PD-1 Is Required To Maintain Stem Cell Properties In Human Dental Pulp Stem Cells

Objective:Mesenchymal stem cells(MSCs)exist in a variety of organs and tissues,including bone marrow,umbilical cord,adipose tissue,skeletal muscle and dental tissues.MSCs from dental pulp(MSC-DP)can be isolated from the dental pulp of exfoliated deciduous teeth or permanent teeth.They are unique population of neural crest-derived stem cells.MSC-DP have a higher self-renewal capacity,compared to the classic bone marrow mesenchymal stem cells(BMMSCs).MSC-DP are multipotent mesenchymal stem cells capable of differentiating into osteo/odontogenic cells,adipocytes,chondrocytes,and neural cells under inductive conditions,and can regenerate dentin-pulp-like tissue in vivo.MSC-DP also possess excellent immunomodulatory properties that can regulate CD4+ T cells and regulatory T cells(Tregs).Systemic infusion of MSC-DP can ameliorate autoimmune disease phenotypes.MSC-DP represent an easily accessible,abundant and promising resource for MSC-based therapy.However,the underlying mechanisms that control MSC-DP function are largely unknown.Inhibitory receptor programmed cell death protein-1(PD-1)is expressed in various immune cells,including activated T cells,B cells,macrophages,dendritic cells and natural killer cells.PD-1-mediated negative immune signaling proceeds through engagement with the ligands.Upon activation,PD-1 suppresses exhausted CD4+ T cells in early phases of T cell activation,leading to immune tolerance.Moreover,PD-1 pathway plays an important role in cancer immunology by inducing tumor-infiltrating CD8+ T cell apoptosis and inhibiting CD8+ T cell function,leading to improvement of escaping from tumor immune-surveillance.So immunotherapies targeting PD-1 pathway have shown significant potential for autoimmune disease and cancer therapy.However,it is generally believed that MSCs fail to express PD-1,and largely unknown whether PD-1 pathway also contributes to stem cell function.In this study,we investigated the role of PD-1 in MSC function,especially MSC-DP,and explored the underlying mechanisms that PD-1 control MSC-DP function.The project will provide the theoretical and experimental evidence for revealing the molecular function of PD-1 in stem cell field.(?)exfoliated deciduous teeth(SHED)and dental pulp stem cells(DPSCs),were isolated and cultured in vitro.2.The PD-1 expression in BMMSCs and MSC-DP was examined,as assessed by Western blotting,qPCR,immunostaining and flow cytometric analysis.3.We knockdown the PD-1 expression by siRNA transfection and isolated PD-1+/PD-1-population by flow cytometric sorting to investigate the functional role of PD-1 in BMMSCs and MSC-DP.4.The detailed mechanisms of PD-1 control MSC-DP function were explored by using PD-1-/-MSC-DP that was generated by CRISPR/Cas9 plasmid,and also by using siRNA transfection and chemical inhibitor.The experimental data were analyzed statistically using SPSS 17.0 software package.P values less than 0.05 were considered statistically significant.Result:1.MSC-DP expressed PD-1 on the cell membrane continuously,while BMMSCs failed to express PD-1.PD-1 was co-expressed with MSC surface markers in MSC-DP and human dental pulp.Flow cytometric analysis was also used to show that 16.76%of SHED were PD-1+/CD73+,17.55%PD-1+/CD90+and 11.86%PD-1+/CD105+double-positive.And the expression level of PD-1 was decreased by passaging MSC-DP.2.PD-1 siRNA treatment significantly reduced the proliferation rate,the number of population doublings and the percentage of S/G2/M phases of MSC-DP compared to the control group(P<0.05).Moreover,PD-1 siRNA treatment markedly increased the multipotential differentiation of MSC-DP,including osteo-/odontogenic differentiation and neurogenic differentiation(P<0.001).When compared with PD-1-MSC-DP,PD-1+MSC-DP showed significantly elevated rates of cell proliferation,population doubling and S/G2/M phases(P<0.001).Also PD-1+ MSC-DP showed reduced capacities of osteo-/odontogenic and neurogenic differentiation(P<0.001).However siPD-1 treatment had no effects on BMMSCs function.3.PD-1 regulated the proliferation rate and population doubling of MSC-DP via SHP2/ERK/Notch cascade for maintaining self-renewal,and regulated the forming of mineralized nodules and the expression levels of osteogenic markers of MSC-DP via SHP2/ERK/?-catenin cascade for inhibiting osteo-/odontogenic differentiation.Conclusion:1.Neural crest-derived MSC-DP express the transmembrane protein PD-1 continuously,that can be used as a new unique surface marker to isolate and characterize MSC-DP.2.PD-1 is required to improve self-renewal and inhibit multipotential differentiation of MSC-DP,that is a key surface molecule controlling stem cell properties in MSC-DP.3.PD-1 regulates cell proliferation via a SHP2/ERK/Notch cascade and regulates osteo-/odontogenic differentiation via a SHP2/ERK/?-catenin cascade.