Nucleus Pulposus-derived Mesenchymal Stem Cells Research Articles

Melatonin alleviates oxidative stress-induced injury to nucleus pulposus-derived mesenchymal stem cells through activating PI3K/Akt pathway

BackgroundThe changes in the microenvironment of degenerative intervertebral discs cause oxidative stress injury and excessive apoptosis of intervertebral disc endogenous stem cells. The purpose of this study was to explore the possible mechanism of the protective effect of melatonin on oxidative stress injury in NPMSCs induced by H2O2. MethodsThe Cell Counting Kit-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of melatonin. ROS content was detected by 2′7′-dichlorofluorescin diacetate (DCFH-DA). Mitochondrial membrane potential (MMP) was detected by the JC-1assay. Transferase mediated d-UTP Nick end labeling (TUNEL) and Annexin V/PI double staining were used to determine the apoptosis rate. Additionally, apoptosis-associated proteins and PI3K/Akt signaling pathway-related proteins were evaluated by immunofluorescence, immunoblotting and PCR. ECMs were evaluated by RT‒PCR and immunofluorescence. In vivo, X-ray, Magnetic resonance imaging (MRI) and Histological analyses were used to evaluate the protective effect of melatonin. ResultsMelatonin had an obvious protective effect on NPMSCs treated with 0–10 μM melatonin for 24 h. In addition, melatonin also had obvious protective effects on mitochondrial dysfunction, decreased membrane potential and cell senescence induced by H2O2. More importantly, melatonin could significantly reduce the apoptosis of nucleus pulposus mesenchymal stem cells induced by H2O2 by regulating the expression of apoptosis-related proteins and decreasing the rate of apoptosis. After treatment with melatonin, the PI3K/Akt pathway was significantly activated in nucleus pulposus mesenchymal stem cells, while the protective effect was significantly weakened after PI3K-IN-1 treatment. In vivo, the results of X-ray, MRI and histological analyses showed that therapy with melatonin could partially reduce the degree of intervertebral disc degeneration. ConclusionOur research demonstrated that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of nucleus pulposus mesenchymal stem cells induced by oxidative stress via the PI3K/Akt pathway, which provides a novel idea for the therapy of intervertebral disc degeneration. The translational potential of this articleThis study indicates that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of NPMSCs through activating the PI3K/Akt pathway. Melatonin might serve as a promising candidate for the prevention and treatment of Intervertebral disc degeneration disease (IVDD) in the future.

Systemic therapeutic investigation of transplanting nucleus pulposus-derived mesenchymal stem cells embedded on Gefitinib-chitosan/collagen blended injectable hydrogels to the intervertebral disc regeneration treatment using a rat model

Systemic therapeutic investigation of transplanting nucleus pulposus-derived mesenchymal stem cells embedded on Gefitinib-chitosan/collagen blended injectable hydrogels to the intervertebral disc regeneration treatment using a rat model

Quercetin ameliorates oxidative stress-induced senescence in rat nucleus pulposus-derived mesenchymal stem cells via the miR-34a-5p/SIRT1 axis.

Intervertebral disc degeneration (IDD) is a main contributor to low back pain. Oxidative stress, which is highly associated with the progression of IDD, increases senescence of nucleus pulposus-derived mesenchymal stem cells (NPMSCs) and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs (IVDs). Quercetin (Que) has been demonstrated to reduce oxidative stress in diverse degenerative diseases. To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism. In vitro, NPMSCs were isolated from rat tails. Senescence-associated β-galactosidase (SA-β-Gal) staining, cell cycle, reactive oxygen species (ROS), real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blot analyses were used to evaluated the protective effects of Que. Meanwhile the relationship between miR-34a-5p and Sirtuins 1 (SIRT1) was evaluated by dual-luciferase reporter assay. To explore whether Que modulates tert-butyl hydroperoxide (TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway, we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression. In vivo, a puncture-induced rat IDD model was constructed, and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo. We found that TBHP can cause NPMSCs senescence changes, such as reduced cell proliferation ability, increased SA-β-Gal activity, cell cycle arrest, the accumulation of ROS, and increased expression of senescence-related proteins. While abovementioned senescence indicators were significantly alleviated by Que treatment. Que decreased the expression levels of senescence-related proteins (p16, p21, and p53) and senescence-associated secreted phenotype (SASP), including IL-1β, IL-6, and MMP-13, and it increased the expression of SIRT1. In addition, the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown. In vivo, X-ray, and histological analyses indicated that Que alleviated IDD in a puncture-induced rat model. In summary, the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway, suggesting that Que may be a potential agent for the treatment of IDD.

