A transcriptomic analysis of dental pulp stem cell senescence in vitro

A transcriptomic analysis of dental pulp stem cell senescence in vitro | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A transcriptomic analysis of dental pulp stem cell senescence in vitro JiDong Xu, Mingchang Hu, Longfei Liu, xuecheng xu, Linlin Xu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4786848/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Oct, 2024 Read the published version in BioMedical Engineering OnLine → Version 1 posted 11 You are reading this latest preprint version Abstract Background/purpose: The utilisation of human dental pulp stem cells (hDPSCs) as autologous stem cells for tissue repair and regenerative techniques represents a significant area of research globally. The objective of this experiment was to investigate the effect of long-term in vitro culture on the multidifferentiation potential of human dental pulp stem cells and the potential molecular mechanisms involved. Materials and methods: The tissue block method was employed to extract hDPSCs from orthodontic minus extraction patients, which were then expanded and cultured in vitro for 12 generations. Stem cells from passages 3, 6, 9 and 12 were selected. Flow cytometry was employed to detect the expression of stem cell surface markers, while CCK-8 was used to assess cell proliferation ability. β-galactosidase staining was employed to detect Cellular senescence, alizarin red S staining was employed to assess osteogenic potential, while Oil Red O staining was used to evaluate lipogenic capacity. RNA sequencing analysis was conducted to identify differentially expressed genes in DPSCs and to investigate potential mechanisms. Results : With increasing passage number, pulp stem cells showed an increase in senescent cells and a decrease in proliferative capacity and osteogenic-lipogenic multidifferentiation potential. The expression of the stem cell surface markers CD34 and CD45 was stable, whereas the expression of CD73, CD90 and CD105 decreased with increasing passages. According to RNA-seq analysis, The differentially expressed genes CFH, WNT16, HSD17B2, IDI and COL5A3 may be associated with stem cell senescence. Conclusion : An increase in in vitro expansion has been observed to induce a state of cellular senescence in pulp stem cells, which in turn results in a reduction in their proliferative capacity and osteogenic-lipogenic differentiation potential. Differential expression of genes such as CFH, Wnt16, HSD17B2, IDI, and COL5A3 may represent a potential mechanism for the induction of cellular senescence in pulp stem cells. DPSCs Senescence Multiple differentiation potential Transcriptome sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The integrity of the alveolar bone is a prerequisite for successful dental treatment. However, bone defects caused by periodontal disease, trauma, tumours and inappropriate orthodontic manipulation have always plagued clinicians. Currently, traditional bone repair techniques such as autologous bone grafting or bone graft substitutes have not been able to achieve satisfactory repair results. [ 1 ] In recent years, tissue-engineered bone repair and regeneration techniques based on autologous stem cells have attracted considerable research interest at home and abroad. Obtaining autologous seed stem cells with good activity and functionality is the key to research. Mesenchymal stem cells (MSCs) are pluripotent cells originating from the mesoderm with fibroblast-like morphology that can be induced to differentiate into osteoblasts, chondrocytes, and adipocytes under appropriate conditions. [ 2 ] Stem cell therapy is the process of applying stem cells of autologous or allogeneic origin and implanting them into the human body after in vitro manipulation such as isolation, identification, purification, expansion, and induction of differentiation. However, the number of stem cells required for each application in research and clinics is considerable, with even the bone marrow, which is rich in stem cells, unable to meet the demand. Consequently, to obtain a large number of mesenchymal stem cells quickly for research or clinical applications, increasing the number of cells through in vitro cell expansion and culture is unavoidable. [ 3 ] Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell (MSC), which are undifferentiated cells with stem cell properties extracted from dental pulp tissue. [ 3 ] They are considered to be the most accessible and promising multifunctional MSCs for tissue engineering and regenerative medicine because of their ease of accessibility, ease of expansion and preservation, and high activity and low tumourigenicity compared with other MSCs in the human body. [ 4 – 9 ] However, the scarcity of DPSCs, coupled with the limited number of human primary DPSCs obtained from in vitro cultures, makes it difficult to meet the demand for tissue engineering and regenerative medicine. In order to obtain sufficient numbers of DPSCs for therapeutic use, in vitro amplification before application is a necessary process. [ 10 ] It is important to note that in the standard culture state, due to the limited number of divisions, stem cells divide to a certain number of generations and enter a proliferative arrest, i.e., enter cellular senescence. [ 11 ] This process leads to functional decline of stem cells, stagnant cell growth, significantly weakened differentiation ability, and increased number of apoptotic cells, which are no longer suitable for tissue engineering therapy and limit its clinical application. [ 12 , 13 ] Consequently, the quality control of stem cells prior to their application in tissue engineering must include an in vitro passaging process. Cellular senescence is strongly regulated at the post-transcriptional level, with the interaction of RNA-binding proteins and non-coding RNAs with senescence-associated messenger RNAs (mRNAs) playing a pivotal role in this regulation. [ 14 ] It has been demonstrated that cellular senescence is affected by specific chemical modifications of mRNAs. [ 14 ] The current research on mRNA modifications has focused on the role of mRNA methylation, with studies investigating its potential involvement in cancer, obesity and other age-related pathologies. [ 15 , 16 ] Indeed, there is accumulating evidence that mRNA modification may be closely related to cellular senescence. The molecular mechanism of cellular senescence resulting from long-term in vitro culture of DPSCs remains unknown, and there are currently no cell culture protocols that can delay or prevent the senescence of DPSCs. The objective of this experiment was to investigate the effects of long-term in vitro expansion culture on the morphology, immunophenotype, proliferation and osteogenic-lipogenic multidifferentiation potential of DPSCs, as well as the changes in mRNA expression of DPSCs during long-term in vitro expansion culture. Furthermore, the experiment aimed to identify the potential molecular mechanism that triggers the senescence of DPSCs. The regulation of DPSCs senescence will play a pivotal role in the clinical application of stem cells. Therefore, an in-depth understanding of DPSCs senescence mechanisms is crucial for the development of strategies to promote the regenerative potential of DPSCs in order to maintain tissue function. RESULTS 1.1 Characterization of DPSCs with different numbers of passages Twenty healthy orthodontic patients (10 males and 10 females) aged 12 to 16 were selected for this study. DPSCs were cultured individually as one sample from one donor. Within 10 days of primary culture, spindle cell-like cells arranged in a swirling pattern were observed free around the pulp tissue mass of all donor samples (Fig. 1A). The cells were expanded up to 12 generations in subsequent in vitro passaging cultures. As the number of in vitro passages increased, the cell morphology exhibited distinguishable replicative senescence changes (Fig. 1A). The cells enlarged and acquired irregular and flattened shapes, while the nuclei became thinner. The cytoplasm showed granularity and contained elevated levels of cellular debris and inclusions. A few cells proliferated without adhering to the wall. Flow cytometry analysis revealed that DPSCs of the P3 expressed CD73, CD90, and CD105 at high levels while expressing CD34 and CD45 at low levels, thereby verifying the DPSCs' immunophenotype (Fig. 1B). As the number of passages increased, the percentage of DPSCs expressing CD34 and CD45 remained stable. However, the percentage of DPSCs expressing CD73, CD90, and CD105 tended to decrease. This change was particularly significant for DPSCs cultured up to P12. (Fig. 1B, Table 1). These findings imply that surface marker expression levels alter to some extent during continuous in vitro cultivation. Table 1. Quantitative profiles of surface marker expression of DPSCs at P3, P6, P9 and P12 (n=20, mean±SD) Marker P3 P6 P9 P12 F P CD34 1.86±2.34 0.67±0.53 3.96±6.47 0.21±0.14 0.9729 0.4375 CD45 2.51±3.79 0.56±0.78 4.89±8.45 0.39±0.76 0.8153 0.5098 CD73 99.30±0.32 96.97±4.84 99.50±0.23 46.26±53.12 3.858 0.0382 CD90 98.96±0.39 96.88±1.48 91.10±11.46 29.38±32.30 15.08 0.0002 CD105 91.29±4.25 84.93±4.87 71.60±4.36 1.14±1.02 446.0 0.05); CD73、CD90 and CD105 showed a decreasing trend in expression with an increasing number of passages (P < 0.05). 1.2 The proliferation of DPSCs with different numbers of passages, changes in senescence DPSCs were cultured for five days, and changes in their proliferative capacity were measured using the CCK-8 method. The results demonstrate that the third generation had the fastest proliferation rate, with subsequent generations exhibiting a progressively slower proliferation rate (Fig. 2A). The study revealed a decline in the proliferative potential of DPSCs with increasing passages during prolonged in vitro culture. SA-β-gal staining was conducted on the third, sixth, ninth, and twelfth generations of DPSCs to examine cell senescence at varying passage numbers. Under microscopic inspection, cells with dark blue staining in their cytoplasm were considered SA-β-gal positive, indicating senescent cells. A minor proportion of blue cells was detected in the third-generational DPSCs (Fig. 2B), which yielded an SA-β-gal positivity rate of 7%. The rate of SA-β-gal positivity was recorded as P6 (10.08±1.08) %, P9 (17.5±1.45) % and P12 (27±2.89) %. Additionally, the number of cells displaying dark blue staining increased gradually with increasing in vitro expansion (Fig. 2B). The statistical significance of the cell differentiation can be observed (P < 0.05). This observation indicates a gradual increase in the proportion of SA-β-gal positive cells during succeeding passages of DPSCs in vitro (Fig. 2C). It can be seen that the cells undergo a senescent phenotype due to multiple in vitro expansions. 1.3 Changes in the multi-differentiation potential of DPSCs with different numbers of passages Results of Osteogenic and Lipogenic Differentiation Experiments: The experiment involved inducing lipogenic and osteogenic differentiation for 14 and 21 days, respectively. The results obtained from alizarin red (ARS) and oil red O (ORO) staining showed that cells from P3 and P6 generations had good osteogenic and lipogenic characteristics. Under the microscope, apparent mineralized nodules and lipid droplets were observed (Fig. 3A, 3B). Stem cells that underwent numerous passages, specifically the P9 and P12, displayed decreased potential for osteogenic and lipogenic differentiation. Additionally, a higher number of apoptotic cells were observed, and there was a shift in cell morphology from an initial long spindle to an oval-like shape. It was challenging to keep apparent mineralized nodules and lipid droplets (Fig. 3A, 3B). The results of quantitative analyses indicate that the osteogenic and lipogenic abilities of DPSCs decrease as the number of in vitro passages increases (Fig. 3C, 3D). These results suggest that continuous passaging in vitro induces cellular senescence in DPSCs. As the number of in vitro passages increased, the heterogeneity of cell morphology increased, the proliferative capacity and multi-differentiation potential decreased, and the proportion of SA-β-gal positive cells increased. 