Abstract
The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased β-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.
Highlights
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic disease mainly characterized by extreme skin fragility caused by mutations in the COL7A1 gene encoding type VII collagen (C7)
The results show thatfrom after a immortalization, fibroblast staining intensity became closer to primary fibroblasts immortalization, fibroblast intensity became closer fibroblasts young donor and, in moststaining cases, significantly lower thantoinprimary the paired primaryfrom cells a young donor and, in most cases, significantly lower than in the paired primary cells (Figure 4B)
High activity of hTERT in these lines ensured the extended lifetime for the panel of fibroblast lines
Summary
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic disease mainly characterized by extreme skin fragility caused by mutations in the COL7A1 gene encoding type VII collagen (C7). Disruption in the function of this protein leads to separation of sublamina densa induced by minor trauma that causes blistering and erosion of the skin, itching and pain. Persistent inflammation accompanying these lesions results in development of skin squamous carcinoma in 30–50-year-old RDEB patients [1]. The availability of patient biomaterials is severely limited, both due to the small number of patients and their health condition. This makes the development of RDEB models an important task required to accelerate the testing of new approaches to treatment. The drawbacks of primary cell lines as a valuable model system are their heterogeneity and patient-specific genetic differences, restricted availability and limited lifespan
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