The inhibitory effect of RADKPS on pyroptosis of nucleus pulposus-derived mesenchymal stem cells.

IIntervertebral disc(IVD) degeneration(IDD)is a primary cause of low back pain(LBP)in people, which is associated with nucleus pulposus-derived mesenchymal stem cells(NPMSCs). In this study, the involvement of lipopolysaccharide(LPS) in the pyroptosis of NPMSCs was investigated, The effect of RADKPS on the pyroptosis of NPMSCs and the underlying mechanism behind the impact of RADKPS on the proliferative capacity of NPMSCs were also studied.Pyroptosis of NPMSCs was induced with 10 μg/ml LPS and its effects on the downstream signaling pathways were explored. The protective effect of RADKPS on NPMSCs under the action of LPS and its possible mechanism were explored. Usinng different techniques such as immunohistochemical analysis, cell proliferation assay, qPCR, and Western blot analysis. Accordingly, Caspase1/p20/p10, a protein associated with pyroptosis, was found to be over-expressed in LPS-challenged NPMSCs, Furthermore, the qPCR results demonstrated that LPS promoted the expression of pyroptosis-related gene IL-1β(P<0.0001) ,while down-regulated the expression of sox-9(p<0.001),which was a gene associated with the extracellular matrix(ECM). The immunohistochemical results identified lowered ERK1/2 expression and phosphorylated (p-)ERK1/2 in the degenerated IVD tissues. In this study, the influence of RADKPS on the proliferative ability of NPMSCs was evaluated using two-dimensional and three-dimensional cultures. It was noted that RADKPS promoted the proliferation of NPMSCs in the two-dimensional and three-dimensional cultures. The findings of the western blot experiments revealed that RADKPS inhibited the expression of pyroptosis-related proteins, while it upregulated the p-ERK1/2 (p < 0.001), RhoA (p < 0.01), collagen II (p < 0.01), and sox-9 (p < 0.01), while ERK inhibitor PD98059 and RhoA signaling pathway inhibitor CCG-1423 inhibited their expression.These findings reveal to us that RADKPS hydrogel may protect NPMSCs from pyroptosis. It was also noted that cell proliferation-related signaling pathways may promote the proliferation of NPMSCs. The results revealed that RADKPS hydrogel could be used as a potential therapeutic approach for IDD.

Myricetin alleviates H2O2-induced senescence and apoptosis in rat nucleus pulposus-derived mesenchymal stem cells.

Transplantation of mesenchymal stem cells (MSCs) has been reported to be a novel promising target for the regeneration of degenerated intervertebral discs (IVDs). However, the culture and survival limitations of MSCs remain challenging for MSC-based biological therapy. Myricetin, a common natural flavonoid, has been suggested to possess antiaging and antioxidant abilities. Therefore, we investigated the biological function of myricetin, and its related mechanisms involving cell senescence in intervertebral disc degeneration (IDD). The nucleus pulposus-derived mesenchymal stem cells (NPMSCs) were isolated from 4-month-old Sprague-Dawley (SD) rats and identified by examining surface markers and multipotent differentiation. Rat NPMSCs were cultured in an MSC culture medium or culture medium with different concentrations of H2O2. Myricetin or the combination of myricetin and EX527 were added to the culture medium to investigate the effects of myricetin. Cell viability was evaluated by cell counting kit-8 assays (CCK-8). The apoptosis rate was determined using Annexin V/PI dual staining. The mitochondrial membrane potential (MMP) was analyzed by a fluorescence microscope after JC-1 staining. The cell senescence was determined by SA-β-Gal staining. MitoSOX green was used to selectively estimate mitochondrial reactive oxygen species (ROS) Apoptosis-associated proteins (Bax, Bcl2, and cleaved caspase-3), senescence markers (p16, p21, and p53), and SIRT1/PGC-1α signaling pathway-related proteins (SIRT1 and PGC-1α) were evaluated by western blotting. The cells isolated from nucleus pulposus (NP) tissues met the criteria for MSCs. Myricetin showed no cytotoxicity up to a concentration of 100 μM in rat NPMSCs cultured for 24 h. Myricetin pretreatment exhibited protective effects against H₂O₂-induced apoptosis. Myricetin could also alleviate H₂O₂-induced mitochondrial dysfunctions of increased mitochondrial ROS production and reduced MMP. Moreover, myricetin pretreatment delayed rat NPMSC senescence, as evidenced by decreased exppression of senescence indicators. Pretreatment of NPMSCs with 10 μM EX527, a selective inhibitor of SIRT1, prior to exposure to 100 μM H2O2, reversed the inhibitory effects of myricetin on cell apoptosis. Myricetin could affect the SIRT1/PGC-1α pathway to protect mitochondrial functions and alleviate cell senescence in H₂O₂-treated NPMSCs.

