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Degeneration of the cartilage endplate (CEP) induces intervertebral disc degeneration (IVDD). Nucleus pulposus cell (NPC) apoptosis is also an important exacerbating factor in IVDD, but the cascade mechanism in IVDD is not clear. We investigated the apoptosis of NPCs and IVDD when stimulated by normal cartilage endplate stem cell (CESC)‐derived exosomes (N‐Exos) and degenerated CESC‐derived exosomes (D‐Exos) in vitro and in vivo. Tert‐butyl hydroperoxide (TBHP) was used to induce inflammation of CESCs. The bioinformatics differences between N‐Exos and D‐Exos were analyzed using mass spectrometry, heat map, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. NPC apoptosis was examined using TUNEL staining. The involvement of the AKT and autophagy signaling pathways was investigated using the signaling inhibitor LY294002. Magnetic resonance imaging, Western blotting, and immunofluorescence staining were used to evaluate the therapeutic effects of N‐Exos in rats with IVDD. TBHP effectively induced inflammation and the degeneration of CEP in rat. N‐Exos were more conducive to autophagy activation than D‐Exos. The apoptotic rate of NPCs decreased obviously after treatment with N‐Exos compared to D‐Exos. N‐Exos inhibited NPCs apoptosis and attenuated IVDD in rat via activation of the AKT and autophagy pathways. These results are the first findings to confirm that CEP delayed the progression of IVDD via exosomes. The therapeutic effects of N‐Exos on NPC apoptosis inhibition and the slowing of IVDD progression were more effective than D‐Exos due to activation of the PI3K/AKT/autophagy pathway, which explained the increase in the incidence of IVDD after inflammation of the CEP., Graphical abstract of the mechanism that cartilage endplate (CEP) inflammation accelerated the progression of intervertebral disc degeneration (IVDD). Normal cartilage endplate stem cell (CESC)‐derived exosomes (N‐Exos) can more effectively inhibit nucleus pulposus cell (NPC) apoptosis than degenerated CEPC‐derived exosomes (D‐Exos) due to the anti‐apoptotic protein carried by exosomes decreasing after the CEP degeneration. Furthermore, N‐Exos also activate the PI3K/AKT signaling pathway in NPC more conducively compared with D‐Exos, enhancing autophagy, alleviating NPC apoptosis in vitro and ameliorating IVDD in vivo

Cartilage endplate (CEP) calcification inhibits the transport of metabolites and nutrients in the intervertebral disk and is an important initiating factor of intervertebral disk degeneration. However, the mechanisms governing CEP degeneration have not been thoroughly elucidated. In this study, we established a mouse CEP degeneration model and showed that autophagy insufficiency caused the degeneration of CEP. We found that the inflammatory cytokine tumor necrosis factor-α (TNF-α) increased the level of intracellular reactive oxygen species (ROS) and caused cell senescence and osteogenic differentiation of cartilage endplate stem cells (CESCs), whereas rapamycin-induced autophagy protected CESCs from TNF-α-induced oxidative stress and cell senescence. Furthermore, rapamycin-induced autophagy helped CESCs maintain the chondrogenic properties and inhibited extracellular matrix protease expression and osteogenic differentiation. Further study revealed that autophagy activated by rapamycin or inhibited by chloroquine influenced the expression and nuclear translocation of Nrf2, thereby controlling the expression of antioxidant proteins and the scavenging of ROS. Taken together, the results indicate that rapamycin-induced autophagy enhances Nrf2/Keap1 signaling and promotes the expression of antioxidant proteins, thereby eliminating ROS, alleviating cell senescence, reducing the osteogenic differentiation of CESCs, and ultimately protecting CEPs from chronic inflammation-induced degeneration. Stem Cells 2019;37:828–840

Previous studies have identified the association between cartilage endplate (CEP) degeneration and abnormal mechanical loading. Several studies have reported that intermittent cyclic mechanical tension (ICMT) regulates CEP degeneration via various biological processes and signaling pathways. However, the functions of microRNAs in regulating the cellular responses of CEP chondrocytes to ICMT remain to be elucidated. The current study determined the differentially expressed microRNAs in human CEP chondrocytes exposed to ICMT using microarray analysis. A total 21 significantly upregulated and 62 downregulated miRNAs were identified compared with the control. The findings were subsequently partially validated by reverse transcription‑quantitative polymerase chain reaction. Potential target genes of the significantly differentially expressed miRNAs were predicted using bioinformatics analysis and were used for Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The present study revealed that the significantly differentially expressed microRNAs were involved in various signaling pathways and biological processes that are crucial to regulating the responses of CEP chondrocytes to ICMT. The current study provided a global view of microRNA expression in CEP chondrocytes under mechanical stimulation, suggesting that microRNAs are important for regulating the mechanical response of CEP chondrocytes. Additionally, it provided a novel insight into the association between mechanical stress and the establishment and progression of intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is typically accompanied by a reduced nutrient supply, which is thought to be a contributor to the apoptosis of nucleus pulposus cells (NPCs). Here, we explored whether Forkhead box O3 (FOXO3), a key transcription factor involved in cellular quality control, could protect NPCs against apoptosis under nutrient deficiency. Firstly, we found that FOXO3 knockdown aggravated nutrient deficiency-induced mitochondrial dysfunction, apoptosis and matrix degradation in NPCs. In addition, the siRNA-mediated downregulation of FOXO3 suppressed mitophagy in starved NPCs. However, when we overexpressed FOXO3 in NPCs by lentivirus transfection, the observed detrimental effects induced by nutrient deprivation were significantly reversed by the FOXO3-activated autophagy. Moreover, by analyzing the human NP samples from different age groups as well as degenerated groups, we found that the FOXO3 protein level decreased with aging and degeneration. Together, our data suggest that FOXO3 plays a vital role in disc degeneration and can be a novel therapeutic target for IDD.

