| Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism regulates boundary formation between cell population, cell proliferation and cell death. Notch signaling plays a crucial role in regulating self-renewal and differentiation of hematopoietic stem cells and progenitors. Notch receptors on progenitors are activated by ligands such as the Jagged and Delta proteins on stroma cells. Notch signaling plays a key role in the progress of tooth regeneration, and the ligand receptor complementary expression is induced following dental injury and associated with differentiation of pulp stem cells into perivascular cells and odontoblasts. It is generally thought that Notch activation promotes self-renewal and inhibits differentiation of the progenitors, as shown in some previous experimental systems. However, the precise function of Notch signaling in dental pulp cells remains to be further elucidated.Dental pulp stem cells (DPSCs) isolated from human dental pulp tissues by Gronthos in 2000 are thought to be the precursors in adult human dental pulp tissues and have potential to differentiate into odontoblasts under normal or pathological condition. Notch signaling expressed in human DPSCs in vitro. In this study, we investigated the effect of Notch ligand Delta1 inhibited by specific RNAi to Delta1 limited by lentivirus vector on growth and differentiation of DPSCs in vitro.Objective:To investigated the crucial effects of Notch ligand Delta1 inhibited by lentivirus-mediated Delta1-RNA interference on proliferation and differentiation of human dental pulp stem cells (DPSCs) in vitro.Methods: 1. Cell culture and identification: DPSCs were isolated from dental pulp of third molars and digested by collagenase typeâ… and dispase digestion, and then cultured. Their morphology and colony-forming efficiency of cell were observed under inverted lighted microscope. Vimentin, GFAP, Nestin, collagen typeâ…¢and STRO-1 were detected to identify the cell phenotype by immunohistochemistry and Flow cytometry. Cells were cultured in an odonto-inductive medium to identifiy their differentiation potential.2. Lentivirus infection: DPSCs were infected by lentivirus-mediated Delta1-RNAi. Downregulation of deficient Delta1 on Notch signaling was confirmed by Western blot analysis of Delta1 protein and RT-PCR assay of Hes-1 mRNA. DPSCs were divided into three groups, including DPSCs/Delta1-RNAi group, DPSCs/wt group and DPSCs/vector group.3. Detection of cell proliferation and differentiation: Changes of proliferation in DPSCs/Delta1-RNAi were examined by cell cycle analysis, CCK-8 assay and Western blot analysis of PCNA. Cells were cultured in odontoblast differentiation-inducing medium, and the differentiation of three group cells was detected with ALP activity assay, calcium concentration measurement, and Western blot analysis of DSPP.Results:1. The dental pulp stem cells exhibited self-renewal and clonogenic cell. Most of them retained spindle-shape morphology or 2-4 processes and high vigor. Colony-forming efficiency of cells derived from dental pulp tissue was 2~15clones/103 cells plated. Immunohistochemical and Flow cytometry analysis showed that DPSCs expressed Vimentin, GFAP, nestin and STRO-1 which express in desmohemoblast stem cells. Cells cultured in odonto-inductive medium express dentin sialophosphoprotein (DSPP) and formed calcified nodules.2. Delta-1 and Delta-1 mRNA expression of DPSCs/Delta1-RNAi group was significantly lower than the DPSCs/vector group and DPSCs/wt group, but there was no different between the DPSCs/vector group and DPSCs/wt group.3. The growth rate and S-cycle of DPSCs/Delta1-RNAi was significantly lower than DPSCs/wt or DPSCs/vector separately, and the PCNA staining was evidently weaker. Comparing with DPSCs/wt group or DPSCs/vector group separately, DPSCs/Delta1-RNAi group had more calcified cell nodules. ALP analysis showed that the expression of ALP in DPSCs/Delta1-RNAi group was significantly higher than the other two control groups, and DSPP expression of DPSCs/Delta1-RNAi group increased markedly. Furthermore, the morphology of DPSCs/Delta1-RNAi also changed after cells were infected by lentivirus-mediated RNAi to Notch ligand Delta1.Conclusions:1. Cells we isolated from adult human dental pulp were clonogenic, rapidly proliferative and had differentiation potential that satisfied the criteria of postnatal somatic stem cell.2. Lentivirus-mediated Delta1-RNAi stably inhibited the expression of Delta1 and Notch signaling.3. The capability of DPSCs/Delta1-RNAi differentiating into odontoblasts in vitro is more easily than DPSCs/wt and DPSCs/vector.4. Notch signaling plays a crucial role in regulating self-renewal and differentiation. Deficient expression of Notch ligand Delta1 inhibited the self-renewal capacity of DPSCs and tended to induce differentiation under odontoblast differentiation-inducing conditions.
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