1,25(OH)2D3 Mitigates Oxidative Stress-Induced Damage to Nucleus Pulposus-Derived Mesenchymal Stem Cells through PI3K/Akt Pathway.

Intervertebral disc degeneration (IVDD) is one of the main causes of low back pain. The local environment of the degenerated intervertebral disc (IVD) increases oxidative stress and apoptosis of endogenous nucleus pulposus-derived mesenchymal stem cells (NPMSCs) and weakens its ability of endogenous repair ability in degenerated IVDs. A suitable concentration of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been certified to reduce oxidative stress and cell apoptosis. The current study investigated the protective effect and potential mechanism of 1,25(OH)2D3 against oxidative stress-induced damage to NPMSCs. The present results showed that 1,25(OH)2D3 showed a significant protective effect on NPMSCs at a concentration of 10−10 M for 24 h. Protective effects of 1,25(OH)2D3 were also exhibited against H2O2-induced NPMSC senescence, mitochondrial dysfunction, and reduced mitochondrial membrane potential. The Annexin V/PI apoptosis detection assay, TUNEL assay, immunofluorescence, western blot, and real-time quantitative polymerase chain reaction assay showed that pretreatment with 1,25(OH)2D3 could alleviate H2O2-induced NPMSC apoptosis, including the apoptosis rate and the expression of proapoptotic-related (Caspase-3 and Bax) and antiapoptotic-related (Bcl-2) proteins. The intracellular expression of p-Akt increased after pretreatment with 1,25(OH)2D3. However, these protective effects of 1,25(OH)2D3 were significantly decreased after the PI3K/Akt pathway was inhibited by the LY294002 treatment. In vivo, X-ray, MRI, and histological analyses showed that 1,25(OH)2D3 treatment relieved the degree of IVDD in Sprague–Dawley rat disc puncture models. In summary, 1,25(OH)2D3 efficiently attenuated oxidative stress-induced NPMSC apoptosis and mitochondrial dysfunction via PI3K/Akt pathway and is a promising candidate treatment for the repair of IVDD.

Urolithin a alleviates oxidative stress-induced senescence in nucleus pulposus-derived mesenchymal stem cells through SIRT1/PGC-1α pathway.

BACKGROUNDIn degenerative intervertebral disc (IVD), an unfavorable IVD environment leads to increased senescence of nucleus pulposus (NP)-derived mesenchymal stem cells (NPMSCs) and the inability to complete the differentiation from NPMSCs to NP cells, leading to further aggravation of IVD degeneration (IDD). Urolithin A (UA) has been proven to have obvious effects in delaying cell senescence and resisting oxidative stress.AIMTo explore whether UA can alleviate NPMSCs senescence and to elucidate the underlying mechanism.METHODSIn vitro, we harvested NPMSCs from rat tails, and divided NPMSCs into four groups: the control group, H2O2 group, H2O2 + UA group, and H2O2 + UA + SR-18292 group. Senescence-associated β-Galactosidase (SA-β-Gal) activity, cell cycle, cell proliferation ability, and the expression of senescence-related and silent information regulator of transcription 1/PPAR gamma coactivator-1α (SIRT1/ PGC-1α) pathway-related proteins and mRNA were used to evaluate the protective effects of UA. In vivo, an animal model of IDD was constructed, and X-rays, magnetic resonance imaging, and histological analysis were used to assess whether UA could alleviate IDD in vivo.RESULTSWe found that H2O2 can cause NPMSCs senescence changes, such as cell cycle arrest, reduced cell proliferation ability, increased SA-β-Gal activity, and increased expression of senescence-related proteins and mRNA. After UA pretreatment, the abovementioned senescence indicators were significantly alleviated. To further demonstrate the mechanism of UA, we evaluated the mitochondrial membrane potential and the SIRT1/PGC-1α pathway that regulates mitochondrial function. UA protected mitochondrial function and delayed NPMSCs senescence by activating the SIRT1/PGC-1α pathway. In vivo, we found that UA treatment alleviated an animal model of IDD by assessing the disc height index, Pfirrmann grade and the histological score.CONCLUSIONIn summary, UA could activate the SIRT1/PGC-1α signaling pathway to protect mitochondrial function and alleviate cell senescence and IDD in vivo and vitro.

Elucidating the role of cell-mediated inflammatory cytokines on allogeneic mouse-derived nucleus pulposus mesenchymal stem cells.