Transplantation of mesenchymal stem cells (MSCs) into the degenerated intervertebral disc (IVD) has shown promise for decelerating or arresting IVD degeneration. Cellular mechanical properties play crucial roles in regulating cell-matrix interactions, potentially reflecting specific changes that occur based on cellular phenotype and behavior. However, the effect of co-culturing of MSCs with nucleus pulposus cells (NPCs) on the mechanical properties of NPCs remains unknown. In our study, we demonstrated that co-culture of degenerated NPCs with MSCs resulted in significantly decreased mechanical moduli (elastic modulus, relaxed modulus, and instantaneous modulus) and increased biological activity (proliferation and expression of matrix genes) in degenerated NPCs, but not normal NPCs. SDF-1, CXCR4 ligand, was highly expressed in MSCs when co-cultured with degenerated NPCs. Inhibition of SDF-1 using CXCR4 antagonist AMD3100 or knocking-down CXCR4 in degenerated NPCs abolished the MSCs-induced decrease in the mechanical moduli and increased biological activity of degenerated NPCs, suggesting a crucial role for SDF-1/CXCR4 signaling. AKT and FAK inhibition attenuated the MSCs- or SDF-1-induced decrease in the mechanical moduli of degenerated NPCs. In conclusion, it was demonstrated in vitro that MSCs regulate the mechanical properties of degenerated NPCs through SDF-1/CXCR4/AKT signaling. These findings highlight a possible mechanical mechanism for MSCs-induced modulation with degenerated NPCs, which may be applicable to MSCs-based therapy for disc degeneration.

Glucose is the major energy supply and a critical metabolite for most cells and is especially important when cell is differentiating. High or low concentrations of glucose enhances or inhibits the osteogenic, chondrogenic and adipogenic differentiation of cell via the insulin, transforming growth factor‐β and peroxisome proliferator‐activated receptor γ pathways, among others. New evidence implicates the hexosamine biosynthetic pathway as a mediator of crosstalk between glucose flux, cellular signalling and epigenetic regulation of cell differentiation. Extracellular glucose flux alters intracellular O‐GlcNAcylation levels through the hexosamine biosynthetic pathway. Signalling molecules that are important for cell differentiation, including protein kinase C, extracellular signal‐regulated kinase, Runx2, CCAAT/enhancer‐binding proteins, are modified by O‐GlcNAcylation. Thus, O‐GlcNAcylation markedly alters cell fate during differentiation via the post‐transcriptional modification of proteins. Furthermore, O‐GlcNAcylation and phosphorylation show complex interactions during cell differentiation: they can either non‐competitively occupy different sites on a substrate or competitively occupy a single site or proximal sites. Therefore, the influence of glucose on cell differentiation via O‐GlcNAcylation offers a potential target for controlling tissue homoeostasis and regeneration in ageing and disease. Here, we review recent progress establishing an emerging relationship among glucose concentration, O‐GlcNAcylation levels and cell differentiation.

