A novel method for banking stem cells from human exfoliated deciduous teeth: lentiviral TERT immortalization and phenotypical analysis

Background: Stem cells from human exfoliated deciduous teeth (SHED) have recently attracted attention as novel multipotential stem cell sources. However, their application is limited due to in vitro replicative senescence. Ectopic expression of telomerase reverse transcriptase (TERT) is a promising strategy for overcoming this replicative senescence. Nevertheless, its potential application and the phenotype as well as tumorigenicity have never been assessed in SHED. Methods: TERT expression was stably restored in SHED (TERT-SHED) isolated from healthy children aged 6-8 years using lentiviral transduction with a puromycin selection marker. The expression of TERT was detected using reverse transcription polymerase chain reaction, Western blot and immunofluorescence. Surface markers of SHED were detected by flow cytometry. Enzyme-linked immunosorbent assay was used to assess senescence-associated beta-galactosidase, while CCK-8 methods were used to examine the proliferation capacity of SHED and TERT-SHED at different passages. Moreover, multilineage differentiation, karyotype, colony formation in soft agar, and tumor formation in nude mice of SHED and TERT-SHED were also examined. Results: Lentiviral transduction induced stable TERT expression even in SHED at the 40th passage. TERT-SHED showed robust proliferation capacity and low concentration of beta-galactosidase. Although they had some different biomarkers than early passage SHED, TERT-SHED at late passage showed similar mutilineage differentiation as TERT at early passage. Moreover, TERT-SHED at late passage showed normal karyotype, no soft agar colony formation, and no tumor formation in nude mice. Conclusions: TERT-immortalized SHED may be a promising resource for stem-cell therapy, although attention should be paid to the biological behavior of the cells.