In this present study, our aim was to evaluate the cell-mediated specific anti-donor antibody and its associated inflammatory cytokine secretion along with its succeeding effects on Nucleus pulposus-derived mesenchymal stem cells (NPMSCs). Tissue from the NP compartment of 12 normal mice was isolated, expanded in cell culture, and the cell phenotypes were confirmed by flow cytometry. Multipotent differentiation and its specific gene expression analysis were confirmed by reverse transcriptase PCR. T and B cells were monitored for both donor and recipient mouse and further analysis of anti-donor antibody secretion was confirmed by lymphocyte crossmatch. In conjunction with anti-donor-specific antibody analysis, the associated inflammatory cytokine expression was analyzed by ELISA. In co-culture, cell-mediated antibody secretion was elevated in T and B cells positive mouse group, when compared to control mouse group. Allogeneic-derived donor NPMSCs were found to be stimulated the secretion of pro-inflammatory cytokines and the level of pro-inflammatory cytokines showed reduced expression in control mouse serum. In co-culture group the concentration of the cell-mediated pro- and anti-inflammatory cytokines found to be increased. PRACTICAL APPLICATIONS: Mesenchymal stem cell exhibit good regeneration capacity for many types of disease, and the mechanism belongs to regeneration is not clear. In intervertebral disc, the nucleus pulposus-derived mesenchymal stem cells showed a better regeneration capacity. On the contrary, the NP cells-based therapy, the Mesenchymal stem cells showed expanded anabolic and reduced catabolic activity together with induced anti-inflammatory effect. In this study, the T & B cells were used to evaluate the anti-donor antibody secretion and also to study how it stimulates the production of anti-donor antibodies against the donor cells. Finally, it was found that T & B cells lead the synthesis of inflammatory cytokines are IL-1, IL-6, and TNF-α. From this study, the results proved that the cell-mediated pro- and anti-inflammatory cytokines to be monitored in allogeneic stem cells-based therapy of intervertebral disc degeneration.

6-gingerol protects nucleus pulposus-derived mesenchymal stem cells from oxidative injury by activating autophagy.

BACKGROUNDTo date, there has been no effective treatment for intervertebral disc degeneration (IDD). Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) showed encouraging results in IDD treatment, but the overexpression of reactive oxygen species (ROS) impaired the endogenous repair abilities of NPMSCs. 6-gingerol (6-GIN) is an antioxidant and anti-inflammatory reagent that might protect NPMSCs from injury.AIMTo investigate the effect of 6-GIN on NPMSCs under oxidative conditions and the potential mechanism.METHODSThe cholecystokinin-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of 6-GIN. ROS levels were measured by 2´7´-dichlorofluorescin diacetate analysis. Matrix metalloproteinase (MMP) was detected by the tetraethylbenzimidazolylcarbocyanine iodide assay. TUNEL assay and Annexin V/PI double-staining were used to determine the apoptosis rate. Additionally, autophagy-related proteins (Beclin-1, LC-3, and p62), apoptosis-associated proteins (Bcl-2, Bax, and caspase-3), and PI3K/Akt signaling pathway-related proteins (PI3K and Akt) were evaluated by Western blot analysis. Autophagosomes were detected by transmission electron microscopy in NPMSCs. LC-3 was also detected by immunofluorescence. The mRNA expression of collagen II and aggrecan was evaluated by real-time polymerase chain reaction (RT-PCR), and the changes in collagen II and MMP-13 expression were verified through an immunofluorescence assay.RESULTS6-GIN exhibited protective effects against hydrogen peroxide-induced injury in NPMSCs, decreased hydrogen peroxide-induced intracellular ROS levels, and inhibited cell apoptosis. 6-GIN could increase Bcl-2 expression and decrease Bax and caspase-3 expression. The MMP, Annexin V-FITC/PI flow cytometry and TUNEL assay results further confirmed that 6-GIN treatment significantly inhibited NPMSC apoptosis induced by hydrogen peroxide. 6-GIN treatment promoted extracellular matrix (ECM) expression by reducing the oxidative stress injury-induced increase in MMP-13 expression. 6-GIN activated autophagy by increasing the expression of autophagy-related markers (Beclin-1 and LC-3) and decreasing the expression of p62. Autophagosomes were visualized by transmission electron microscopy. Pretreatment with 3-MA and BAF further confirmed that 6-GIN-mediated stimulation of autophagy did not reduce autophagosome turnover but increased autophagic flux. The PI3K/Akt pathway was also found to be activated by 6-GIN. 6-GIN inhibited NPMSC apoptosis and ECM degeneration, in which autophagy and the PI3K/Akt pathway were involved.CONCLUSION6-GIN efficiently decreases ROS levels, attenuates hydrogen peroxide-induced NPMSCs apoptosis, and protects the ECM from degeneration. 6-GIN is a promising candidate for treating IDD.

