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Characterization of human telomerase reverse transcriptase immortalized anterior cruciate ligament cell lines.

ABSTRACT: BACKGROUND:The anterior-cruciate-ligament (ACL) contains mesenchymal stem cells (ACL-MSCs), suggesting the feasibility of regenerative treatments of this tissue. The immortalization of isolated cells results in cell-lines applicable to develop cell-based therapies. Immortal cell lines eliminate the need for frequent cell isolation from donor tissues. The objective of this study was to characterize cell lines that were generated from isolated ACL-MSCs using TERT gene transfer. METHODS:We isolated ACL-MSCs from human ACLs derived at the time of ACL reconstruction surgery or total knee arthroplasty. We generated cell lines and compared them to non-immortalized ACL-MSCs. We assessed the cellular morphology and we detected surface antigen expression. The resistance to senescence was inferred using the beta galactosidase activity. Histology, immunohistochemistry, and reverse transcriptase polymerase chain reaction (RT-PCR) were used to evaluate the multilineage differentiation capacity. RESULTS:The morphology of hTERT-ACL-MSCs was similar to ACL up to the last assessment at passage eight. We detected a strong surface expression of CD44, CD90, CD105, and STRO-1 in hTERT-ACL-MSCs. No substantial reduction in the ATP activity was observed in hTERT-ACL-MSCs. CONCLUSION:Cell lines generated from ACL-MSCs maintain their morphology, surface antigen expression profile, and proliferative capacity; while markers of senescence appear to be reduced. These cell-lines maintained their multilineage differentiation capacity. The demonstrated model systems can be used for further development of new cell-based regenerative approaches in anterior cruciate ligament research, which may lead to new therapeutic strategies in the future.

SUBMITTER: Prager P

PROVIDER: S-EPMC6962762 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Characterization of human telomerase reverse transcriptase immortalized anterior cruciate ligament cell lines.

Prager Patrick P Schieker Matthias M Jakob Franz F Docheva Denitsa D Konrads Christian C Steinert Andre A

Biomedical journal 20191223 6

<h4>Background</h4>The anterior-cruciate-ligament (ACL) contains mesenchymal stem cells (ACL-MSCs), suggesting the feasibility of regenerative treatments of this tissue. The immortalization of isolated cells results in cell-lines applicable to develop cell-based therapies. Immortal cell lines eliminate the need for frequent cell isolation from donor tissues. The objective of this study was to characterize cell lines that were generated from isolated ACL-MSCs using TERT gene transfer.<h4>Methods< ...[more]

PMID: 31948601

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Project description:The aims of this study were to generate periodontal ligament (PDL) cells that have adenovirus- or lentivirus-mediated overexpression of human telomerase reverse transcriptase (hTERT) and to compare the osteogenic and proliferative abilities of the two cell lines to establish an efficient and stable cell model that will be more suitable for studies of PDL regeneration. After construction of the recombinant adenovirus plasmid pAd-pshuttle-cmv-hTERT, human PDL cells were infected by packaged adenovirus and lentivirus particles to establish two PDL cell lines. The expression levels of hTERT and mRNA for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, core-binding factor (runt-related transcription factor 2), and type I collagen were assessed for each cell line. After culture in osteoinductive culture medium for 14 days, the PDL cells were stained with alizarin red to observe formation of mineralized nodules, and proliferation activity was measured with a CCK-8 kit. A quantitative polymerase chain reaction assay indicated that the two transduced cell lines expressed hTERT levels that were significantly higher than that seen for normal PDL cells. Expression of all osteogenic genes tested, with the exception of osteopontin, was higher for both the adenovirus- and lentivirus-transduced cells relative to normal PDL cells. The expression of bone sialoprotein, osteocalcin, and runt-related transcription factor 2 in adenovirus-transduced cells was significantly higher than that for lentivirus-transduced cells. Alizarin red staining showed that the adenovirus-transduced cell line produced more mineralized nodules than the lentivirus-transduced cell line, whereas a CCK-8 test showed that the adenovirus-transduced cell line had higher proliferation activity than lentivirus-transduced cells. In conclusion, a PDL cell line established by adenovirus transduction had superior osteogenic differentiation and proliferative activity compared to the cell line produced by lentivirus transduction. The results indicate that PDL cells having adenovirus-mediated expression of hTERT would be a more suitable model for studies of PDL regeneration.

Dataset Information

Characterization of human telomerase reverse transcriptase immortalized anterior cruciate ligament cell lines.

Publications

Characterization of human telomerase reverse transcriptase immortalized anterior cruciate ligament cell lines.

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