1.4 Differential gene expression profiles of DPSCs during aging in vitro 16 samples of 4 groups of DPSCs from P3, P6, P9 and P12 were subjected to mRNA sequencing (mRNA-seq). This sequencing detected 33638 expressed genes and differentially expressed DE mRNA were screened by the |log2FC| > =1&P-value < 0.05 criteria. Compared with P3, there were 283 DE mRNA in P6, of which 98 were up-regulated and 185 down-regulated (Fig. 4A), 536 DE mRNA in P9, of which 347 were up-regulated and 189 down-regulated (Fig. 4B), and 1159 DE mRNA in P12, of which 692 were up-regulated and 467 down-regulated (Fig. 4C), DE mRNA was depicted by heat polygraph (Fig. 4D), in which the most significant changes in DE mRNA were observed in the P12, Table 2 shows the top 10 mRNAs with the largest up-regulation or down-regulation in the P12 compared with the P3, including CFH, WNT16, IDI1.etc, and these differentially altered RNAs were closely associated with the signaling pathways that regulate stem cell pluripotency. Table 2.Characteristics of the mRNA with the most significant fold change between P12 and P3 (n=4, mean±SD) Gene ID Gene name Log2FC[P12/P3] Regulate ENSG00000086696 HSD17B2 2.542375093 up ENSG00000002745 WNT16 1.808346941 up ENSG00000000971 CFH 1.438485741 up ENSG00000080573 COL5A3 -1.055618779 down ENSG00000067064 IDI1 -1.033491011 down 1.5 mRNA GO and KEGG analysis The sequencing results showed the most pronounced differential changes in the P12 and P3, and we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on the P12 and P3 DE mRNA. The top 20 GO terms are related to biological processes, cellular components, and molecular functions (Fig. 5A-5C). The top 10 GO terms related to bioprocesses and DE mRNA with the information associated with these terms are shown in Table 3 (P < 0.05), and these bioprocess-related mRNAs are closely related in the regulation of stress response, cell proliferation, cellular bioprocess-regulation, and organ development. KEGG analysis of DE mRNA revealed that 35 pathways were significantly altered (P < 0.05) in DPSCs during in vitro senescence. The top 20 enriched pathways are shown in Figure 5D. The top-ranked pathways include signaling pathways related to viral myocarditis, pertussis, coronavirus disease (COVID-19), and basal cell carcinoma, closely associated with human diseases. Table 3. Top 10 terms with the most significant differences in DE mRNAs' GO biological process analysis. Term Count Rich ratio P value Candidate gene Regulation of response to stress 21 0.143836 2.99E-07 FIGNL1;MET;EDNRB;CXCL6;USP1;RADX;POLR3G;UBAS-H3B;C1QTNF1;DUSP1;HBEGF;STK26;FOXC2;CFH;TNFAIP-6;RASSF2;HLAF;GPRC5B;WNT16;MMP3 Regulation of cell population proliferation 22 0.150685 4.53E-07 EFNB2;EDNRB;PLAAT4;ODC1;DUSP1;SGK1;GRPR;ADGR-G1;SPRY2;CD24;TIAM1;SGPP2;CHRNA7;BST2;HBEGF;PTG-S1;RAC2;RTKN2;COL18A1;E2F7;CDC6;HSF4 Regulation of multicellular organismal process 30 0.205479 5.46E-07 EFNB2;SCN2B;PLAAT4;ZFHX2;C1QTNF1;NSUN5P1;EDNR-B;POLR3G;SGK1;HLAF;L3MBTL1;TIAM1;CAPN3;UBASH3-B;SPRY2;CD24;CASP1;LAMA2;CD14;FABP5;KCNJ2;CHRN-A7;BST2;HBEGF;AMIGO2;RASSF2;GPRC5B;FOXC2;MET;C-XCL6 Cell adhesion 17 0.116438 5.72E-07 BOC;EFNB2;TNFAIP6;PCDHGB1;TIAM1;COL5A3;ENTPD1;ADGRG1;UBASH3B;COL18A1;ARVCF;NCAM1;CD24;PCD-HGA4;RAC2;AMIGO2;LAMA2 Animal organ development 20 0.136986 7.04E-07 RTKN2;TCF21;CAPN3;MET;HSD17B2;ADGRG1;SLC6A17;ODC1;HSD11B1;HBEGF;COL5A3;GAS6;L3MBTL1;E2F7;AMI-GO2;LCP1;FOXC2;LAMA2;HSF4;ILDR2 Regulation of signal transduction 29 0.198630 7.70E-07 C1QTNF1;GREM2;L3MBTL1;TIAM1;ADGRG1;PAQR3;CAP-N3;UBASH3B;SPRY2;CD24;CASP1;CD14;FABP5;CHRNA7;BST2;DUSP1;HBEGF;GRIA3;RAC2;RASSF2;GPRC5B;RTKN-2;TCF21;FIGNL1;MET;SECTM1;NCAM1;RBBP8;WNT16 Regulation of response to stimulus 45 0.308219 8.04E-07 EFNB2;TNFAIP6;TIAM1;C1QTNF1;GREM2;EDNRB;POLR3-G;HLAF;L3MBTL1;FABP5;ADGRG1;PAQR3;CAPN3;UBAS-H3B;SPRY2;CXCL6;CD24;CASP1;STK26;NCAM1;CFB;CD14;C2;CHRNA7;CFH;RADX;BST2;DUSP1;HBEGF;MMP3;GRI-A3;RAC2;RASSF2;GPRC5B;FOXC2;RTKN2;TCF21;FIGNL1;MET;SECTM1;RBBP8;USP1;HLAC;WNT16 Positive regulation of intracellular signal transduction 17 0.116438 8.39E-07 RTKN2;MET;CD14;TIAM1;ADGRG1;SECTM1;C1QTNF1;BST2;HBEGF;SPRY2;CD24;RAC2;CASP1;RASSF2;CHRNA7;G-PRC5B;WNT16 Response to biotic stimulus 20 0.136986 8.47E-07 APOL1;GRPR;EDNRB;POLR3G;CD14;C2;CXCL6;ISG20;OD-C1;CFB;BST2;IFI44L;CASP1;MX2;CD24;FER1L6;CFH;OAS1-;HLAC Positive regulation of macromolecule metabolic process 32 0.219178 9.67E-07 EDNRB;C1QTNF1;NSUN5P1;CYTL1;POLR3G;HLAF;MYBL1;ZFHX2;CAPN3;SPRY2;EBF4;CD24;CASP1;STK26;CD14;TIAM1;ZNF711;WNT5A;CHRNA7;HBEGF;RASSF2;GPRC5B;FOXC2;TCF21;MET;GAS6;HIVEP3;HSF4;E2F7;CDC6;WNT16 1.6 PPI and co-expression network analysis Compared with the P3 of DPSCs, a total of 70 nodes and 85 interaction pairs were identified in the DE mRNA PPI network in the P12 (Fig. 6). Nodes with high topological scores may play an essential role in the aging process of DPSCs in vitro. In this study, bone morphogenetic protein 4 (BMP4), interferon-stimulated exonuclease gene 20 (ISG20), type II transmembrane glycoprotein (BST2), and other DE mRNA interactions such as WNT family member 5A (WNT5A) were identified as essential genes in this network. 1.7 Validation of the vital RNA expression To verify the RNA-seq data and significant findings from computational analysis, we performed quantitative real-time PCR for some critical RNAs selected from the RNA networks. The expression levels of those RNAs were measured in 4 human DPSCs in a different passage. The qPCR results showed that the expression of WNT16、CFH and HSD17B2 were upregulated and that the expression of COL5A3 and IDI were downregulated (Fig. 7). All qPCR results were consistent with the RNA-seq results, confirming the reliability of the sequencing results. Discussion DPSCs exhibit multidirectional differentiation potential, self-renewal replication and immunomodulation, and have promising applications in tissue engineering and cell therapy. [ 17 ] DPSCs, as a type of mesenchymal stem cell, have a higher proliferation rate, greater in vivo osteoinductive formation and multidirectional differentiation potential compared to bone marrow mesenchymal stem cells. [ 18 ] Furthermore, DPSCs can be obtained from wisdom teeth or teeth that require extraction for orthodontic purposes, which is a straightforward process, less traumatic to patients, and involves fewer ethical considerations. [ 19 ] A study by Nela et al. demonstrated that even after thawing after one year of cryopreservation at -80°C, the cell viability, proliferative capacity, and differentiation ability of DPSCs remained unaffected. [ 20 ] These advantages have expanded the clinical applications of DPSCs. Studies have demonstrated that DPSCs must be expanded and cultured in vitro to meet the needs of clinical treatment. [ 21 ] Nevertheless, prolonged in vitro expansion and culture will result in cellular senescence. The current research on cell senescence resulting from prolonged in vitro expansion and culture primarily focuses on other types of stem cells, with fewer studies on the senescence of DPSCs. Further research is required to elucidate the related aspects of DPSCs and the underlying mechanisms of senescence. This will facilitate the determination of the optimal number of in vitro expansion generations to ensure that the cell activity and number meet the clinical therapeutic application requirements. [ 21 ] In this experiment, we successfully established a senescence model of DPSCs by extracting primary DPSCs and culturing them to 12 generations by in vitro expansion under standard culture conditions. DPSCs cultured to the third generation were selected and flow cytometry was performed to detect the expression of antigenic markers on their surfaces as well as for osteogenic-lipogenic induction. The results demonstrated that the negative markers CD34 and CD45 were expressed at low levels, while the positive markers CD73, CD90 and CD105 were highly expressed. Furthermore, the flow cytometry results were consistent with the surface immunostaining profiles of MSCs, as previously reported in the literature. [ 22 ] These findings indicated that the cells extracted exhibited the potential for osteoblastic and lipogenic differentiation, which confirmed that the cells were indeed MSCs. Following a prolonged period of in vitro passaging and expansion culture, it was observed that the proliferative capacity of the cells declined, accompanied by a gradual increase in the proportion of SA-β-gal-positive cells with each additional passage. This is a marker of cellular senescence, which suggests that the DPSCs exhibited a gradual process of cellular senescence with the increase in the number of passages. [ 23 ] Although DPSCs continued to exhibit low expression of CD34 and CD45, there was a tendency for CD73, CD90 and CD105 expression to decline with the number of passages, with the most significant decrease observed following in vitro expansion to 12 generations. The results of osteogenic-lipogenic induction experiments demonstrated that the osteogenic-lipogenic differentiation potential of DPSCs was reduced with the increase in the number of in vitro amplifications. These experimental results were consistent with those of previous studies. [ 24 , 25 ] The above experimental results suggest that DPSCs with reduced polydifferentiation potential after aging may no longer be suitable for clinical treatment. However, there is a divergence of opinion regarding the changes in stem cell surface markers with passaging. Kim et al. studied 55 generations of bone marrow MSCs cultured in vitro and found that the expression of stem cell surface markers tended to stabilise with increasing number of passages. [ 26 ] In contrast, Bakuplu et al.expanded DPSCs in vitro up to 12 generations and found that the expression of CD105, a surface marker of DPSCs, decreased with increasing number of passages, while the expression of CD73 and CD90 remained relatively stable. [ 27 , 28 ] This is not exactly the same as our experimental results. The experimental results may be affected by various factors, including the source of pulp tissue, the stage of tooth development and the age of the patient. In addition, heterogeneity may increase during the culture process due to differences in culture time, medium used and number of passages. There are fewer studies on the expression of surface markers in DPSCs with different numbers of passages, which remains to be further explored. There are no specific surface markers for DPSCs. [ 28 , 29 ] The typical markers for MSCs, including CD73, CD90 and CD105, do not appear to provide sufficient or specific indications to maintain the ‘stemness’ of DPSCs. Therefore, additional stem cell markers and other multiparametric immunophenotyping should be employed to validate DPSCs stemness. The mRNA expression profiles reflect the biological behaviour and function of dental pulp stem cells (DPSCs) cultured in vitro over a long period of time. Changes in these mRNA expression profiles are closely related to functional changes during in vitro ageing. This experimental study identified a series of mRNAs associated with the senescence of DPSCs and their associated molecular mechanisms, which may be potential mediators of changes in biological properties such as reduced proliferation and differentiation after long-term in vitro expansion and culture. A total of 1159 differentially expressed (DE) mRNAs were identified between P12 and P3. Further analysis of these DE mRNAs revealed that changes in CFH, Wnt16, IDI1, COL5A3, and HSD17B2 mRNAs were closely associated with cellular senescence. CFH is a complement inhibitor that plays a key role in complement homeostasis. [ 30 ] The sequencing results demonstrated that the CFH gene exhibited elevated expression in senescent DPSCs subjected to long-term expansion and in vitro culture. Furthermore, mutations and variants of the CFH gene have been demonstrated to be significantly associated with a number of human age-related diseases, including cancer and age-related macular degeneration. [ 30 – 32 ] Wnt16, a member of the Wnt family, has been shown to be closely associated with osteogenic differentiation, cellular senescence and tumourigenesis. [ 33 – 35 ] Additionally, it has been implicated in the proliferation and differentiation of stem cells. [ 36 ] The results of RNA-seq sequencing demonstrated that: Wnt16 gene expression was observed to be upregulated, while the results of the osteogenic induction assay indicated that the osteogenic capacity of stem cells exhibited a gradual decline with increasing passages. This suggests that the elevated expression of the Wnt16 gene may potentially inhibit osteogenic differentiation. Jiang et al.demonstrated that the Wnt/β-catenin protein signalling pathway inhibits the osteogenic differentiation of human mesenchymal stem cells and MC3T3-E1 cells (mouse embryonic osteoblasts). [ 37 – 39 ] This assertion is corroborated by the findings of our own experiments. However, Carolyn et al.have proposed that the Wnt16 gene stimulates the osteogenic differentiation of perivascular stem cells (PSC). [ 40 ] These disparate findings indicate that the Wnt signalling pathway plays a pivotal role in the regulatory network of endosteal homeostasis. Both overactivation and inactivation of Wnt signalling can result in skeletal deformities, bone diseases and cartilage loss. [ 41 ] The number of studies investigating the Wnt16 gene and osteogenic differentiation of stem cells is limited, and the role of this gene in osteogenesis in DPSCs cells remains to be further elucidated. Isopentenyl diphosphate isomerase 1 (IDI1) is an enzyme that encodes a peroxisomal localization, which removes toxic hydrogen peroxide produced by different oxidative enzymes in the peroxisomal respiratory pathway. [ 42 ] It is involved in processes such as cell division and proliferation and is associated with age-related diseases. [ 43 ] The experimental results demonstrated that the expression of IDI1 was reduced in senescent DPSCs. This reduction in the synthesis of peroxisome-localised enzymes and the catabolism of hydrogen peroxide leads to an exacerbation of cellular senescence. The collagen type V alpha 3 chain (COL5A3) is a member of the collagen family that is closely associated with osteogenesis and tumourigenesis. The experimental results indicate that cellular senescence is associated with a reduction in COL5A3 expression. Pavitra K et al.demonstrated that hypoxia stimulates the expression of COL1A1, COL5A1 and COL5A3 in osteoblasts, which plays a role in maintaining bone volume by promoting collagen production. [ 44 ] Chen et al.observed that collagen inhibits immune signalling in the tumour microenvironment (chemokines) production, thereby inhibiting anti-tumour immune responses. [ 45 ] The loss of collagen results in an increase in chemokine levels, which in turn facilitates the proliferation of cancer cells. 