Heme Oxygenase-1-Mediated Autophagy Protects against Oxidative Damage in Rat Nucleus Pulposus-Derived Mesenchymal Stem Cells.

Although endogenous nucleus pulposus-derived mesenchymal stem cell- (NPMSC-) based regenerative medicine has provided promising repair strategy for intervertebral disc (IVD) degeneration, the hostile microenvironments in IVD, including oxidative stress, can negatively affect the survival and function of the NPMSCs and severely hinder the endogenous repair process. Therefore, it is of great importance to reveal the mechanisms of the endogenous repair failure caused by the adverse microenvironments in IVD. The aim of this study was to investigate the effect of oxidative stress on the rat NPMSCs and its underlying mechanism. Our results demonstrated that oxidative stress inhibited cell viability, induced apoptosis, and increased the production of reactive oxygen species (ROS) in NPMSCs. In addition, the results showed that the expression level of heme oxygenase-1 (HO-1) increased at an early stage but decreased at a late stage when NPMSCs were exposed to oxidative stress, and the oxidative damages of NPMSCs could be partially reversed by promoting the expression of HO-1. Further mechanistic analysis indicated that the protective effect of HO-1 against oxidative damage in NPMSCs was mediated by the activation of autophagy. Taken together, our study revealed that oxidative stress could inhibit cell viability, induce apoptosis, and increase ROS production in NPMSCs, and HO-1-mediated autophagy might act as a protective response to the oxidative damage. These findings might enhance our understanding on the mechanism of the endogenous repair failure during IVD degeneration and provide novel research direction for the endogenous repair of IVD degeneration.

The protective effects of PI3K/Akt pathway on human nucleus pulposus mesenchymal stem cells against hypoxia and nutrition deficiency

BackgroundTo study the effects of hypoxia and nutrition deficiency mimicking degenerated intervertebral disc on the biological behavior of human nucleus-derived pulposus mesenchymal stem cells (hNP-MSCs) and the role of PI3K/Akt pathway in the process in vitro.MethodshP-MSCs were isolated from lumbar disc and were further identified by their immunophenotypes and multilineage differentiation. Then, cells were divided into the control group, hypoxia and nutrition deficiency group, the LY294002 group, and insulin-like growth factor 1 (IGF-1) group. Then cell apoptosis, the cell viability, the caspase 3 activity, and the expression of PI3K, Akt, and functional genes (aggrecan, collagen I, and collagen II) were evaluated.ResultOur work showed that isolated cells met the criteria of International Society for cellular Therapy. Therefore, cells obtained from degenerated nucleus pulposus were definitely hNP-MSCs. Our results showed that hypoxia and nutrition deficiency could significantly increase cell apoptosis, the caspase 3 activity, and inhibit cell viability. Gene expression results demonstrated that hypoxia and nutrition deficiency could increase the relative expression of PI3K and Akt gene and inhibit the expression of functional genes. However, when the PI3K/Akt pathway was inhibited by LY294002, the cell apoptosis and caspase 3 activity significantly increased while the cell viability was obviously inhibited. Quantitative real-time PCR results showed that the expression of functional genes was more significantly inhibited. Our study further verified that the above-mentioned biological activities of hNP-MSCs could be significantly improved by IGF1.ConclusionsPI3K/Akt signal pathway may have protective effects on human nucleus pulposus-derived mesenchymal stem cells against hypoxia and nutrition deficiency.

The effect of high glucose on the biological characteristics of nucleus pulposus-derived mesenchymal stem cells.

Diabetes mellitus (DM) is a dependent risk factor in the progression of intervertebral disc degeneration (IVDD). High glucose supply has negative effects on nucleus pulpous (NP) cell and mesenchymal stem cell (MSC) biology. However, the effect of hyperglycaemia on the biological characterization of nucleus pulpous-derived mesenchymal stem cell (NPMSC) has not been investigated previously. Therefore, further exploration of the effects of DM-associated hyperglycaemia on NPMSC biology is important to better understand and develop endogenous repair strategies of DM patient-associated IVDD. Therefore, the cell biological characteristics were compared between NPMSC cultured in media with low glucose concentration (LG-NPMSC) and high glucose concentration (HG-NPMSC). The results demonstrated that HG-NPMSC showed significantly decreased cell proliferation, colony formation ability, migration and wound-healing capability compared with those of LG-NPMSC. HG-NPMSC also showed significantly decreased expressions of stemness genes and mRNA and protein expressions of silent information regulator protein 1 (SIRT1), SIRT6, hypoxia inducible factor-1α (HIF-1α) and glucose transporter 1 (GLUT-1), whereas increased cell apoptosis, cell senescence and caspase-3 expression. These results suggest that high glucose may decrease proliferation and stemness maintenance ability and increase apoptosis and senescence of NPMSC. SIGNIFICANCE OF THE STUDY: We found that high glucose concentration significantly decreased cell proliferation, colony formation ability, migration and wound-healing capability of nucleus pulposus-derived mesenchymal stem cells. Moreover, high glucose cultured nucleus pulposus-derived mesenchymal stem cells showed significantly decreased expression of stemness genes, related mRNA and protein, whereas increased cell apoptosis, cell senescence and expression of caspase-3. The present study indicated that better control of high concentration glucose in the early stage of diabetes mellitus should be recommended to prevent or limit intervertebral disc degeneration.

