Effect Of TERT-YAP On An Immortalized Periodontal Ligament Stem Cell Line

ObjectivesThe purpose of this study was to research the effect of YAP on an immortalized periodontal ligament stem cell line.Using periodontal tissue of human extracted orthodontic teeth to isolate and culture human periodontal ligament stem cells in vitro.The ability of cellsproliferation,apoptosis,senescence rate and cell differentiation ability of periodontal ligament stem cells in different passages were tested in vitro.On the basis,virus containing TERT gene wasused to transfect hPDLSCs to establish an immortalized human periodontal ligament stem cell line.The proliferation ability,cell apoptosis rate and cell senescence rateof periodontal ligament stem cellsafter TERT gene was overexpressed.The expression level of some proteins in the Hippo/YAP signaling pathway was detected after TERT gene was overexpressed.On this basis,YAP inhibitor Verteporfinwas added.Then detect the changes of cells biological characters.We can see from the results that the Hippo/YAP signaling pathway mediating the establishment of immortalized periodontal ligament stem cellline and the mechanism.To establish an immortalized human periodontal ligament stem cell line(hPDLSCs)and investigate whether and how YAP mediates the establishment of the stem cell line.Methods:Primary hPDLSCs were cultured and transfected with lentivirus containing the telomerase reverse transcriptase(TERT)gene.The expression of TERT was detected viathe polymerase chain reaction(PCR)and real-time quantitative PCR(qRT-PCR).Flow cytometry was employed to detect surface markers of hPDLSCs and TERT-hPDLSCs.The cell counting kit-8(CCK-8)and 5-ethynyl-2'-deoxyuridine(EdU)methods were used to examine the proliferation ability of the cells.Flow cytometry and TUNEL staining were employed to examine the cell apoptosis rate.The P-galactosidase staining assay was used to assess the rate of cell senescence.The osteogenic differentiation ability of the cellswas detected viaalkaline phosphatase(ALP)staining and Alizarin red staining assays.BALB/c mice were employed to determine the tumorigenicity of TERT-hPDLSCs.The expression levels of YAP and other proteins in the Hippo signaling pathway were detected by Western blotting.Verteporfin was used to inhibit the binding of YAP to the downstream target gene TEAD.Results:TERT-hPDLSCs showed stable high expression of TERT,even at the thirtieth passage after transfection with lentivirus containing the TERT gene.Compared with primary hPDLSCs,TERT-hPDLSCs exhibited a stronger proliferation abilityand lower cell apoptosis and senescencerates while maintaining the same osteogenetic differentiation ability as primary hPDLSCs.The transfection of hPDLSCs with lentivirus containing the TERT gene did not lead to tumorigenesis in nude mice.YAP and other key proteins in the Hippo signaling pathway showed higher expression levels in TERT-hPDLSCs than those in hPDLSCs.The proliferation of TERT-hPDLSCs decreased,while the apoptosis and senescencerates of these cells increased after YAP was inhibited with verteporfin.However,TERT-hPDLSCs still showed a stronger proliferation ability and lower cell apoptosisand senescencerates than hPDLSCs treated with verteporfin at the same concentration.Conclusions:Overexpression of TERT in hPDLSCs resulted in the successful establishment of an immortalized periodontal ligament stem cell line.TERT may regulate the biological characteristics of hPDLSCs through the Hippo/YAP signaling pathway.hPDLSCs could be a feasible resource for stemcell research and a promising resource for stem cell therapy.