17-β-hydroxysteroid dehydrogenase type 2 (HSD17B2) is a protein associated with estrogen synthesis and regulates estradiol (E2). [ 46 ] The experimental results indicated that HSD17B2 expression was upregulated, suggesting that it may be associated with cellular senescence. Lu et al.demonstrated that the inhibition of HSD17B2 activity suppressed E2 inactivation, increased endogenous estrogen levels, and improved bone metabolism-related indicators. [ 46 ] In conclusion, these DE mRNA changes are closely associated with cellular senescence, disease development, and osteogenic differentiation, among other factors. One limitation of this experiment is that we did not perform gene knockdown. To study the effect of gene knockdown on dental pulp stem cell senescence, further studies are needed in subsequent experiments. KEGG analysis has identified numerous signalling pathways associated with human diseases, including viral myocarditis, pertussis, coronavirus disease (COVID-19) and basal cell carcinoma. These pathways play a pivotal role in the functional changes induced by ageing, thereby underscoring the significance of DPSCs ageing in the pathogenesis and clinical treatment of cancer, cancer and metabolic diseases. Among the interactions between DE-encoded proteins, BMP4, ISG20, BST2 and Wnt5a were identified as key genes interacting with many other DE mRNAs in this network.BMP4, a member of the TGF-β superfamily, is involved in a variety of biologically regulated processes such as cell proliferation and cellular differentiation. [ 47 ] BMP4 plays a key role in bone formation, and the gene may promote alveolar bone development by increasing the expression levels of Runx2, BSP and OCN. [ 48 ] ISG20 has been associated with certain RNA virus-induced diseases, and a study of ISG20 elucidated its involvement in antiviral mechanisms, which found that overexpression of recombinant ISG20 in cultured cells increased cellular resistance to infection with certain RNA genomes of viruses. [ 49 , 50 ] BST2, also known as CD317, is involved in a variety of physiological and pathological processes, including inflammation, immune regulation and tumourigenesis, and BST2 is overexpressed in various malignant tumours, suggesting that BST2 may be associated with certain tumour disorders induced by cellular senescence. [ 36 , 51 – 53 ] Wnt5a is a representative Wnt protein of the non-classical Wnt signalling pathway, which plays an important role in the development and maturation of various tissues and organs, and is closely related to a variety of diseases such as infectious diseases, cancer and metabolic disorders. [ 54 ] Studies on Wnt5a and related aspects of its signalling pathway may provide new ideas for the diagnosis and treatment of human diseases. [ 55 ] In summary, these proteins are closely related to the biological changes of DPSCs after long-term in vitro amplification and culture. However, whether these proteins can be used as markers for clinical detection remains to be further investigated. In addition, DPSCs are derived from neural crest cells and have unique advantages in nerve repair, cartilage formation and corneal reconstruction. [ 56 – 59 ] Furthermore, in addition to its use in repairing teeth and maxillofacial bone tissue, it can also be used to repair tissues outside the oral cavity, such as nerves, cartilage and other systems. Dental pulp stem cells are also receiving increasing attention in the field of regenerative medicine. Autologous stem cell therapy for diabetes and myocardial infarction have likewise shown promising applications. [60.61] Dental pulp stem cells extracted by the pulp tissue block method exhibited low CD34 and CD45 expression, high CD73, CD90 and CD105 expression, and demonstrated osteogenic and lipogenic differentiation potential. The number of in vitro expansions was found to induce cellular senescence of pulp stem cells, which resulted in a reduction in proliferative capacity and osteogenic and lipogenic differentiation potential. It is possible that changes in mRNA induced by prolonged expansion in vitro may be a potential mechanism for the senescence of dental pulp stem cells. The differential expression of genes, including CFH, WNT16, HSD17B2, IDI and COL5A3, may be of significant importance in the context of pulp stem cell senescence.It is crucial to explore the molecular mechanism behind the senescence of DPSCs in order to establish a more robust theoretical basis for the clinical application of DPSCs. Materials and methods 3.1 Cell isolation and passaging This study was evaluated by the Ethics Committee of Qingdao Stomatological Hospital, Qingdao University (2022KQYX023) and begins on 1 October 2023 and ends on 1 May 2024. Pulp stem cells were obtained from teeth lacking dental and periodontal tissue diseases and taken from orthodontic patients aged 12–16 without systemic diseases via the tissue block methodology. The cells were passaged at 80% cell density and grown in vitro , expanding culture for up to 12 generations. This study employed DPSCs from 3rd, 6th, 9th, and 12th generations. The DPSCs were cultured in α-MEM medium (Pricella, Wuhan, China) with varying concentrations of fetal bovine serum (FBS, Pricella, Wuhan, China) and 1% penicillin-streptomycin (BioIndustries, Israel) according to the specific experimental conditions. All cells were maintained at 37°C under constant humidity of 5% CO 2 . 3.2 Flow cytometry Flow cytometry was utilized to assess the presence of positive markers (CD73, CD90, and CD105) and negative markers (CD34 and CD45) in DPSCs. They were digested and centrifuged to prepare the DPSCs (P3, P6, P9, and P12). The cell density was subsequently standardized to 1×10 4 cells/mL, and 100 µL of cell suspension was introduced to the centrifuge tube. Anti-human antibodies CD34 (E-AB-F1143C), CD45 (E-AB-F1137C), CD73 (E-AB-F1242D), CD90 (E-AB-F1167D), and CD105 (E-AB-F1143D) (Elabscience, Wuhan, China) were added in a ratio of 50:1. PBS (Priscilla, Wuhan, China) was used as a control. The supernatant was centrifuged and discarded after a one-hour dark incubation at room temperature, followed by two washes with PBS and resuspension in 500 µL of PBS. The expression levels of various antibodies were then determined by flow cytometry using the DxFLEX machine from Beckman Coulter (Suzhou, China). 3.3 Cell proliferation Use the Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China) to measure cell proliferation according to the manufacturer's instructions. DPSCs at P3, P6, P9, and P12 were seeded at a density of 1×10 4 cells/well in 96-well culture plates and incubated for 1 to 5 days. At each specific time point, the complete medium was exchanged with new α-MEM basal medium supplemented with a 10% CCK-8 solution. This solution was then incubated under dark conditions for 2 hours at 37°C. Finally, cell proliferation capacity was determined by detecting absorbance at 450nm using an enzyme label reader (SynergyH1/H1M, Bio-Tek, China). 3.4 Cellular senescence Cellular senescence was detected through β-galactosidase staining analysis. DPSCs at P3, P6, P9, and P12 were introduced into 6-well plates at a density of 1×10 5 cells/well. The SA-β-gal activity was then evaluated by the instructions provided by the β-galactosidase staining kit manual (Beyotime, Shanghai, China). Senescent cells were stained blue by SA-β-gal, and SA-β-gal positive cells were evaluated as a percentage of all cells based on three randomly selected bright fields for observation and calculation. 3.5 Osteogenic differentiation DPSCs of P3, P6, P9, and P12 were plated in 6-well dishes at a 2×10 5 cells/well density until they reached 70% confluence. The original medium was subsequently aspirated and replaced with Osteogenic Differentiation Inducing Medium (Pricella, Wuhan, China). The cells were cultured for 21 days, with a medium change every 3 days. After fixing with 4% paraformaldehyde, the samples were stained with Alizarin Red S following washing with PBS. Images were collected by observing under an optical microscope (OLYMPUS, Japan). Then, 10% Cetylpyridinium Chloride solution was added to the wells, and the solution dissolved for 30 min at room temperature. The decolorized solution was then collected, and the absorbance at 562 nm was measured with an enzyme label reader. 3.6 Lipogenic differentiation DPSCs (P3, P6, P9, and P12) were seeded in 6-well plates at 2×10 5 cells/well density. After the cells reached 100% confluence, the original medium was removed, and lipid induction was carried out for 14 days using a Lipid-forming differentiation-inducing medium (Pricella, Wuhan, China) following the manual instructions. All abbreviations used in the text are explained in their first usage. The cells were immobilized using a 4% paraformaldehyde solution (Elabscience, Wuhan, China), washed with PBS, and then stained with Oil Red O. Following observation and capture of the images under a light microscope, the stained lipid droplets were dissolved in isopropanol (Sigma-Aldrich) at room temperature, the decolorized solution collected, and the absorbance at 510 nm measured using an enzyme marker. 3.7 Library construction and high-throughput sequencing 3.7.1 RNA extraction Total RNA was extracted from P3, P6, P9 and P12 generation stem cells (n = 4) using TRIzol reagent (Pricella, Wuhan, China) according to the manufacturer's instructions. RNA quality was then determined using a 5300 Bioanalyzer (Agilent) and quantified using an ND-2000 (NanoDrop Technologies). High-quality RNA samples (OD260/280 = 1.8 ~ 2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, > 1 µg) were used to construct sequencing libraries. 3.7.2 Library preparation and sequencing RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co, Ltd, Shanghai, China. RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co, Ltd, Shanghai, China, according to the manufacturer's (Illumina, San Diego, CA) instructions. The mRNA-seq transcriptome libraries were prepared with 1 µg of total RNA following the Illumina® Stranded mRNA Prep, Ligation method provided by Illumina (San Diego, CA). First, messenger RNA was isolated by polyA selection using oligo (dT) beads, followed by fragmentation with fragmentation buffer. Double-stranded cDNA was synthesized using the SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) and random hexamer primers (Illumina). According to Illumina's library construction protocol, the synthesized cDNAs were subjected to end repair, phosphorylation, and 'A' base addition. The 300 bp cDNA target fragment was size-screened on 2% Low Range Ultra Agarose, followed by 15 PCR cycles of PCR amplification using Phusion DNA polymerase (NEB). After quantification with Qubit 4.0, paired-end RNA-seq sequencing libraries were sequenced with a NovaSeq 6000 sequencer (2×150 bp read length). 3.7.3 Quality control and reading mapping Raw paired-end reads were trimmed and quality-controlled using fast and default parameters. Clean reads were individually aligned to the reference genome in targeted mode using HISAT2 software. Mapped reads for each sample were assembled on a reference basis using StringTie software. 3.7.4 Differential expression analysis and functional enrichment To identify differentially expressed genes (DEGs) between two different samples, the expression level of each transcript was calculated based on the transcripts per million reads (TPM) method. RSEM was used to quantify gene abundance. Differential expression analysis was performed using DESeq2 or DEGseq.|DEGs with log2FC| ≥ 1 and FDR < 0.05 (DESeq2) or FDR < 0.001 (DEGseq) were considered as significantly differentially expressed genes. Functional enrichment analyses, including GO and KEGG, were also performed, and DEGs were significantly enriched in GO terms and metabolic pathways (Bonferroni-corrected P- value < 0.05). GO functional enrichment and KEGG pathway analysis were done by Goatools and Python scipy, respectively. 3.7.5 Quantitative real-time PCR Total RNA was extracted from all groups mentioned above, and RNA concentration was assessed using a micro drop ultra-microspectrophotometer Micro Drop (Bio DL, Texas, USA). All samples used GAPDH (Abcam, Cambridge, Britain) as an internal reference, and the mRNA expression levels of WNT16 (Sangon Biotech, China), CFH (Sangon Biotech, China), COL5A3 (Sangon Biotech, China), HSD17B2 (Sangon Biotech, China) and IDI (Sangon Biotech, China) were quantified 2 −△△CT method. The primers used were WNT16: 22 ATC GGA AAC ACC ACG GGC AAA G (forward) and 24 CAG CGG CAG TCT ACT GAC ATC AAC (reverse); CFH: 132 GAA GGA TGT GTA TAA GGC GGG TGA G (forward) and 132 CAG GAG GTG TCT CTG CAT GTT GG (reverse); COL5A3: 19 ACC CGC ACC TGC CTG ATG G (forward) and 21 TCT CCT CCC GCC GTG AAG TTG (reverse); HSD17B2: 22 AGT CTG CCT GCT CAT CCT GTC C (forward) and 21 CCG CAA TCA CCA CCT GTC ACC (reverse); IDI: 117 CCG CAT CCA CTA CGC AGA CAC (forward) and 117 ACC TCA CTG ACC TCG TTG CTA TTT G (reverse). 