Influence of simvastatin on the biological behavior of nucleus pulposus-derived mesenchymal stem cells.

Objective(s):This research is to study the influences of different concentrations of simvastatin on the biological activities of nucleus pulposus-derived mesenchymal stem cells (NPMSC).Materials and Methods: NPMSC were cultured with different concentrations of simvastatin (0, 0.01, 0.1, and 1 μM) and assessed to determine the possible effects of simvastatin. The cell proliferation was assessed with CCK-8 assay. The flowcytometry and multilineage differentiation were also performed to identify the stem characterization of the cells. The mRNA expressions of aggrecan, collagen type II, glucose transporter 1 (GLUT-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were determined by qRT-PCR.Results: The results demonstrated that the cells isolated from nucleus pulposus of healthy Sprague-Dawley (SD) rat met the criteria of MSC. NPMSC could form sunflower-like colonies and strongly expressed stem cell-related genes. In addition, NPMSC showed strong ability of chondrogenic, adipogenic and osteogenic differentiation. Simvastatin at certain range concentrations (0.01 μM-0.1 μM)) significantly promoted colony-forming rate and cell proliferation, and inhibited cell apoptosis. Simvastatin could promote expressions of aggrecan, collagen type II, HIF-1α, VEGF and GLUT-1, while 0.1 μmol/l concentration reached the maximum effect. Our study further demonstrated that HIF-1α-intermediated signaling pathway might participate in regulating the biological activities of NPMSC. Conclusion: Proper concentration of simvastatin can promote the biological behavior of NPMSC, and HIF-1α-intermediated signaling pathway might participate in the mechanism.

Injectable Hydrogel Combined with Nucleus Pulposus-Derived Mesenchymal Stem Cells for the Treatment of Degenerative Intervertebral Disc in Rats.

Stem cell-based tissue engineering in treating intervertebral disc (IVD) degeneration is promising. An appropriate cell scaffold can maintain the viability and function of transplanted cells. Injectable hydrogel has the potential to be an appropriate cell scaffold as it can mimic the condition of the natural extracellular matrix (ECM) of nucleus pulposus (NP) and provide binding sites for cells. This study was aimed at investigating the effect of injectable hydrogel-loaded NP-derived mesenchymal stem cells (NPMSC) for the treatment of IVD degeneration (IDD) in rats. In this study, we selected injectable 3D-RGD peptide-modified polysaccharide hydrogel as a cell transplantation scaffold. In vitro, the biocompatibility, microstructure, and induced differentiation effect on NPMSC of the hydrogel were studied. In vivo, the regenerative effect of hydrogel-loaded NPMSC on degenerated NP in a rat model was evaluated. The results showed that NPMSC was biocompatible and able to induce differentiation in hydrogel in vivo. The disc height index (almost 87%) and MRI index (3313.83 ± 227.79) of the hydrogel-loaded NPMSC group were significantly higher than those of other groups at 8 weeks after injection. Histological staining and immunofluorescence showed that the hydrogel-loaded NPMSC also partly restored the structure and ECM content of degenerated NP after 8 weeks. Moreover, the hydrogel could support long-term NPMSC survival and decrease cell apoptosis rate of the rat IVD. In conclusion, injectable hydrogel-loaded NPMSC transplantation can delay the level of IDD and promote the regeneration of the degenerative IVD in the rat model.