Background Adult stem cells exist in a quiescent state (G0) within the in vivo niche; the loss of quiescence often leads to a decrease in the number and function of adult stem cells, impairing tissue regeneration and repair. Endogenous repair by nucleus pulposus-derived stem cells has recently shown promising regenerative potential for the treatment of intervertebral disc degeneration (IDD). However, the number and function of nucleus pulposus stem cells (NPSCs) declined throughout the process of IDD. This effect may have a specific relationship with quiescence. However, the biology of the quiescent NPSCs has not been reported. Methods First, we established an in vitro model for NPSC quiescence with serum starvation. The induction of G0 was confirmed by flow cytometry analyses of dual staining with Hoechst 33342 and Pyronin Y, immunofluorescent staining with Ki67 and Western blot analysis of P27 expression. NPSCs were cultured under serum starvation conditions for a long time period (21 days). To examine the functional phenotype of quiescent NPSCs, the cells were reactivated with 10% serum and differentiated into osteogenic and chondrogenic lineages in vitro. The number of colony-forming units was also estimated. To elucidate the role of autophagy in the quiescence of NPSCs, we activated and inhibited autophagy in starved cells with rapamycin and chloroquine, respectively. Then, the expression of P27 was evaluated by Western blot analysis, and the immunofluorescence of Ki67 was assessed. Finally, we assessed the role of P27 siRNA in NPSC quiescence by flow cytometry analyses and 5-ethynyl-20-deoxyuridine incorporation assays under normal and serum-starved conditions. Results NPSC quiescence was induced by 48 h of serum starvation, and they maintained quiescence for up to 21 days. Upon reactivation with serum, the quiescent NPSCs re-entered the cell cycle and exhibited enhanced clonogenic self-renewal, osteogenic differentiation and chondrogenic differentiation potentials compared to control NPSCs under normal culture conditions. We also found that autophagy underlay serum starvation-induced NPSC quiescence. Further study demonstrated that autophagy mediated the quiescence of NPSCs by regulating P27. Conclusions Serum starvation efficiently induces quiescence in NPSCs. Quiescent NPSCs maintain stem cell properties. Our study reveals that autophagy plays a role in maintaining NPSC quiescence and that autophagy mediates the quiescence of NPSCs by regulating P27. We conclude that the induction of quiescence in cultured NPSCs provides a useful model for the analysis of mechanisms that might be relevant to the biology of NPSCs in vivo.

Background Radiotherapy to cancer patients is inevitably accompanied by normal tissue injury, and the bone is one of the most commonly damaged tissues. Damage to bone marrow mesenchymal stem cells (BM-MSCs) induced by radiation is thought to be a major cause of radiation-induced bone loss. Exosomes exhibit great therapeutic potential in the treatment of osteoporosis, but whether exosomes are involved in radiation-induced bone loss has not been thoroughly elucidated to date. The main purpose of this study is to investigate the role of exosomes derived from BM-MSCs in restoring recipient BM-MSC function and alleviating radiation-induced bone loss. Methods BM-MSC-derived exosomes were intravenously injected to rats immediately after irradiation. After 28 days, the left tibiae were harvested for micro-CT and histomorphometric analysis. The effects of exosomes on antioxidant capacity, DNA damage repair, proliferation, and cell senescence of recipient BM-MSCs were determined. Osteogenic and adipogenic differentiation assays were used to detect the effects of exosomes on the differentiation potential of recipient BM-MSCs, and related genes were measured by qRT-PCR and Western blot analysis. β-Catenin expression was detected at histological and cytological levels. Results BM-MSC-derived exosomes can attenuate radiation-induced bone loss in a rat model that is similar to mesenchymal stem cell transplantation. Exosome-treated BM-MSCs exhibit reduced oxidative stress, accelerated DNA damage repair, and reduced proliferation inhibition and cell senescence-associate protein expression compared with BM-MSCs that exclusively received irradiation. Following irradiation, exosomes promote β-catenin expression in BM-MSCs and restore the balance between adipogenic and osteogenic differentiation. Conclusions Our findings indicate that BM-MSC-derived exosomes take effects by restoring the function of recipient BM-MSCs. Therefore, exosomes may represent a promising cell-free therapeutic approach for the treatment of radiation-induced bone loss. Electronic supplementary material The online version of this article (10.1186/s13287-018-1121-9) contains supplementary material, which is available to authorized users.

Summary Degenerative cartilage endplate (CEP) shows decreased chondrification and increased ossification. Cartilage endplate stem cells (CESCs), with the capacity for chondro-osteogenic differentiation, are responsible for CEP restoration. CEP is avascular and hypoxic, while the physiological hypoxia is disrupted in the degenerated CEP. Hypoxia promoted chondrogenesis but inhibited osteogenesis in CESCs. This tissue-specific differentiation fate of CESCs in response to hypoxia was physiologically significant with regard to CEP maintaining chondrification and refusing ossification. MIF, a downstream target of HIF1A, is involved in cartilage and bone metabolisms, although little is known about its regulatory role in differentiation. In CESCs, MIF was identified as a key point through which HIF1A regulated the chondro-osteogenic differentiation. Unexpectedly, unlike the traditionally recognized mode, increased nuclear-expressed MIF under hypoxia was identified to act as a transcriptional regulator by interacting with the promoter of SOX9 and RUNX2. This mode of HIF1A/MIF function may represent a target for CEP degeneration therapy., Graphical Abstract, Highlights • The hypoxic microenvironment is disrupted in degenerative CEP • Hypoxia promotes chondrogenesis but inhibits osteogenesis in CESCs • Hypoxia regulates chondro-osteogenesis through HIF1A/MIF pathway • MIF acts as a transcriptional regulator under hypoxia, In this article, Zhou, Huang, Liu, and colleagues show how the HIF1A/MIF pathway regulated the chondro-osteogenic differentiation of CESCs and suggest the role of MIF as a transcriptional regulator under hypoxia.