3.8 Statistical analysis The experiments were conducted thrice in each group, and the outcomes were represented as mean ± standard deviation. To compare various groups, we utilized a one-way analysis of variance (ANOVA) test and statistically analyzed data using Graph Pad Prism 10 software. Differences between groups were significant at P < 0.05. Declarations Author Contributions : Jidong Xu and Yu Song conceptualized the study. Jidong Xu and Mingchang Hu designed the method. Jidong Xu and Longfei Liu performed data analysis. Linlin Xu and Xuecheng Xu provided resources and interpreted the data. Jidong Xu and Linlin Xu performed data acquisition. Jidong Xu wrote the original draft preparation, and Yu Song reviewed and edited the manuscript. Yu Song supervised the study . All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Qingdao Chinese Medicine Science and Technology Project (2022-ZYYQ04), Qingdao Key Health Discipline Development Fund (2020–2022), and Qingdao Clinical Research Center for Oral Diseases (22-3-7-lczx-7-nsh). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Qingdao Stomatological Hospital (2022KQYX023, September 2022). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study . 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Cite Share Download PDF Status: Published Journal Publication published 18 Oct, 2024 Read the published version in BioMedical Engineering OnLine → Version 1 posted Editorial decision: Revision requested 18 Aug, 2024 Reviews received at journal 18 Aug, 2024 Reviews received at journal 15 Aug, 2024 Reviewers agreed at journal 03 Aug, 2024 Reviewers agreed at journal 31 Jul, 2024 Reviews received at journal 31 Jul, 2024 Reviewers agreed at journal 31 Jul, 2024 Reviewers invited by journal 31 Jul, 2024 Editor assigned by journal 24 Jul, 2024 Submission checks completed at journal 24 Jul, 2024 First submitted to journal 23 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4786848","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":341752030,"identity":"9a086321-4eda-4396-9c55-bc8fd08057e7","order_by":0,"name":"JiDong Xu","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"JiDong","middleName":"","lastName":"Xu","suffix":""},{"id":341752032,"identity":"0d7ca18e-fddb-4245-861e-7ffc778b00c8","order_by":1,"name":"Mingchang Hu","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Mingchang","middleName":"","lastName":"Hu","suffix":""},{"id":341752034,"identity":"235eae2b-a53c-4c0f-8775-a3a05ce86e48","order_by":2,"name":"Longfei Liu","email":"","orcid":"","institution":"QIngdao Engineering Vocational College","correspondingAuthor":false,"prefix":"","firstName":"Longfei","middleName":"","lastName":"Liu","suffix":""},{"id":341752038,"identity":"972a760c-6a2e-4aed-930f-f70774e6725a","order_by":3,"name":"xuecheng xu","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"xuecheng","middleName":"","lastName":"xu","suffix":""},{"id":341752041,"identity":"fdbbafb8-908b-4235-8133-ec9fc9f94909","order_by":4,"name":"Linlin Xu","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Linlin","middleName":"","lastName":"Xu","suffix":""},{"id":341752044,"identity":"35e265d5-419a-413b-aa8b-1ec964861016","order_by":5,"name":"Yu Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBACAzBZIcHDz8DA+CChooZYLWds5CQbGJgNHpw5RqQWxrY0Y4MDDGySD1uYCWsxFztj/JmH7XBiw/kzZhWJDWwM/O3dCXi1WM7OMZPm4Tmc2Dgjx+xG4g4ZBokzZzfgd9jtHDNmHonDic0SPEAtZ9gYDCRyCWoBOszgcGIb/xmzgsQ2ZqK0GEjzJKQZ8zDkmDEQpcVydlqZ5JwDNnISEmnFEglnjvEQ9Iu5dPLmD2//SfDYnz+88eOPiho5/vZe/FpAgIkHTHGA44iHoHIQYPwBptgfEKV6FIyCUTAKRh4AALfRSUyAG+2mAAAAAElFTkSuQmCC","orcid":"","institution":"Qingdao Stomatological Hospital Affiliated to Qingdao University","correspondingAuthor":true,"prefix":"","firstName":"Yu","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2024-07-23 08:17:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4786848/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4786848/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12938-024-01298-w","type":"published","date":"2024-10-18T15:56:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63298480,"identity":"5869df0b-5466-4c8d-8408-3f234affd237","added_by":"auto","created_at":"2024-08-26 15:48:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":864788,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological and immunophenotypic changes in primary and passaged cells. A: Morphology of primary cells (P0). Morphology of passaged cells (P3, P6, P9, and P12). B: Surface marker expression for passaged cells (P3, P6, P9 and P12) of DPSCs.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4786848/v1/ac08232b08e2f31d30f427b0.png"},{"id":63298474,"identity":"fc44a026-417b-41f4-b883-823192d1560a","added_by":"auto","created_at":"2024-08-26 15:48:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":768650,"visible":true,"origin":"","legend":"\u003cp\u003eVariations in the proliferation and senescence of dental pulp stem cells in generations P3, P6, P9, and P12. A: Differences in the proliferative ability of dental pulp MSCs with varying numbers of passages established through the CCK-8 method. B, C: β-Galactosidase staining and the corresponding percentage of staining of dental pulp MSCs with diverse numbers of passages. (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, n = 20).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4786848/v1/59808a80e89b4443d3f4ec0e.png"},{"id":63298477,"identity":"9ecea455-e707-43cd-a4fb-da8784bd484a","added_by":"auto","created_at":"2024-08-26 15:48:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2153595,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of the multi-differentiation capacity of P3, P6, P9, and P12 generation dental pulp mesenchymal stem cells. A: ARS staining for osteogenic differentiation. B: ORO staining for lipogenic differentiation. C and D: Quantitative analysis of osteogenic lipogenesis in P3, P6, P9, and P12 dental pulp mesenchymal stem cells. (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n=20)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4786848/v1/1473f10e92157a6ecb22c226.png"},{"id":63299601,"identity":"ebd339ca-373c-4e6f-a5af-eab01a4e82a9","added_by":"auto","created_at":"2024-08-26 15:56:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1119287,"visible":true,"origin":"","legend":"\u003cp\u003eDE mRNA volcanograms and DE mRNA thermoclusters of P3, P6, P9, and P12 generation DPSCs. A: P6 vs. P3 DE mRNA volcanograms. B: P9 vs. P3 DE mRNA volcanograms. C: P12 vs. P3 DE mRNA volcanograms. D: DE mRNA thermoclusters. (n=4)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4786848/v1/a592dc7c09ea920373c0c6bd.png"},{"id":63298475,"identity":"bc7956be-4281-4d54-af69-e3794d4e6104","added_by":"auto","created_at":"2024-08-26 15:48:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":755189,"visible":true,"origin":"","legend":"\u003cp\u003eGraph of Go and KEGG analysis of DE mRNA between P12 and P3. A, B, and C: Top 20 items showing biological processes, cellular components, and cellular functions, respectively. D: KEGG analysis clustering of DE mRNA showing top 20 pathways. (n=4)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4786848/v1/1f69c140a1de4c4a953738f1.png"},{"id":63298479,"identity":"302e59c4-6c9c-4b10-96d6-c0c118fad834","added_by":"auto","created_at":"2024-08-26 15:48:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":485105,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the protein interaction between P12 and P3. Nodes indicate genes, and edges indicate the existence of interactions between two genes; the more edges connected to this node, the greater its degree, and the larger the node, indicating the more significant the importance of the gene in this network.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4786848/v1/98dd86528aa096b760a5b7a0.png"},{"id":63298473,"identity":"f1398180-cfc1-48de-8777-80723f3f4fd1","added_by":"auto","created_at":"2024-08-26 15:48:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":39502,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of the critical RNAs between P12 and P3. Comparison of log2FC in three DE mRNAs between RNA-seq and quantitative real-time PCR, WNT16 and HSD17B2 were upregulated, and COL5A3 was downregulated (n = 4).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4786848/v1/4bfd250148639d75f04604fb.png"},{"id":67148685,"identity":"b78539e1-3aee-4364-9d27-7e25d07ba671","added_by":"auto","created_at":"2024-10-21 16:06:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7754152,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4786848/v1/ea86e794-cf8d-452b-aed2-21b3c2e42508.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A transcriptomic analysis of dental pulp stem cell senescence in vitro","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe integrity of the alveolar bone is a prerequisite for successful dental treatment. However, bone defects caused by periodontal disease, trauma, tumours and inappropriate orthodontic manipulation have always plagued clinicians. Currently, traditional bone repair techniques such as autologous bone grafting or bone graft substitutes have not been able to achieve satisfactory repair results.\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e In recent years, tissue-engineered bone repair and regeneration techniques based on autologous stem cells have attracted considerable research interest at home and abroad. Obtaining autologous seed stem cells with good activity and functionality is the key to research.\u003c/p\u003e \u003cp\u003eMesenchymal stem cells (MSCs) are pluripotent cells originating from the mesoderm with fibroblast-like morphology that can be induced to differentiate into osteoblasts, chondrocytes, and adipocytes under appropriate conditions.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e Stem cell therapy is the process of applying stem cells of autologous or allogeneic origin and implanting them into the human body after in vitro manipulation such as isolation, identification, purification, expansion, and induction of differentiation. However, the number of stem cells required for each application in research and clinics is considerable, with even the bone marrow, which is rich in stem cells, unable to meet the demand. Consequently, to obtain a large number of mesenchymal stem cells quickly for research or clinical applications, increasing the number of cells through in vitro cell expansion and culture is unavoidable.\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDental pulp stem cells (DPSCs) are a type of mesenchymal stem cell (MSC), which are undifferentiated cells with stem cell properties extracted from dental pulp tissue.\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e They are considered to be the most accessible and promising multifunctional MSCs for tissue engineering and regenerative medicine because of their ease of accessibility, ease of expansion and preservation, and high activity and low tumourigenicity compared with other MSCs in the human body.\u003csup\u003e[\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e However, the scarcity of DPSCs, coupled with the limited number of human primary DPSCs obtained from in vitro cultures, makes it difficult to meet the demand for tissue engineering and regenerative medicine. In order to obtain sufficient numbers of DPSCs for therapeutic use, in vitro amplification before application is a necessary process.\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e It is important to note that in the standard culture state, due to the limited number of divisions, stem cells divide to a certain number of generations and enter a proliferative arrest, i.e., enter cellular senescence.\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e This process leads to functional decline of stem cells, stagnant cell growth, significantly weakened differentiation ability, and increased number of apoptotic cells, which are no longer suitable for tissue engineering therapy and limit its clinical application.\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e Consequently, the quality control of stem cells prior to their application in tissue engineering must include an in vitro passaging process.\u003c/p\u003e \u003cp\u003eCellular senescence is strongly regulated at the post-transcriptional level, with the interaction of RNA-binding proteins and non-coding RNAs with senescence-associated messenger RNAs (mRNAs) playing a pivotal role in this regulation.\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e It has been demonstrated that cellular senescence is affected by specific chemical modifications of mRNAs.\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e The current research on mRNA modifications has focused on the role of mRNA methylation, with studies investigating its potential involvement in cancer, obesity and other age-related pathologies.\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e Indeed, there is accumulating evidence that mRNA modification may be closely related to cellular senescence.