Naringin alleviates H2O2-induced apoptosis via the PI3K/Akt pathway in rat nucleus pulposus-derived mesenchymal stem cells

ABSTRACT Purpose: To investigate the protective effect of naringin (Nar) on H2O2-induced apoptosis of nucleus pulposus-derived mesenchymal stem cells (NPMSC) and the potential mechanism in this process. Methods: Rat NPMSC were cultured in MSC culture medium or culture medium with different concentrations of H2O2. Nar or the combination of Nar and LY294002 was added into the culture medium to investigate the effects of Nar. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. The apoptosis rate was determined using Annexin V/PI dual staining and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays. Additionally, the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. ATP level in NPMSC was analyzed via ATP detection kit. Mitochondrial ultrastructure change was observed through transmission electron microscope (TEM). Levels of apoptosis-associated molecules (cleaved caspase-3, Bax and Bcl-2) were evaluated via RT-PCR and western blot, respectively. Results: The cells isolated from NP met the criteria for MSC. H2O2 significantly promoted NPMSC apoptosis in a dose and time-dependent manner. Nar showed no cytotoxicity effect on NPMSC up to a concentration of 100 μM for 24 h. Nar exhibited protective effects against H2O2-induced NPMSC apoptosis including apoptosis rate, expressions of proapoptosis and antiapoptosis related genes and protein. Nar could also alleviate H2O2-induced mitochondrial dysfunction of increased mitochondrial ROS production, reduced MMP, decreased intracellular ATP and mitochondrial ultrastructure change. However, these protected effects were inhibited after LY294002 treatment. Conclusions: Our results demonstrated that Nar efficiently attenuated H2O2-induced NPMSC apoptosis and mitochondrial dysfunction. The activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.

Comparison of different methods for the isolation and purification of rat nucleus pulposus-derived mesenchymal stem cells

ABSTRACT Purpose Recently, nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have been identified and have shown good prospects for the repair of degenerative intervertebral discs. However, there is no consensus about the methods for the isolation and purification of NPMSCs. Therefore, a reliable and efficient isolation and purification method is potentially needed. We aimed to compare different methods and to identify an optimal method for isolating and purifying NPMSCs. Methods NPMSCs were isolated and purified using two common methods (a low-density culture (LD) method and a mesenchymal stem cell complete medium culture (MSC-CM) method) and two novel methods (a cloning cylinder (CC) method and a combination of the CC and MSC-CM methods (MSC-CM+CC)). The morphology, MSC-specific surface markers (CD44, CD73, CD90, CD105, CD34 and HLA-DR), multiple-lineage differentiation potential, colony formation ability, and stemness gene (Oct4, Nanog, and Sox2) expression were evaluated and compared. Results NPMSCs isolated from nucleus pulposus (NP) tissues via the four methods met the criteria stated by the International Society of Cell Therapy (ISCT) for MSCs, including adherent growth ability, MSC-specific surface antigen expression, and multi-lineage differentiation potential. In particular, the MSC-CM+CC method yielded a relatively higher quality of NPMSCs in terms of cell surface markers, multiple-lineage differentiation potential, colony formation ability, and stemness gene expression. Conclusions Our results indicated that NPMSCs can be obtained via all four methods and that the MSC-CM+CC method is more reliable and efficient than the other three methods. The findings from this study provide an alternative option for isolating and purifying NPMSCs.

The Differential Effects of Leukocyte-Containing and Pure Platelet-Rich Plasma on Nucleus Pulposus-Derived Mesenchymal Stem Cells: Implications for the Clinical Treatment of Intervertebral Disc Degeneration.

Background Platelet-rich plasma (PRP) is a promising strategy for intervertebral disc degeneration. However, the potential harmful effects of leukocytes in PRP on nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have seldom been studied. This study aimed at comparatively evaluating effects of pure platelet-rich plasma (P-PRP) and leukocyte-containing platelet-rich plasma (L-PRP) on rabbit NPMSCs in vitro. Methods NPMSCs isolated from rabbit NP tissues were treated with L-PRP or P-PRP in vitro, and then cell proliferation and expression of stem cell markers, proinflammatory cytokines (TNF-α, IL-1β), production of ECM (extracellular matrix-related protein), and NF-κB p65 protein were validated by CCK-8 assay, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and western blot respectively. Results NPMSCs differentiate into nucleus pulposus-like cells after treatment of PRPs (P-PRP and L-PRP), and NPMSCs exhibited maximum proliferation at a 10% PRP dose. L-PRP had observably higher concentration of leukocytes, TNF-α, and IL-1β than P-PRP. Furthermore, compared to P-PRP, L-PRP induced the differentiated NPMSCs to upregulate the expression of TNF-α and IL-1β, enhanced activation of the NF-κB pathway, increased the expression of MMP-1 and MMP-13, and produced less ECM in differentiated NPMSCs. Conclusions Both P-PRP and L-PRP can induce the proliferation and NP-differentiation of NPMSCs. Compared to L-PRP, P-PRP can avoid the activation of the NF-κB pathway, thus reducing the inflammatory and catabolic responses.

