Human TERT Lentivirus (CMV)

Human exfoliated deciduous teeth (SHED) stem cells 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. Here, researchers established a method to immortalize SHEDs by ectopic stable expression of TERT via a lentiviral vector. Lentiviral transduction induced stable TERT expression even in SHEDs at the 40th passage. TERT-SHEDs exhibited robust proliferation capacity and low concentrations of β-galactosidase. Although they had some different biomarkers from early-passage SHEDs, late-passage TERT-SHEDs exhibited multi-lineage differentiation similar to early-passage TERTs. In addition, late-passage TERT-SHEDs exhibited normal karyotypes, no soft agar colony formation, and no tumor formation in nude mice.

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 of Alizarin Red S, Oil Red O, and Toluidine Blue, respectively (Figure 1a-c). In addition, TERT-SHED P20 showed similar differentiation ability as 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)