Human TERT Lentivirus (CMV, Puro)

Human exfoliated deciduous tooth stem cells (SHEDs) have recently attracted attention as a novel source of multipotent stem cells. However, their application is limited due to replicative senescence in vitro. Ectopic expression of telomerase reverse transcriptase (TERT) is a promising strategy to overcome this replicative senescence. Using lentiviral transduction with a puromycin selection marker, TERT expression was stably restored in SHEDs isolated from healthy children aged 6-8 years (TERT-SHEDs). The study showed that lentiviral transduction induced stable TERT expression even in SHEDs at passage 40. TERT-SHEDs showed robust proliferation capacity and low concentrations of β-galactosidase. Although they had some different biomarkers from early-passage SHEDs, late-passage TERT-SHEDs showed multi-lineage differentiation similar to early-passage TERTs. In addition, late-passage TERT-SHEDs had normal karyotypes, no soft agar colony formation, and no tumor formation in nude mice. These data suggest that TERT-immortalized SHEDs may be a promising resource for stem cell therapy.

The multidirectional differentiation potential of SHED was measured by differentiation induction assay. SHED P4 could differentiate into osteogenic, adipogenic, and chondrogenic lineages, with positive staining by Alizarin Red S, Oil Red O, and Toluidine Blue, respectively (Figure 1a-c). TERT-SHED P20 showed similar differentiation ability to SHED P4 (Figure 1e-g). In addition, real-time PCR analysis confirmed that TERT-SHED P20 and SHED P4 had similar expression of osteogenic (ALP and BSP), adipogenic (LPL and PPAR-γ), and chondrogenic (ACAN and COL2A1) differentiation markers (Figure 1d and h).

Figure 1. Multilineage differentiation assay of SHED P4 and TERT-SHED P20. (Yin Z, et al., 2016)

β-GAL activity at pH 6 is a known characteristic of senescent cells, whereas this activity was not found in pre-senescent, quiescent, or immortalized cells. We first detected the senescence marker β-GAL in SHED and TERT-SHED at different passages. As shown in Figure 2a, the concentration of β-GAL in passage 20 SHED (SHED P20) was 120 times higher than that in SHED P4, but the concentration of β-GAL in passage 40 TERT-SHED (TERT-SHED P40) was still at a very low level. The proliferation capacity of SHED was detected by CCK-8 assay. As shown in Figure 2b, the proliferation capacity of late passage (P20) SHED was significantly decreased, while the proliferation potential of TERT-SHED P40 was similar to that of early passage (P4) SHED.

Figure 2. Comparison of β-GAL expression and proliferation ability between SHED and TERT-SHED of different passages. (Yin Z, et al., 2016)