Effect of Compression Loading on Human Nucleus Pulposus-Derived Mesenchymal Stem Cells.

Purpose Mechanical loading plays a vital role in the progression of intervertebral disc (IVD) degeneration, but little is known about the effect of compression loading on human nucleus pulposus-derived mesenchymal stem cells (NP-MSCs). Thus, this study is aimed at investigating the effect of compression on the biological behavior of NP-MSCs in vitro. Methods Human NP-MSCs were isolated from patients undergoing lumbar discectomy for IVD degeneration and were identified by immunophenotypes and multilineage differentiation. Then, cells were cultured in the compression apparatus at 1.0 MPa for different times (0 h, 24 h, 36 h, and 48 h). The viability-, differentiation-, and differentiation-related genes (Runx2, APP, and Col2) and colony formation-, migration-, and stem cell-related proteins (Sox2 and Oct4) were evaluated. Results The results showed that the isolated cells fulfilled the criteria of MSC stated by the International Society for Cellular Therapy (ISCT). And our results also indicated that compression loading significantly inhibited cell viability, differentiation, colony formation, and migration. Furthermore, gene expression suggested that compression loading could downregulate the expression of stem cell-related proteins and lead to NP-MSC stemness losses. Conclusions Our results suggested that the biological behavior of NP-MSCs could be inhibited by compression loading and therefore enhanced our understanding on the compression-induced endogenous repair failure of NP-MSCs during IVDD.

The Influence of Hyperosmolarity in the Intervertebral Disc on the Proliferation and Chondrogenic Differentiation of Nucleus Pulposus-Derived Mesenchymal Stem Cells

Nucleus pulposus-derived mesenchymal stem cells (NP-MSCs) are suitable cell candidates for intervertebral disc (IVD) regeneration. However, little work has been done to determine the proliferation and chondrogenic differentiation of NP-MSCs in the hyperosmotic microenvironment of IVD. This study aimed to investigate the influence of the hyperosmolarity of IVD on the proliferation and chondrogenic differ­entiation of NP-MSCs. NP-MSCs were cultured in media of 300, 400, 430, and 500 mOsm/L, mimicking the osmotic pressures of serious degenerative, moderately degenerative, and healthy IVD. Cell proliferation was measured by CCK-8 assay. The expression of aggrecan, collagen I, and collagen II were measured by gene and protein expression analysis. Alcian blue and dimethylmethylene blue assay were used to investigate the accumulation of sulfate glycosaminoglycan. The regulation role of extracellular signal-regulated kinase (ERK) pathway was also analyzed. The results showed that, compared to 300 mOsm/L, hyperosmolarity of healthy IVD (430 and 500 mOsm/L) inhibited the proliferation and chondrogenic differentiation of NP-MSCs. The relative hypoosmotic condition of moderately degenerative IVD (400 mOsm/L) led to great proliferation and chondrogenic differentiation capacity. The ERK pathway was activated by the hyperosmolarity; inhibition of the ERK pathway abolished the difference in cell proliferation between the 300 mOsm/L and the hyperosmotic conditions, and enhanced chondrogenic differentiation. In conclusion, hyperosmolarity of IVD had a significant impact on the proliferation and chondrogenic differentiation of NP-MSCs. The ERK pathway was involved in the inhibition of proliferation and chondrogenic differentiation of NP-MSCs by the hyperosmolarity of IVD. The relative hypo-osmotic condition prevailing in degenerative discs offers a more permissive microenvironment for NP-MSCs.

Icariin Improves the Viability and Function of Cryopreserved Human Nucleus Pulposus-Derived Mesenchymal Stem Cells.

Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have shown a good prospect in the regeneration of intervertebral disc (IVD) tissues. However, fresh NPMSCs are not always readily available for basic research and clinical applications. Therefore, there is a need for an effective long-term cryopreservation method for NPMSCs. The aim of this study was to determine whether adding icariin (ICA) to the conventional cryoprotectant containing dimethyl sulfoxide (DMSO) had a better cryoprotective effect for NPMSCs. The results showed that the freezing solution containing ICA along with DMSO significantly increased the postthawed cell viability, decreased the apoptosis rate, improved cell adherence, and maintained the mitochondrial functions, as compared to the freezing solution containing DMSO alone. And the inhibition of oxidative stress and upregulation of heat shock proteins (HSPs) in the presence of ICA also confirmed the beneficial effect of ICA. Furthermore, ICA had no cytotoxicity and did not alter the characteristics of postthawed NPMSCs. In conclusion, these results suggested that the addition of ICA to the conventional freezing medium could improve the viability and function of the cryopreserved human NPMSCs and provided an optimal formulated freezing solution for human NPMSC cryopreservation.