\u003c/p\u003e \u003cp\u003eThe molecular mechanism of cellular senescence resulting from long-term in vitro culture of DPSCs remains unknown, and there are currently no cell culture protocols that can delay or prevent the senescence of DPSCs. The objective of this experiment was to investigate the effects of long-term in vitro expansion culture on the morphology, immunophenotype, proliferation and osteogenic-lipogenic multidifferentiation potential of DPSCs, as well as the changes in mRNA expression of DPSCs during long-term in vitro expansion culture. Furthermore, the experiment aimed to identify the potential molecular mechanism that triggers the senescence of DPSCs. The regulation of DPSCs senescence will play a pivotal role in the clinical application of stem cells. Therefore, an in-depth understanding of DPSCs senescence mechanisms is crucial for the development of strategies to promote the regenerative potential of DPSCs in order to maintain tissue function.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003ch2\u003e1.1 Characterization of DPSCs with different numbers of passages\u003c/h2\u003e\n\u003cp\u003eTwenty healthy orthodontic patients (10 males and 10 females) aged 12 to 16 were selected for this study. DPSCs were cultured individually as one sample from one donor. Within 10 days of primary culture, spindle cell-like cells arranged in a swirling pattern were observed free around the pulp tissue mass of all donor samples (Fig. 1A). The cells were expanded up to 12 generations in subsequent in vitro passaging cultures. As the number of in vitro passages increased, the cell morphology exhibited distinguishable replicative senescence changes (Fig. 1A). The cells enlarged and acquired irregular and flattened shapes, while the nuclei became thinner. The cytoplasm showed granularity and contained elevated levels of cellular debris and inclusions. A few cells proliferated without adhering to the wall. Flow cytometry analysis revealed that DPSCs of the P3 expressed CD73, CD90, and CD105 at high levels while expressing CD34 and CD45 at low levels, thereby verifying the DPSCs\u0026apos; immunophenotype (Fig. 1B). As the number of passages increased, the percentage of DPSCs expressing CD34 and CD45 remained stable. However, the percentage of DPSCs expressing CD73, CD90, and CD105 tended to decrease. This change was particularly significant for DPSCs cultured up to P12. (Fig. 1B, Table 1). These findings imply that surface marker expression levels alter to some extent during continuous in vitro cultivation.\u003c/p\u003e\n\u003cp\u003eTable 1. Quantitative profiles of surface marker expression of DPSCs at P3, P6, P9 and P12 (n=20, mean\u0026plusmn;SD)\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMarker\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eP3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eP6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eP9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eP12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eCD34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e1.86\u0026plusmn;2.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e0.67\u0026plusmn;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e3.96\u0026plusmn;6.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e0.21\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e0.9729\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e0.4375\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eCD45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e2.51\u0026plusmn;3.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e0.56\u0026plusmn;0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e4.89\u0026plusmn;8.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e0.39\u0026plusmn;0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e0.8153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e0.5098\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eCD73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e99.30\u0026plusmn;0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e96.97\u0026plusmn;4.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e99.50\u0026plusmn;0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e46.26\u0026plusmn;53.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e3.858\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e0.0382\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eCD90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e98.96\u0026plusmn;0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e96.88\u0026plusmn;1.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e91.10\u0026plusmn;11.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e29.38\u0026plusmn;32.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e15.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eCD105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e91.29\u0026plusmn;4.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e84.93\u0026plusmn;4.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e71.60\u0026plusmn;4.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e1.14\u0026plusmn;1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e446.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: There was no significant difference in the expression of DPSCs in CD34 and CD45 with different numbers of passages (P\u0026nbsp;\u0026gt; 0.05); CD73、CD90 and CD105 showed a decreasing trend in expression with an increasing number of passages (P \u0026lt; 0.05).\u003c/p\u003e\n\u003ch2\u003e1.2 The proliferation of DPSCs with different numbers of passages, changes in senescence\u003c/h2\u003e\n\u003cp\u003eDPSCs were cultured for five days, and changes in their proliferative capacity were measured using the CCK-8 method. The results demonstrate that the third generation had the fastest proliferation rate, with subsequent generations exhibiting a progressively slower proliferation rate (Fig. 2A). The study revealed a decline in the proliferative potential of DPSCs with increasing passages during prolonged in vitro culture. SA-\u0026beta;-gal staining was conducted on the third, sixth, ninth, and twelfth generations of DPSCs to examine cell senescence at varying passage numbers. Under microscopic inspection, cells with dark blue staining in their cytoplasm were considered SA-\u0026beta;-gal positive, indicating senescent cells. A minor proportion of blue cells was detected in the third-generational DPSCs (Fig. 2B), which yielded an SA-\u0026beta;-gal positivity rate of 7%. The rate of SA-\u0026beta;-gal positivity was recorded as P6 (10.08\u0026plusmn;1.08) %, P9 (17.5\u0026plusmn;1.45) % and P12 (27\u0026plusmn;2.89) %. Additionally, the number of cells displaying dark blue staining increased gradually with increasing in vitro expansion (Fig. 2B). The statistical significance of the cell differentiation can be observed (P\u0026nbsp;\u0026lt; 0.05). This observation indicates a gradual increase in the proportion of SA-\u0026beta;-gal positive cells during succeeding passages of DPSCs in vitro (Fig. 2C). It can be seen that the cells undergo a senescent phenotype due to multiple in vitro expansions.\u003c/p\u003e\n\u003ch2\u003e1.3 Changes in the multi-differentiation potential of DPSCs with different numbers of passages\u003c/h2\u003e\n\u003cp\u003eResults of Osteogenic and Lipogenic Differentiation Experiments: The experiment involved inducing lipogenic and osteogenic differentiation for 14 and 21 days, respectively. The results obtained from alizarin red (ARS) and oil red O (ORO) staining showed that cells from P3 and P6 generations had good osteogenic and lipogenic characteristics. Under the microscope, apparent mineralized nodules and lipid droplets were observed (Fig. 3A, 3B). Stem cells that underwent numerous passages, specifically the P9 and P12, displayed decreased potential for osteogenic and lipogenic differentiation. Additionally, a higher number of apoptotic cells were observed, and there was a shift in cell morphology from an initial long spindle to an oval-like shape. It was challenging to keep apparent mineralized nodules and lipid droplets (Fig. 3A, 3B). The results of quantitative analyses indicate that the osteogenic and lipogenic abilities of DPSCs decrease as the number of in vitro passages increases (Fig. 3C, 3D).\u003c/p\u003e\n\u003cp\u003eThese results suggest that continuous passaging in vitro induces cellular senescence in DPSCs. As the number of in vitro passages increased, the heterogeneity of cell morphology increased, the proliferative capacity and multi-differentiation potential decreased, and the proportion of SA-\u0026beta;-gal positive cells increased.\u003c/p\u003e\n\u003ch2\u003e1.4 Differential gene expression profiles of DPSCs during aging in vitro\u003c/h2\u003e\n\u003cp\u003e16 samples of 4 groups of DPSCs from P3, P6, P9 and P12 were subjected to mRNA sequencing (mRNA-seq). This sequencing detected 33638 expressed genes and differentially expressed DE mRNA were screened by the |log2FC| \u0026gt; =1\u0026amp;P-value \u0026lt; 0.05 criteria. Compared with P3, there were 283 DE mRNA in P6, of which 98 were up-regulated and 185 down-regulated (Fig. 4A), 536 DE mRNA in P9, of which 347 were up-regulated and 189 down-regulated (Fig. 4B), and 1159 DE mRNA in P12, of which 692 were up-regulated and 467 down-regulated (Fig. 4C), DE mRNA was depicted by heat polygraph (Fig. 4D), in which the most significant changes in DE mRNA were observed in the P12, Table 2 shows the top 10 mRNAs with the largest up-regulation or down-regulation in the P12 compared with the P3, including CFH, WNT16, IDI1.etc, and these differentially altered RNAs were closely associated with the signaling pathways that regulate stem cell pluripotency.\u003c/p\u003e\n\u003cp\u003eTable 2.Characteristics of the mRNA with the most significant fold change between P12 and P3 (n=4, mean\u0026plusmn;SD)\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.280423280423282%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLog2FC[P12/P3]\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.929453262786595%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRegulate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003eENSG00000086696\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.280423280423282%\"\u003e\n \u003cp\u003eHSD17B2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003e2.542375093\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.929453262786595%\"\u003e\n \u003cp\u003eup\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003eENSG00000002745\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.280423280423282%\"\u003e\n \u003cp\u003eWNT16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003e1.808346941\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.929453262786595%\"\u003e\n \u003cp\u003eup\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003eENSG00000000971\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.280423280423282%\"\u003e\n \u003cp\u003eCFH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003e1.438485741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.929453262786595%\"\u003e\n \u003cp\u003eup\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003eENSG00000080573\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.280423280423282%\"\u003e\n \u003cp\u003eCOL5A3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003e-1.055618779\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.929453262786595%\"\u003e\n \u003cp\u003edown\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003eENSG00000067064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.280423280423282%\"\u003e\n \u003cp\u003eIDI1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\"\u003e\n \u003cp\u003e-1.033491011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.929453262786595%\"\u003e\n \u003cp\u003edown\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch2\u003e1.5 mRNA GO and KEGG analysis\u003c/h2\u003e\n\u003cp\u003eThe sequencing results showed the most pronounced differential changes in the P12 and P3, and we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on the P12 and P3 DE mRNA. The top 20 GO terms are related to biological processes, cellular components, and molecular functions (Fig. 5A-5C). The top 10 GO terms related to bioprocesses and DE mRNA with the information associated with these terms are shown in Table 3 (P \u0026lt; 0.05), and these bioprocess-related mRNAs are closely related in the regulation of stress response, cell proliferation, cellular bioprocess-regulation, and organ development. KEGG analysis of DE mRNA revealed that 35 pathways were significantly altered (P \u0026lt; 0.05) in DPSCs during in vitro senescence. The top 20 enriched pathways are shown in Figure 5D. The top-ranked pathways include signaling pathways related to viral myocarditis, pertussis, coronavirus disease (COVID-19), and basal cell carcinoma, closely associated with human diseases.\u003c/p\u003e\n\u003cp\u003eTable 3. Top 10 terms with the most significant differences in DE mRNAs\u0026apos; GO biological process analysis.