| Background:Periodontal ligament stem cells(PDLSCs)are odontogenic mesenchymal stem cells(MSCs)that were first identified in and isolated from periodontal ligament tissue by Seo et al in 2004.PDLSCs were reported to have the ability to self-renew and the potential to differentiate into various specialized cell types,such as osteoblasts,fibroblasts and cementoblasts et al.Further evidence showed that PDLSCs had certain immune regulation function and little immunogenicity,were easy to obtain from deciduous teeth,permanent third molars or premolars extracted for orthodontics,demonstrating a promising application in bone tissue engineering.However,as a complex and heterogeneous population,PDLSCs even within the same condition may reflect different biological characteristics,which restricts their utilization as stem cells.During tooth development,ectomesenchymal stem cells(EMSCs)originating from the cranial neural crest(CNC)differentiate into various mesenchymal cell lines,ultimately giving rise to pulp,dentine,cementum and periodontal ligaments.Some scholars have suggested that a population of the CNC-derived progenitor cells remain in the periodontal ligaments and become a more ancestral subgroup in PDLSCs after the completion of tooth development.More accurate isolation of this subgroup will promote the successful application of PDLSCs in clinical treatment in the future.p75 neurotrophin receptor(p75NTR)is a well-conserved transmembrane neurotrophin/proneurotrophin receptor that belongs to the tumour necrosis factor receptor superfamily.The role of p75NTR is not limited to the nervous system,as it may have multifarious biological functions in non-neuronal tissues during development and differentiation.In addition,p75NTR is highly expressed in CNC-derived cells and proved to be a reliable cell surface marker for stem cells from the CNC.In our previous study,p75NTR was used as a cell surface marker to isolate CNC derived p75NTR~+EMSCs from rats.Further research found that these cells displayed superior multilineage differentiation potential and that p75NTR was involved in the positive regulation of osteogenic differentiation of EMSCs.However,few studies that isolate PDLSCs based on p75NTR expression have been reported,and the effect and mechanism of p75NTR on the biological characteristics of PDLSCs are also unclear.The present study aimed to use p75NTR as a cell surface marker to isolate PDLSCs and to further investigate the effect and underlying mechanism of p75NTR on the regenerative potential of PDLSCs.We expected to find an optimized cell surface marker for the identification and purification of PDLSCs,thus promoting their applications in bone tissue engineering.Methods:Part?:Fluorescence-activated cell sorting of PDLSCs using p75NTRAfter obtaining approval from hospital ethics committee and informed consent from the patient,the periodontal ligament tissue of healthy premolars from 8-to 25-year-old patients undergoing orthodontic treatment were collected,and the primary PDLSCs were isolated by enzyme digestion method and further cultured to the third-passage of cells in vitro.P75NTR was used as a cell surface marker,and PDLSCs were sorted by fluorescence-activated cell sorting to obtain p75NTR~+PDLSCs and P75NTR~-PDLSCs.The fourth-passage of p75NTR~+,P75NTR~-and unsorted PDLSCs were analysed by flow cytometry to detect the expression of p75NTR and MSCs-related surface markers,and the expression of p75NTR in the three types of cells was verified by immunofluorescence.Part?:Studies on biological characteristics of p75NTR~+,p75NTR~-and unsorted PDLSCsThe fourth-passage of p75NTR~+,p75NTR~-and unsorted PDLSCs were collected:(1)After routine culture for 10 days,the clone formation units of the three kinds of cells were observed by crystal violet staining,and the clone formation rate was calculated.(2)The proliferation ability of three kinds of cells was detected by CCK-8 kit every day.(3)The apoptosis of three kinds of cells was detected by flow cytometry.(4)After 21 days of adipogenic induction,oil red O staining was used to observe the adipogenic differentiation levels of the three kinds of cells.