Dataset Information

HTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination.

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.

SUBMITTER: Evtushenko NA

PROVIDER: S-EPMC8067634 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination.

Evtushenko Nadezhda A NA Beilin Arkadii K AK Dashinimaev Erdem B EB Ziganshin Rustam H RH Kosykh Anastasiya V AV Perfilov Maxim M MM Rippa Alexandra L AL Alpeeva Elena V EV Vasiliev Andrey V AV Vorotelyak Ekaterina A EA Gurskaya Nadya G NG

International journal of molecular sciences 20210407 8

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 secretio ...[more]

PMID: 33916959

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Project description:Following a certain type-specific number of mitotic divisions, terminally differentiated cells undergo proliferative senescence, thwarting efforts to expand different cell populations in vitro for the needs of scientific research or medical therapies. The primary cause of this phenomenon is the progressive shortening of the telomeres and the subsequent activation of cell cycle control pathways leading to a block of cell proliferation. Restoration of telomere length by transgenic expression of telomerase reverse transcriptase (TERT) usually results in bypassing of the replicative senescence and ultimately in cell immortalization. To date, there have not been any reports regarding immortalization of cells from common marmoset (Callithrix jacchus), an important non-human primate model for various human diseases, with the use of exogenous human TERT (hTERT). In this study, marmoset fibroblasts were successfully immortalized with transposon-integrated transgenic hTERT and expanded in vitro for over 500 population doublings. Calculation of population doubling levels (PDL) showed that the derived hTERT-transgenic lines had significantly higher proliferation potential than the wild-type fibroblasts, which reached only a maximum of 46 doublings. However, the immortalized cells exhibited differences in the morphology compared with the control fibroblasts and transcriptome analysis also revealed changes in the gene expression patterns. Finally, the karyotypes of all hTERT-transgenic cell lines showed various aberrations such as presence of extra Chromosome 17, isochromosome 21q, or tetraploidy. By single-cell expansion of the least affected monoclonal immortalized line, one sub-clonal line with normal karyotype was established, suggesting the possibility to derive immortal marmoset cells with normal karyotypes. The results of this study are an important step towards the development and optimization of methods for the production of immortalized cells from common marmoset monkeys.

Project description:Human papillomaviruses (HPV) from the genus beta have been implicated in the development of cutaneous squamous cell cancer in epidermodysplasia verruciformis and organ transplant patients. In contrast to alpha-high-risk HPV, which cause ano-genital and oropharyngeal cancers, beta-HPV replication is not well understood. The beta-HPV49 transcriptome was analyzed by RNA sequencing using stable keratinocyte cell lines maintaining high levels of extrachromosomally replicating E8- genomes, which can be established due to a lack of the viral E8^E2 repressor protein. This analysis indicated the presence of four transcription start sites, two polyadenylation signals, and splice donor (SD) and acceptor sites consistent with the conserved gene expression patterns of animal and human PV. Surprisingly, a novel SD in the E6 oncogene (SD217) was identified resembling the SD in E6 of carcinogenic alpha-HPV. Mutation of SD217 enhanced E6 protein expression but had no influence on the growth of keratinocytes transduced with retroviral HPV49 E6 and E7 expression vectors. Inactivation of SD217 in the context of the HPV49 wild-type genome did not enable immortalization and prevented immortalization in the context of the E8- genome. The analysis of SD217 mutant genomes revealed a strong down-regulation of SD217 usage, but only weak effects on other viral transcripts. This suggests that SD217 does not contribute to immortalization by modulating viral gene expression. Usage of SD217 is increased in immortalized E8- cell lines compared with transiently transfected cells, which may indicate that long-term extrachromosomal maintenance requires reduced E6 protein levels.IMPORTANCEHigh-risk (hr) human papillomaviruses (HPV) from the genus alpha cause ano-genital and oropharyngeal cancers, whereas beta-HPV have been implicated to cause skin cancer in epidermodysplasia verruciformis and organ transplant patients. In contrast to alpha hr-HPV, the replication cycle of beta-HPV is not very well understood. Transcriptional profiling of beta-HPV49 by RNA sequencing reveals transcription start sites and splice sites conserved among HPV. Surprisingly, a splice donor site in the E6 oncogene (SD217), previously only described for hr-HPV, was identified that controls E6 oncoprotein levels and is required for immortalization of keratinocytes by the HPV49 genome.

Dataset Information

HTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination.

Publications

hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination.

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