\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"116%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"23%\"\u003e\n \u003cp\u003eTerm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.142857142857142%\"\u003e\n \u003cp\u003eCount\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.142857142857142%\"\u003e\n \u003cp\u003eRich ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.428571428571429%\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"47.285714285714285%\"\u003e\n \u003cp\u003eCandidate gene\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23%\"\u003e\n \u003cp\u003eRegulation of response to stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.142857142857142%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.142857142857142%\"\u003e\n \u003cp\u003e0.143836\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.428571428571429%\"\u003e\n \u003cp\u003e2.99E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"47.285714285714285%\"\u003e\n \u003cp\u003eFIGNL1;MET;EDNRB;CXCL6;USP1;RADX;POLR3G;UBAS-H3B;C1QTNF1;DUSP1;HBEGF;STK26;FOXC2;CFH;TNFAIP-6;RASSF2;HLAF;GPRC5B;WNT16;MMP3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23%\"\u003e\n \u003cp\u003eRegulation of cell population proliferation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.142857142857142%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.142857142857142%\"\u003e\n \u003cp\u003e0.150685\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.428571428571429%\"\u003e\n \u003cp\u003e4.53E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"47.285714285714285%\"\u003e\n \u003cp\u003eEFNB2;EDNRB;PLAAT4;ODC1;DUSP1;SGK1;GRPR;ADGR-G1;SPRY2;CD24;TIAM1;SGPP2;CHRNA7;BST2;HBEGF;PTG-S1;RAC2;RTKN2;COL18A1;E2F7;CDC6;HSF4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23%\"\u003e\n \u003cp\u003eRegulation of multicellular organismal process\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.142857142857142%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.142857142857142%\"\u003e\n \u003cp\u003e0.205479\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.428571428571429%\"\u003e\n \u003cp\u003e5.46E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"47.285714285714285%\"\u003e\n \u003cp\u003eEFNB2;SCN2B;PLAAT4;ZFHX2;C1QTNF1;NSUN5P1;EDNR-B;POLR3G;SGK1;HLAF;L3MBTL1;TIAM1;CAPN3;UBASH3-B;SPRY2;CD24;CASP1;LAMA2;CD14;FABP5;KCNJ2;CHRN-A7;BST2;HBEGF;AMIGO2;RASSF2;GPRC5B;FOXC2;MET;C-XCL6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23%\"\u003e\n \u003cp\u003eCell adhesion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.142857142857142%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.142857142857142%\"\u003e\n \u003cp\u003e0.116438\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.428571428571429%\"\u003e\n \u003cp\u003e5.72E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"47.285714285714285%\"\u003e\n \u003cp\u003eBOC;EFNB2;TNFAIP6;PCDHGB1;TIAM1;COL5A3;ENTPD1;ADGRG1;UBASH3B;COL18A1;ARVCF;NCAM1;CD24;PCD-HGA4;RAC2;AMIGO2;LAMA2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23%\"\u003e\n \u003cp\u003eAnimal organ development\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.142857142857142%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.142857142857142%\"\u003e\n \u003cp\u003e0.136986\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.428571428571429%\"\u003e\n \u003cp\u003e7.04E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"47.285714285714285%\"\u003e\n \u003cp\u003eRTKN2;TCF21;CAPN3;MET;HSD17B2;ADGRG1;SLC6A17;ODC1;HSD11B1;HBEGF;COL5A3;GAS6;L3MBTL1;E2F7;AMI-GO2;LCP1;FOXC2;LAMA2;HSF4;ILDR2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23%\"\u003e\n \u003cp\u003eRegulation of signal transduction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.142857142857142%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.142857142857142%\"\u003e\n \u003cp\u003e0.198630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.428571428571429%\"\u003e\n \u003cp\u003e7.70E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"47.285714285714285%\"\u003e\n \u003cp\u003eC1QTNF1;GREM2;L3MBTL1;TIAM1;ADGRG1;PAQR3;CAP-N3;UBASH3B;SPRY2;CD24;CASP1;CD14;FABP5;CHRNA7;BST2;DUSP1;HBEGF;GRIA3;RAC2;RASSF2;GPRC5B;RTKN-2;TCF21;FIGNL1;MET;SECTM1;NCAM1;RBBP8;WNT16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23%\"\u003e\n \u003cp\u003eRegulation of response to stimulus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.142857142857142%\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.142857142857142%\"\u003e\n \u003cp\u003e0.308219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.428571428571429%\"\u003e\n \u003cp\u003e8.04E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"47.285714285714285%\"\u003e\n \u003cp\u003eEFNB2;TNFAIP6;TIAM1;C1QTNF1;GREM2;EDNRB;POLR3-G;HLAF;L3MBTL1;FABP5;ADGRG1;PAQR3;CAPN3;UBAS-H3B;SPRY2;CXCL6;CD24;CASP1;STK26;NCAM1;CFB;CD14;C2;CHRNA7;CFH;RADX;BST2;DUSP1;HBEGF;MMP3;GRI-A3;RAC2;RASSF2;GPRC5B;FOXC2;RTKN2;TCF21;FIGNL1;MET;SECTM1;RBBP8;USP1;HLAC;WNT16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23%\"\u003e\n \u003cp\u003ePositive regulation of intracellular signal transduction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.142857142857142%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.142857142857142%\"\u003e\n \u003cp\u003e0.116438\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.428571428571429%\"\u003e\n \u003cp\u003e8.39E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"47.285714285714285%\"\u003e\n \u003cp\u003eRTKN2;MET;CD14;TIAM1;ADGRG1;SECTM1;C1QTNF1;BST2;HBEGF;SPRY2;CD24;RAC2;CASP1;RASSF2;CHRNA7;G-PRC5B;WNT16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23%\"\u003e\n \u003cp\u003eResponse to biotic stimulus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.142857142857142%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.142857142857142%\"\u003e\n \u003cp\u003e0.136986\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.428571428571429%\"\u003e\n \u003cp\u003e8.47E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"47.285714285714285%\"\u003e\n \u003cp\u003eAPOL1;GRPR;EDNRB;POLR3G;CD14;C2;CXCL6;ISG20;OD-C1;CFB;BST2;IFI44L;CASP1;MX2;CD24;FER1L6;CFH;OAS1-;HLAC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23%\"\u003e\n \u003cp\u003ePositive regulation of macromolecule metabolic process\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.142857142857142%\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.142857142857142%\"\u003e\n \u003cp\u003e0.219178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.428571428571429%\"\u003e\n \u003cp\u003e9.67E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"47.285714285714285%\"\u003e\n \u003cp\u003eEDNRB;C1QTNF1;NSUN5P1;CYTL1;POLR3G;HLAF;MYBL1;ZFHX2;CAPN3;SPRY2;EBF4;CD24;CASP1;STK26;CD14;TIAM1;ZNF711;WNT5A;CHRNA7;HBEGF;RASSF2;GPRC5B;FOXC2;TCF21;MET;GAS6;HIVEP3;HSF4;E2F7;CDC6;WNT16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch2\u003e1.6 PPI and co-expression network analysis\u003c/h2\u003e\n\u003cp\u003eCompared with the P3 of DPSCs, a total of 70 nodes and 85 interaction pairs were identified in the DE mRNA PPI network in the P12 (Fig. 6). Nodes with high topological scores may play an essential role in the aging process of DPSCs in vitro. In this study, bone morphogenetic protein 4 (BMP4), interferon-stimulated exonuclease gene 20 (ISG20), type II transmembrane glycoprotein (BST2), and other DE mRNA interactions such as WNT family member 5A (WNT5A) were identified as essential genes in this network.\u003c/p\u003e\n\u003ch2\u003e1.7 Validation of the vital RNA expression\u003c/h2\u003e\n\u003cp\u003eTo verify the RNA-seq data and significant findings from computational analysis, we performed quantitative real-time PCR for some critical RNAs selected from the RNA networks. The expression levels of those RNAs were measured in 4 human DPSCs in a different passage. The qPCR results showed that the expression of WNT16、CFH and HSD17B2 were upregulated and that the expression of COL5A3 and IDI were downregulated (Fig. 7). All qPCR results were consistent with the RNA-seq results, confirming the reliability of the sequencing results.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDPSCs exhibit multidirectional differentiation potential, self-renewal replication and immunomodulation, and have promising applications in tissue engineering and cell therapy.\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e DPSCs, as a type of mesenchymal stem cell, have a higher proliferation rate, greater in vivo osteoinductive formation and multidirectional differentiation potential compared to bone marrow mesenchymal stem cells. \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e Furthermore, DPSCs can be obtained from wisdom teeth or teeth that require extraction for orthodontic purposes, which is a straightforward process, less traumatic to patients, and involves fewer ethical considerations.\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e A study by Nela et al. demonstrated that even after thawing after one year of cryopreservation at -80\u0026deg;C, the cell viability, proliferative capacity, and differentiation ability of DPSCs remained unaffected.\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e These advantages have expanded the clinical applications of DPSCs. Studies have demonstrated that DPSCs must be expanded and cultured in vitro to meet the needs of clinical treatment.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e Nevertheless, prolonged in vitro expansion and culture will result in cellular senescence. The current research on cell senescence resulting from prolonged in vitro expansion and culture primarily focuses on other types of stem cells, with fewer studies on the senescence of DPSCs. Further research is required to elucidate the related aspects of DPSCs and the underlying mechanisms of senescence. This will facilitate the determination of the optimal number of in vitro expansion generations to ensure that the cell activity and number meet the clinical therapeutic application requirements.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this experiment, we successfully established a senescence model of DPSCs by extracting primary DPSCs and culturing them to 12 generations by in vitro expansion under standard culture conditions. DPSCs cultured to the third generation were selected and flow cytometry was performed to detect the expression of antigenic markers on their surfaces as well as for osteogenic-lipogenic induction. The results demonstrated that the negative markers CD34 and CD45 were expressed at low levels, while the positive markers CD73, CD90 and CD105 were highly expressed. Furthermore, the flow cytometry results were consistent with the surface immunostaining profiles of MSCs, as previously reported in the literature.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e These findings indicated that the cells extracted exhibited the potential for osteoblastic and lipogenic differentiation, which confirmed that the cells were indeed MSCs. Following a prolonged period of in vitro passaging and expansion culture, it was observed that the proliferative capacity of the cells declined, accompanied by a gradual increase in the proportion of SA-β-gal-positive cells with each additional passage. This is a marker of cellular senescence, which suggests that the DPSCs exhibited a gradual process of cellular senescence with the increase in the number of passages. \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e Although DPSCs continued to exhibit low expression of CD34 and CD45, there was a tendency for CD73, CD90 and CD105 expression to decline with the number of passages, with the most significant decrease observed following in vitro expansion to 12 generations. The results of osteogenic-lipogenic induction experiments demonstrated that the osteogenic-lipogenic differentiation potential of DPSCs was reduced with the increase in the number of in vitro amplifications. These experimental results were consistent with those of previous studies.\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e The above experimental results suggest that DPSCs with reduced polydifferentiation potential after aging may no longer be suitable for clinical treatment.\u003c/p\u003e \u003cp\u003eHowever, there is a divergence of opinion regarding the changes in stem cell surface markers with passaging. Kim et al. studied 55 generations of bone marrow MSCs cultured in vitro and found that the expression of stem cell surface markers tended to stabilise with increasing number of passages. \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e In contrast, Bakuplu et al.expanded DPSCs in vitro up to 12 generations and found that the expression of CD105, a surface marker of DPSCs, decreased with increasing number of passages, while the expression of CD73 and CD90 remained relatively stable.\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e This is not exactly the same as our experimental results. The experimental results may be affected by various factors, including the source of pulp tissue, the stage of tooth development and the age of the patient. In addition, heterogeneity may increase during the culture process due to differences in culture time, medium used and number of passages. There are fewer studies on the expression of surface markers in DPSCs with different numbers of passages, which remains to be further explored. There are no specific surface markers for DPSCs. \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e The typical markers for MSCs, including CD73, CD90 and CD105, do not appear to provide sufficient or specific indications to maintain the \u0026lsquo;stemness\u0026rsquo; of DPSCs. Therefore, additional stem cell markers and other multiparametric immunophenotyping should be employed to validate DPSCs stemness.