(5)After 21 days of chondrogenic induction,alcian blue staining was used to observe the chondrogenic differentiation levels of the three kinds of cells.(6)After 7 and 21 days of osteogenic induction,alkaline phosphatase(ALP)staining and alizarin red staining was used to observe the osteogenic differentiation levels of the three kinds of cells respectively.(7)Before osteogenic induction and after 7 days of osteogenic induction,the m RNA and protein expression of p75NTR,ALP and Runt related transcription factor 2(RUNX2)of the three kinds of cells were detected by fluorescent quantitation reverse transcription-polymerase chain reaction(q RT-PCR)and Western blot respectively.Part ?:RNA sequencing(RNA-seq)and differential analysis of p75NTR~+,p75NTR~-and unsorted PDLSCsThe fourth-passage of p75NTR~+,p75NTR~-and unsorted PDLSCs were collected,and the total RNA of the three kinds of cells was collected after routine culture for 3days.After RNA quality control,library construction,quality control,Illumina sequencing,sequencing data quality control and gene quantitative analysis,DESeq2 software was used for statistical analysis of the data to obtain the differential gene expression profiles of the three kinds of cells.Furthermore,the Cluster Profiler software was used for enrichment analysis of differentially expressed genes in p75NTR~+and p75NTR~-PDLSCs to screen out the key signal pathways with differences.Finally,5 kinds of genes related to this signal pathways,Laminin alpha 1(LAMA1),collagen type?alpha 1(COL4A1),integrin alpha 1(ITGA1),integrin alpha 7(ITGA7)and integrin alpha 8(ITGA8),were selected from the differential gene expression profiles and their m RNA expression was detected by q RT-PCR to verify the results of RNA-seq.Part ?:Discussion on mechanism of p75NTR optimizes the osteogenic potential of PDLSCs through ITGA1ITGA1 was selected as the key differentially expressed gene related to osteogenic differentiation in p75NTR~+and p75NTR~-PDLSCs.The fourth-passage of p75NTR~+PDLSCs were collected:(1)Small interfering RNA was used to transfect cells to silence ITGA1,and the silencing effect was verified by immunofluorescence.(2)After ITGA1 silencing and 3days of osteogenic induction,the osteogenic differentiation levels of cells were observe by ALP stain,the m RNA expression of ITGA1,p75NTR,ALP,RUNX2,ITGA7,ITGA8,LAMA1 and COL4A1 of cells were detected by q RT-PCR,and the protein expression of ITGA1,p75NTR,ALP and RUNX2 of cells were detected by Western blot.The fourth-passage of p75NTR~+PDLSCs were selected:(1)Adenovirus was used to transfect cells to overexpress ITGA1,and the overexpression effect was verified by immunofluorescence.(2)After ITGA1 overexpression and 3 days of osteogenic induction,the osteogenic differentiation levels of cells were observe by ALP stain,the m RNA expression of ITGA1,p75NTR,ALP,RUNX2,ITGA7,ITGA8,LAMA1 and COL4A1 of cells were detected by q RT-PCR,and the protein expression of ITGA1,p75NTR,ALP and RUNX2 of cells were detected by Western blot.Results:Part?:Fluorescence-activated cell sorting showed that p75NTR~+PDLSCs accounted for0.99%±0.38%of the isolated PDLSCs.p75NTR~+,p75NTR~-and unsorted PDLSCs showed a long spindle morphology,which is morphologically characteristic of MSCs.Flow cytometry analysis after cell sorting showed that the expression rates of p75NTR were 60.59%in p75NTR~+PDLSCs,0.55%in p75NTR~-PDLSCs and 1.31%in unsorted PDLSCs.Moreover,MSC markers(CD44,CD73,CD90 and CD105)were highly expressed in p75NTR~+,p75NTR~-and unsorted PDLSCs,while MSC negative markers(CD45,CD34,CD11b,CD19and HLA-DR)were expressed at low levels in the three kinds of cells.Immunofluorescence assay showed that the expression of p75NTR in p75NTR~+PDLSCs was stronger than that in p75NTR~-and unsorted PDLSCs,while there was no significant difference in p75NTR~-and unsorted PDLSCs.Part ?:The crystal violet staining among p75NTR~+,p75NTR~-and unsorted PDLSCs showed that there was no significant difference in the clonal formation rates of the three