-associated keratin mutations show faster scratch wound closure than wild-type cells, 25 possibly a consequence of the constitutively stressed state of the mutant cells which has the effect of priming them for a wound response. We examined the effect of desmin supplementation on this surrogate indicator of epithelial wound healing.…”

“…Keratinocyte culture conditions were as described previously. 25 (b) Desmin and K14 expression levels in KEB-7 (a), KEB-7.Des (b) and wild-type NEB-1 (c) were detected by immunoblotting with NCL-DES-DERII mouse monoclonal antibody against desmin (Novocastra; dilution 1:100, track B) and LL001 mouse monoclonal antibody to K14. 26 The expression levels of endogenous K14 and transfected desmin are comparable (b).…”

“…The wound area was microscopically measured using AxioVision 3.0 software (Carl Zeiss Vision GmbH), and the reduction in scratch wound area over the 8 h was calculated. 25 The extent of scratch wound closure was measured as the reduction of the scratch area after 8 h. Images above show phase-contrast micrographs of the extent of scratch values for each cell line (n ¼ 48). KEB-7 cells migrate most quickly into the scratch area; KEB-7.Des wound closure is significantly slower and approaches the speed of the wild-type NEB-1 cells.…”