The functions of mesenchymal stem cells (MSCs) appear to decline with age due to cellular senescence, which could reduce the efficacy of MSCs-based therapies. Recently, MSCs have been identified in the nucleus pulposus, which offers great potential for intervertebral disc (IVD) repair. However, this potential might be affected by the senescence of nucleus pulposus MSCs (NPMSCs), but whether or not this exists remains unknown. The aim of this study was to investigate the age-related changes in NPMSCs. NPMSCs isolated from young (3-month-old) and old (14-month-old) Sprague-Dawley rats were cultured in vitro. Differences in morphology, proliferation, colony formation, multilineage differentiation, cell cycle, and expression of β-galactosidase (SA-β-gal) and senescent markers (p53, p21, and p16) were compared between groups. Both young and old NPMSCs fulfilled the criteria for definition as MSCs. Moreover, young NPMSCs presented better proliferation, colony-forming, and multilineage differentiation capacities than old NPMSCs. Old NPMSCs displayed senescent features, including significantly increased G0/G1 phase arrest, increased SA-β-gal expression, decreased S phase entry, and significant p53-p21-pRB pathway activation. Therefore, this is the first study demonstrating that senescent NPMSCs accumulate in IVD with age. The efficacy of NPMSCs is compromised by donor age, which should be taken into consideration prior to clinical application.

Objectives To evaluate the effects of the limiting dilution method and plating density in rat nucleus pulposus mesenchymal stem/progenitor cells (NPMSCs). Materials and Methods Nucleus pulposus tissues were isolated from 12-week-old male Sprague-Dawley rats and NPMSCs were isolated using limiting dilution method. Cells were then classified into 3 groups according to plating density. Cell morphologies were observed, and colony-forming units, migration abilities, proliferative capacities, cell cycle percentages, multilineage differentiation capacities, stem cell biomarker expression levels, and immunophenotyping were also examined in each group. Results Low density group (LD) had higher morphological homogeneity, stronger colony-forming ability, higher cell proliferation capacity, and enhanced cell migration ability relative to the other two groups (p < 0.05). Moreover, LD had more cells entering S phase, with fewer cells arrested in G0/G1 phase (p < 0.05). While all three density groups showed a multilineage differentiation potential, LD showed a higher degree of observed and semiquantified lineage specific staining (p < 0.05). Furthermore, LD displayed higher expression levels of stem cell biomarkers (Nanog, Oct4, and Sox2) and showed higher percentages of CD29+, CD44+, and CD90+ cells (p < 0.05) following flow cytometry analysis. Conclusions Limiting dilution method is suggested when isolating NPMSCs as a means of improving cell activity and plasticity.

Nucleus pulposus cells (NPCs) play a vital role in maintaining the homeostasis of the intervertebral disc (IVD). Previous studies have discovered that NPCs exhibited malfunction due to cellular senescence during disc aging and degeneration; this might be one of the key factors of IVD degeneration. Thus, we conducted this study in order to investigate the altered biofunction and the underlying genes and pathways of senescent NPCs. We isolated and identified NPCs from the tail discs of young (2 months) and old (24 months) SD rats and confirmed the senescent phenotype through SA-β-gal staining. CCK-8 assay, transwell assay, and cell scratch assay were adopted to detect the proliferous and migratory ability of two groups. Then, a rat Gene Chip Clariom™ S array was used to detect differentially expressed genes (DEGs). After rigorous bioinformatics analysis of the raw data, totally, 1038 differentially expressed genes with a fold change>1.5 were identified out of 23189 probes. Among them, 617 were upregulated and 421 were downregulated. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted and revealed numerous number of enriched GO terms and signaling pathways associated with senescence of NPCs. A protein-protein interaction (PPI) network of the DEGs was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) database and Cytoscape software. Module analysis was conducted for the PPI network using the MCODE plugin in Cytoscape. Hub genes were identified by the CytoHubba plugin in Cytoscape. Derived 5 hub genes and most significantly up- or downregulated genes were further verified by real-time PCR. The present study investigated underlying mechanisms in the senescence of NPCs on a genome-wide scale. The illumination of molecular mechanisms of NPCs senescence may assist the development of novel biological methods to treat degenerative disc diseases.

Nucleus pulposus (NP) mesenchymal stem cells (NPMSCs) are a potential cell source for intervertebral disc (IVD) regeneration; however, little is known about their response to tumor necrosis factor-α (TNF-α), a critical inflammation factor contributing to accelerating IVD degeneration. Accordingly, the aim of this study was to investigate the regulatory effects of TNF-α at high and low concentrations on the biological behaviors of healthy rat NPMSCs, including proliferation, migration, and NP differentiation. In this study, NPMSCs were treated with different concentration of TNF-α (0-200 ng/mL). Then we used annexin V/propidium iodide flow cytometry analysis to detect the apoptosis rate of NPMSCs. Cell Counting Kit-8, Edu assay, and cell cycle test were used to examine the proliferation of NPMSCs. Migration ability of NPMSCs was detected by wound healing assay and transwell migration assay. Pellets method was used to induce NP differentiation of NPMSCs, and immunohistochemical staining, real-time polymerase chain reaction, and Western blot analysis were used to examine the NPC phenotypic genes and proteins. The cells were further treated with the nuclear factor-κB (NF-κB) pathway inhibitor Bay 11-7082 to determine the role of the NF-κB pathway in the mechanism underlying the differentiation process. Results showed that treatment with a high concentration of TNF-α (50-200 ng/mL) could induce apoptosis of NPMSCs, whereas a relatively low TNF-α concentration (0.1-10 ng/mL) promoted the proliferation and migration of NPMSCs, but inhibited their differentiation toward NP cells.Moreover, we identified that the NF-κB signaling pathway is activated during the TNF-α-inhibited differentiation of NPMSCs, and the NF-κB signal inhibitor Bay 11-7082 could partially eliminate the adverse effect of TNF-α on the differentiation of NPMSCs. Therefore, our findings provide important insight into the dynamic biological behavior reactivity of NPMSCs to TNF-α during IVD degeneration process, thus may help us understanding the underlying mechanism of IVD degeneration.