\u003c/p\u003e \u003cp\u003eThe mRNA expression profiles reflect the biological behaviour and function of dental pulp stem cells (DPSCs) cultured in vitro over a long period of time. Changes in these mRNA expression profiles are closely related to functional changes during in vitro ageing. This experimental study identified a series of mRNAs associated with the senescence of DPSCs and their associated molecular mechanisms, which may be potential mediators of changes in biological properties such as reduced proliferation and differentiation after long-term in vitro expansion and culture. A total of 1159 differentially expressed (DE) mRNAs were identified between P12 and P3. Further analysis of these DE mRNAs revealed that changes in CFH, Wnt16, IDI1, COL5A3, and HSD17B2 mRNAs were closely associated with cellular senescence. CFH is a complement inhibitor that plays a key role in complement homeostasis.\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e The sequencing results demonstrated that the CFH gene exhibited elevated expression in senescent DPSCs subjected to long-term expansion and in vitro culture. Furthermore, mutations and variants of the CFH gene have been demonstrated to be significantly associated with a number of human age-related diseases, including cancer and age-related macular degeneration.\u003csup\u003e[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e Wnt16, a member of the Wnt family, has been shown to be closely associated with osteogenic differentiation, cellular senescence and tumourigenesis.\u003csup\u003e[\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e Additionally, it has been implicated in the proliferation and differentiation of stem cells.\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e The results of RNA-seq sequencing demonstrated that: Wnt16 gene expression was observed to be upregulated, while the results of the osteogenic induction assay indicated that the osteogenic capacity of stem cells exhibited a gradual decline with increasing passages. This suggests that the elevated expression of the Wnt16 gene may potentially inhibit osteogenic differentiation. Jiang et al.demonstrated that the Wnt/β-catenin protein signalling pathway inhibits the osteogenic differentiation of human mesenchymal stem cells and MC3T3-E1 cells (mouse embryonic osteoblasts).\u003csup\u003e[\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e This assertion is corroborated by the findings of our own experiments. However, Carolyn et al.have proposed that the Wnt16 gene stimulates the osteogenic differentiation of perivascular stem cells (PSC).\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e These disparate findings indicate that the Wnt signalling pathway plays a pivotal role in the regulatory network of endosteal homeostasis. Both overactivation and inactivation of Wnt signalling can result in skeletal deformities, bone diseases and cartilage loss.\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e The number of studies investigating the Wnt16 gene and osteogenic differentiation of stem cells is limited, and the role of this gene in osteogenesis in DPSCs cells remains to be further elucidated. Isopentenyl diphosphate isomerase 1 (IDI1) is an enzyme that encodes a peroxisomal localization, which removes toxic hydrogen peroxide produced by different oxidative enzymes in the peroxisomal respiratory pathway. \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e It is involved in processes such as cell division and proliferation and is associated with age-related diseases.\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e The experimental results demonstrated that the expression of IDI1 was reduced in senescent DPSCs. This reduction in the synthesis of peroxisome-localised enzymes and the catabolism of hydrogen peroxide leads to an exacerbation of cellular senescence. The collagen type V alpha 3 chain (COL5A3) is a member of the collagen family that is closely associated with osteogenesis and tumourigenesis. The experimental results indicate that cellular senescence is associated with a reduction in COL5A3 expression. Pavitra K et al.demonstrated that hypoxia stimulates the expression of COL1A1, COL5A1 and COL5A3 in osteoblasts, which plays a role in maintaining bone volume by promoting collagen production.\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e Chen et al.observed that collagen inhibits immune signalling in the tumour microenvironment (chemokines) production, thereby inhibiting anti-tumour immune responses.\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e The loss of collagen results in an increase in chemokine levels, which in turn facilitates the proliferation of cancer cells. 17-β-hydroxysteroid dehydrogenase type 2 (HSD17B2) is a protein associated with estrogen synthesis and regulates estradiol (E2).\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e The experimental results indicated that HSD17B2 expression was upregulated, suggesting that it may be associated with cellular senescence. Lu et al.demonstrated that the inhibition of HSD17B2 activity suppressed E2 inactivation, increased endogenous estrogen levels, and improved bone metabolism-related indicators.\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e In conclusion, these DE mRNA changes are closely associated with cellular senescence, disease development, and osteogenic differentiation, among other factors. One limitation of this experiment is that we did not perform gene knockdown. To study the effect of gene knockdown on dental pulp stem cell senescence, further studies are needed in subsequent experiments.\u003c/p\u003e \u003cp\u003eKEGG analysis has identified numerous signalling pathways associated with human diseases, including viral myocarditis, pertussis, coronavirus disease (COVID-19) and basal cell carcinoma. These pathways play a pivotal role in the functional changes induced by ageing, thereby underscoring the significance of DPSCs ageing in the pathogenesis and clinical treatment of cancer, cancer and metabolic diseases.\u003c/p\u003e \u003cp\u003eAmong the interactions between DE-encoded proteins, BMP4, ISG20, BST2 and Wnt5a were identified as key genes interacting with many other DE mRNAs in this network.BMP4, a member of the TGF-β superfamily, is involved in a variety of biologically regulated processes such as cell proliferation and cellular differentiation.\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e BMP4 plays a key role in bone formation, and the gene may promote alveolar bone development by increasing the expression levels of Runx2, BSP and OCN.\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e ISG20 has been associated with certain RNA virus-induced diseases, and a study of ISG20 elucidated its involvement in antiviral mechanisms, which found that overexpression of recombinant ISG20 in cultured cells increased cellular resistance to infection with certain RNA genomes of viruses.\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e BST2, also known as CD317, is involved in a variety of physiological and pathological processes, including inflammation, immune regulation and tumourigenesis, and BST2 is overexpressed in various malignant tumours, suggesting that BST2 may be associated with certain tumour disorders induced by cellular senescence.\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e Wnt5a is a representative Wnt protein of the non-classical Wnt signalling pathway, which plays an important role in the development and maturation of various tissues and organs, and is closely related to a variety of diseases such as infectious diseases, cancer and metabolic disorders.\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e Studies on Wnt5a and related aspects of its signalling pathway may provide new ideas for the diagnosis and treatment of human diseases.\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e In summary, these proteins are closely related to the biological changes of DPSCs after long-term in vitro amplification and culture. However, whether these proteins can be used as markers for clinical detection remains to be further investigated.\u003c/p\u003e \u003cp\u003eIn addition, DPSCs are derived from neural crest cells and have unique advantages in nerve repair, cartilage formation and corneal reconstruction.\u003csup\u003e[\u003cspan additionalcitationids=\"CR57 CR58\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e Furthermore, in addition to its use in repairing teeth and maxillofacial bone tissue, it can also be used to repair tissues outside the oral cavity, such as nerves, cartilage and other systems. Dental pulp stem cells are also receiving increasing attention in the field of regenerative medicine. Autologous stem cell therapy for diabetes and myocardial infarction have likewise shown promising applications.\u003csup\u003e[60.61]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDental pulp stem cells extracted by the pulp tissue block method exhibited low CD34 and CD45 expression, high CD73, CD90 and CD105 expression, and demonstrated osteogenic and lipogenic differentiation potential. The number of in vitro expansions was found to induce cellular senescence of pulp stem cells, which resulted in a reduction in proliferative capacity and osteogenic and lipogenic differentiation potential. It is possible that changes in mRNA induced by prolonged expansion in vitro may be a potential mechanism for the senescence of dental pulp stem cells. The differential expression of genes, including CFH, WNT16, HSD17B2, IDI and COL5A3, may be of significant importance in the context of pulp stem cell senescence.It is crucial to explore the molecular mechanism behind the senescence of DPSCs in order to establish a more robust theoretical basis for the clinical application of DPSCs.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Cell isolation and passaging\u003c/h2\u003e \u003cp\u003e This study was evaluated by the Ethics Committee of Qingdao Stomatological Hospital, Qingdao University (2022KQYX023) and begins on 1 October 2023 and ends on 1 May 2024. Pulp stem cells were obtained from teeth lacking dental and periodontal tissue diseases and taken from orthodontic patients aged 12\u0026ndash;16 without systemic diseases via the tissue block methodology. The cells were passaged at 80% cell density and grown \u003cem\u003ein vitro\u003c/em\u003e, expanding culture for up to 12 generations. This study employed DPSCs from 3rd, 6th, 9th, and 12th generations. The DPSCs were cultured in α-MEM medium (Pricella, Wuhan, China) with varying concentrations of fetal bovine serum (FBS, Pricella, Wuhan, China) and 1% penicillin-streptomycin (BioIndustries, Israel) according to the specific experimental conditions. All cells were maintained at 37\u0026deg;C under constant humidity of 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Flow cytometry\u003c/h2\u003e \u003cp\u003eFlow cytometry was utilized to assess the presence of positive markers (CD73, CD90, and CD105) and negative markers (CD34 and CD45) in DPSCs. They were digested and centrifuged to prepare the DPSCs (P3, P6, P9, and P12). The cell density was subsequently standardized to 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL, and 100 \u0026micro;L of cell suspension was introduced to the centrifuge tube. Anti-human antibodies CD34 (E-AB-F1143C), CD45 (E-AB-F1137C), CD73 (E-AB-F1242D), CD90 (E-AB-F1167D), and CD105 (E-AB-F1143D) (Elabscience, Wuhan, China) were added in a ratio of 50:1. PBS (Priscilla, Wuhan, China) was used as a control. The supernatant was centrifuged and discarded after a one-hour dark incubation at room temperature, followed by two washes with PBS and resuspension in 500 \u0026micro;L of PBS. The expression levels of various antibodies were then determined by flow cytometry using the DxFLEX machine from Beckman Coulter (Suzhou, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Cell proliferation\u003c/h2\u003e \u003cp\u003eUse the Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China) to measure cell proliferation according to the manufacturer's instructions. DPSCs at P3, P6, P9, and P12 were seeded at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well in 96-well culture plates and incubated for 1 to 5 days. At each specific time point, the complete medium was exchanged with new α-MEM basal medium supplemented with a 10% CCK-8 solution. This solution was then incubated under dark conditions for 2 hours at 37\u0026deg;C. Finally, cell proliferation capacity was determined by detecting absorbance at 450nm using an enzyme label reader (SynergyH1/H1M, Bio-Tek, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Cellular senescence\u003c/h2\u003e \u003cp\u003eCellular senescence was detected through β-galactosidase staining analysis. DPSCs at P3, P6, P9, and P12 were introduced into 6-well plates at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003ecells/well. The SA-β-gal activity was then evaluated by the instructions provided by the β-galactosidase staining kit manual (Beyotime, Shanghai, China). Senescent cells were stained blue by SA-β-gal, and SA-β-gal positive cells were evaluated as a percentage of all cells based on three randomly selected bright fields for observation and calculation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Osteogenic differentiation\u003c/h2\u003e \u003cp\u003eDPSCs of P3, P6, P9, and P12 were plated in 6-well dishes at a 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well density until they reached 70% confluence. The original medium was subsequently aspirated and replaced with Osteogenic Differentiation Inducing Medium (Pricella, Wuhan, China). The cells were cultured for 21 days, with a medium change every 3 days. After fixing with 4% paraformaldehyde, the samples were stained with Alizarin Red S following washing with PBS. Images were collected by observing under an optical microscope (OLYMPUS, Japan). Then, 10% Cetylpyridinium Chloride solution was added to the wells, and the solution dissolved for 30 min at room temperature. The decolorized solution was then collected, and the absorbance at 562 nm was measured with an enzyme label reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Lipogenic differentiation\u003c/h2\u003e \u003cp\u003eDPSCs (P3, P6, P9, and P12) were seeded in 6-well plates at 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well density. After the cells reached 100% confluence, the original medium was removed, and lipid induction was carried out for 14 days using a Lipid-forming differentiation-inducing medium (Pricella, Wuhan, China) following the manual instructions. All abbreviations used in the text are explained in their first usage. The cells were immobilized using a 4% paraformaldehyde solution (Elabscience, Wuhan, China), washed with PBS, and then stained with Oil Red O. Following observation and capture of the images under a light microscope, the stained lipid droplets were dissolved in isopropanol (Sigma-Aldrich) at room temperature, the decolorized solution collected, and the absorbance at 510 nm measured using an enzyme marker.