Nadezhda A. Evtushenko,1,† Arkadii K. Beilin,1,2,† Erdem B. Dashinimaev,1,2 Rustam H. Ziganshin,3 Anastasiya V. Kosykh,1,2 Maxim M. Perfilov,3 Alexandra L. Rippa,2 Elena V. Alpeeva,2 Andrey V. Vasiliev,2 Ekaterina A. Vorotelyak,2 and Nadya G. Gurskaya1,3
1Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Ostrovityanova 1, 117997 Moscow, Russia;
2Koltzov Institute of Developmental Biology of Russian Academy of Sciences, 26 Vavilova Str., 119334 Moscow, Russia;
3Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
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.
CoolLED pE-300white (CoolLED Ltd., Andover, UK) with illumination intensity of about 0.04–0.47 W/cm2 for a 20× lens and 0.18–2.0 W/cm2 for a 40× lens was used as a light source.
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Int. J. Mol. Sci.
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