types of cells.The CCK-8 assay showed that there was no significant difference in proliferation capacity of the three types of cells for 7 consecutive days.Flow cytometry showed that there was no significant difference in apoptosis of the three types of cells.The oil red O staining and alcian blue staining showed that all three kinds of cells have the potential to differentiate into adipogenic and chondrogenic cell lines.The ALP staining and alizarin red staining showed that all three kinds of cells have the potential to differentiate into osteogenic cell lines,and p75NTR~+PDLSCs displayed stronger osteogenic differentiation than p75NTR~-and unsorted PDLSCs.q RT-PCR and Western blot test showed that before osteogenic induction,the m RNA and protein levels of p75NTR were higher in p75NTR~+PDLSCs than in p75NTR~-and unsorted PDLSCs,while m RNA and protein levels of ALP and RUNX2 were no significant difference in the three types of cells were not significantly different.In addition,after 7 days of osteogenic induction,the m RNA and protein levels of p75NTR,ALP and RUNX2 were increased in p75NTR~+,p75NTR~-and unsorted PDLSCs,although the m RNA and protein levels of p75NTR,ALP and RUNX2 elevated more in p75NTR~+PDLSCs than in p75NTR~-and unsorted PDLSCs.Part ?:The cluster analysis showed that compared with p75NTR~-PDLSCs,713 genes of p75NTR~+PDLSCs were up-regulated and 754 genes were down-regulated.Compared with unsorted PDLSCs,458 genes of p75NTR~+PDLSCs were up-regulated and 410 genes were down-regulated.Compared with unsorted PDLSCs,214 genes of p75NTR~-PDLSCs were up-regulated and 196 genes were down-regulated.KEGG pathway analysis showed that differentially expressed genes between p75NTR~+and p75NTR~-PDLSCs were involved several signaling pathways,among which the ECM-receptor interaction signaling pathway might be closely related to the osteogenic differentiation of PDLSCs.q RT-PCR test showed that the m RNA levels of ITGA1,ITGA7,ITGA8,LAMA1 and COL4A1 in p75NTR~+PDLSCs were higher than in p75NTR~-PDLSCs,which was consistent with RNA-seq results.Part ?:After ITGA1 silencing in p75NTR~+PDLSCs,immunofluorescence assay confirmed that the expression of ITGA1 was decreased.The ALP staining showed that the osteogenic differentiation level was decreased.q RT-PCR test showed that the m RNA levels of ITGA1,p75NTR,ALP and RUNX2 were decreased,but the m RNA levels of ITGA7,ITGA8,LAMA1 and COL4A1 were not significantly changed.Western blot test showed that the protein levels of ITGA1,p75NTR,ALP and RUNX2 were decreased.After ITGA1overexpression in p75NTR~-PDLSCs,immunofluorescence assay confirmed that the expression of ITGA1 was increased.The ALP staining showed that the osteogenic differentiation level was increased.q RT-PCR test showed that the m RNA levels of ITGA1,p75NTR,ALP and RUNX2 were increased,but the m RNA levels of ITGA7,ITGA8,LAMA1and COL4A1 were not significantly changed.Western blot test showed that the protein levels of ITGA1,p75NTR,ALP and RUNX2 were increased.Conclusion:1.p75NTR can be used to isolate PDLSCs to obtain CNC-derived stem cell subpopulations.2.p75NTR~+PDLSCs demonstrated superior osteogenic potential than p75NTR~-and unsorted PDLSCs.3.Differentially expressed genes between p75NTR~+and p75NTR~-PDLSCs are involved in the ECM-receptor interaction signaling pathway,and p75NTR~+PDLSCs expressed higher ITGA1 levels than p75NTR~-PDLSCs.4.As a key receptor in the ECM-receptor interaction signaling pathway,ITAG1 can positively regulate the osteogenic differentiation of PDLSCs under the environment of osteogenic induction.To sum up,the present study first demonstrated that p75NTR can be used to isolate homogeneous PDLSCs with superior osteogenic potential.Moreover,p75NTR optimizes the osteogenic potential of PDLSCs by up-regulating ITGA1 expression via the ECM-receptor interaction signalling pathway.These findings suggest that p75NTR can be used as a novel cell surface marker to identify and purify PDLSCs,thus promoting their applications in bone tissue engineering. |
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