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Library construction and high-throughput sequencing\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.7.1 RNA extraction\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from P3, P6, P9 and P12 generation stem cells (n\u0026thinsp;=\u0026thinsp;4) using TRIzol reagent (Pricella, Wuhan, China) according to the manufacturer's instructions. RNA quality was then determined using a 5300 Bioanalyzer (Agilent) and quantified using an ND-2000 (NanoDrop Technologies). High-quality RNA samples (OD260/280\u0026thinsp;=\u0026thinsp;1.8\u0026thinsp;~\u0026thinsp;2.2, OD260/230\u0026thinsp;\u0026ge;\u0026thinsp;2.0, RIN\u0026thinsp;\u0026ge;\u0026thinsp;6.5, 28S:18S\u0026thinsp;\u0026ge;\u0026thinsp;1.0, \u0026gt;\u0026thinsp;1 \u0026micro;g) were used to construct sequencing libraries.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.7.2 Library preparation and sequencing\u003c/h2\u003e \u003cp\u003eRNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co, Ltd, Shanghai, China. RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co, Ltd, Shanghai, China, according to the manufacturer's (Illumina, San Diego, CA) instructions. The mRNA-seq transcriptome libraries were prepared with 1 \u0026micro;g of total RNA following the Illumina\u0026reg; Stranded mRNA Prep, Ligation method provided by Illumina (San Diego, CA). First, messenger RNA was isolated by polyA selection using oligo (dT) beads, followed by fragmentation with fragmentation buffer. Double-stranded cDNA was synthesized using the SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) and random hexamer primers (Illumina). According to Illumina's library construction protocol, the synthesized cDNAs were subjected to end repair, phosphorylation, and 'A' base addition. The 300 bp cDNA target fragment was size-screened on 2% Low Range Ultra Agarose, followed by 15 PCR cycles of PCR amplification using Phusion DNA polymerase (NEB). After quantification with Qubit 4.0, paired-end RNA-seq sequencing libraries were sequenced with a NovaSeq 6000 sequencer (2\u0026times;150 bp read length).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.7.3 Quality control and reading mapping\u003c/h2\u003e \u003cp\u003eRaw paired-end reads were trimmed and quality-controlled using fast and default parameters. Clean reads were individually aligned to the reference genome in targeted mode using HISAT2 software. Mapped reads for each sample were assembled on a reference basis using StringTie software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.7.4 Differential expression analysis and functional enrichment\u003c/h2\u003e \u003cp\u003eTo identify differentially expressed genes (DEGs) between two different samples, the expression level of each transcript was calculated based on the transcripts per million reads (TPM) method. RSEM was used to quantify gene abundance. Differential expression analysis was performed using DESeq2 or DEGseq.|DEGs with log2FC| \u0026ge; 1 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (DESeq2) or FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (DEGseq) were considered as significantly differentially expressed genes. Functional enrichment analyses, including GO and KEGG, were also performed, and DEGs were significantly enriched in GO terms and metabolic pathways (Bonferroni-corrected \u003cem\u003eP-\u003c/em\u003evalue\u0026thinsp;\u0026lt;\u0026thinsp;0.05). GO functional enrichment and KEGG pathway analysis were done by Goatools and Python scipy, respectively.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.7.5 Quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from all groups mentioned above, and RNA concentration was assessed using a micro drop ultra-microspectrophotometer Micro Drop (Bio DL, Texas, USA). All samples used GAPDH (Abcam, Cambridge, Britain) as an internal reference, and the mRNA expression levels of WNT16 (Sangon Biotech, China), CFH (Sangon Biotech, China), COL5A3 (Sangon Biotech, China), HSD17B2 (Sangon Biotech, China) and IDI (Sangon Biotech, China) were quantified 2\u003csup\u003e\u0026minus;△△CT\u003c/sup\u003e method. The primers used were WNT16: 22 ATC GGA AAC ACC ACG GGC AAA G (forward) and 24 CAG CGG CAG TCT ACT GAC ATC AAC (reverse); CFH: 132 GAA GGA TGT GTA TAA GGC GGG TGA G (forward) and 132 CAG GAG GTG TCT CTG CAT GTT GG (reverse); COL5A3: 19 ACC CGC ACC TGC CTG ATG G (forward) and 21 TCT CCT CCC GCC GTG AAG TTG (reverse); HSD17B2: 22 AGT CTG CCT GCT CAT CCT GTC C (forward) and 21 CCG CAA TCA CCA CCT GTC ACC (reverse); IDI: 117 CCG CAT CCA CTA CGC AGA CAC (forward) and 117 ACC TCA CTG ACC TCG TTG CTA TTT G (reverse).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe experiments were conducted thrice in each group, and the outcomes were represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. To compare various groups, we utilized a one-way analysis of variance (ANOVA) test and statistically analyzed data using Graph Pad Prism 10 software. Differences between groups were significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: Jidong Xu and Yu Song conceptualized the study. Jidong Xu and Mingchang Hu designed the method. Jidong Xu and Longfei Liu performed data analysis. Linlin Xu and Xuecheng Xu provided resources and interpreted the data. Jidong Xu and Linlin Xu performed data acquisition. Jidong Xu wrote the original draft preparation, and Yu Song reviewed and edited the manuscript. Yu Song supervised the study . All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by Qingdao Chinese Medicine Science and Technology Project (2022-ZYYQ04), Qingdao Key Health Discipline Development Fund (2020\u0026ndash;2022), and Qingdao Clinical Research Center for Oral Diseases (22-3-7-lczx-7-nsh).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Qingdao Stomatological Hospital (2022KQYX023, September 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e Informed consent was obtained from all subjects involved in the study . Written informed consent has been obtained from the patient(s) to publish this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhao, R., et al., Bone Grafts and Substitutes in Dentistry: A Review of Current Trends and Developments. Molecules, 2021. 26(10).\u003c/li\u003e\n\u003cli\u003eAl-Azab, M., et al., Aging of mesenchymal stem cell: machinery, markers, and strategies of fighting. Cell Mol Biol Lett, 2022. 27(1): p. 69.\u003c/li\u003e\n\u003cli\u003eMorsczeck, C., Cellular senescence in dental pulp stem cells. 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Stem Cells, 2008. 26(3): p. 638-45.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biomedical-engineering-online","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmeo","sideBox":"Learn more about [BioMedical Engineering OnLine](http://biomedical-engineering-online.biomedcentral.com/)","snPcode":"12938","submissionUrl":"https://submission.nature.com/new-submission/12938/3","title":"BioMedical Engineering OnLine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"DPSCs, Senescence, Multiple differentiation potential, Transcriptome sequencing","lastPublishedDoi":"10.21203/rs.3.rs-4786848/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4786848/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground/purpose:\u003c/strong\u003e The utilisation of human dental pulp stem cells (hDPSCs) as autologous stem cells for tissue repair and regenerative techniques represents a significant area of research globally. The objective of this experiment was to investigate the effect of long-term in vitro culture on the multidifferentiation potential of human dental pulp stem cells and the potential molecular mechanisms involved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and methods:\u003c/strong\u003e The tissue block method was employed to extract hDPSCs from orthodontic minus extraction patients, which were then expanded and cultured in vitro for 12 generations. Stem cells from passages 3, 6, 9 and 12 were selected. Flow cytometry was employed to detect the expression of stem cell surface markers, while CCK-8 was used to assess cell proliferation ability. β-galactosidase staining was employed to detect Cellular senescence, alizarin red S staining was employed to assess osteogenic potential, while Oil Red O staining was used to evaluate lipogenic capacity. RNA sequencing analysis was conducted to identify differentially expressed genes in DPSCs and to investigate potential mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: With increasing passage number, pulp stem cells showed an increase in senescent cells and a decrease in proliferative capacity and osteogenic-lipogenic multidifferentiation potential. The expression of the stem cell surface markers CD34 and CD45 was stable, whereas the expression of CD73, CD90 and CD105 decreased with increasing passages. According to RNA-seq analysis, The differentially expressed genes CFH, WNT16, HSD17B2, IDI and COL5A3 may be associated with stem cell senescence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: An increase in in vitro expansion has been observed to induce a state of cellular senescence in pulp stem cells, which in turn results in a reduction in their proliferative capacity and osteogenic-lipogenic differentiation potential. Differential expression of genes such as CFH, Wnt16, HSD17B2, IDI, and COL5A3 may represent a potential mechanism for the induction of cellular senescence in pulp stem cells.\u003c/p\u003e","manuscriptTitle":"A transcriptomic analysis of dental pulp stem cell senescence in vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-26 15:47:55","doi":"10.21203/rs.3.rs-4786848/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-18T16:37:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-18T09:05:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-15T08:51:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190145621530991462185762183557572815506","date":"2024-08-03T13:38:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"208759292931767815589453347913996767109","date":"2024-08-01T02:55:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-31T16:53:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179925645929756144285130914173188946929","date":"2024-07-31T16:38:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-31T13:11:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-24T23:25:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-24T23:25:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioMedical Engineering OnLine","date":"2024-07-23T08:15:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biomedical-engineering-online","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmeo","sideBox":"Learn more about [BioMedical Engineering OnLine](http://biomedical-engineering-online.biomedcentral.com/)","snPcode":"12938","submissionUrl":"https://submission.nature.com/new-submission/12938/3","title":"BioMedical Engineering OnLine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b2f3de09-fe0c-4563-9e7e-d21731bc4ba8","owner":[],"postedDate":"August 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T15:58:40+00:00","versionOfRecord":{"articleIdentity":"rs-4786848","link":"https://doi.org/10.1186/s12938-024-01298-w","journal":{"identity":"biomedical-engineering-online","isVorOnly":false,"title":"BioMedical Engineering OnLine"},"publishedOn":"2024-10-18 15:56:53","publishedOnDateReadable":"October 18th, 2024"},"versionCreatedAt":"2024-08-26 15:47:55","video":"","vorDoi":"10.1186/s12938-024-01298-w","vorDoiUrl":"https://doi.org/10.1186/s12938-024-01298-w","workflowStages":[]},"version":"v1","identity":"rs-4786848","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4786848","identity":"rs-4786848","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}