Nucleus Pulposus-derived Stem Cells Research Articles

The most influential articles on stem cells in intervertebral disc degeneration

BackgroundStem cell-related studies have been increasingly conducted to facilitate the regeneration of degenerative discs. However, analyses of high-impact articles focused on this topic are rare. This study aimed to determine and summarize the most-cited studies examining stem cells in the context of intervertebral disc degeneration (IDD).MethodsWe searched the Web of Science (WoS) database for stem cell-related articles in IDD, and the 50 highest-cited papers were summarized. A correlation analysis was conducted to determine the relationship among WoS citations, Altmetric Attention Score (AAS), and Dimensions.ResultsThe number of citations of the top 50 manuscripts ranged from 92 to 370. The top three countries were the United States (14), China (10), and Japan (9). Spine (12) was the most prevalent journal, and this was followed by Biomaterials (6). Bone marrow-derived stem cells were the most common subject (38), and they were followed by nucleus pulposus-derived stem cells (4) and annulus fibrosus-derived stem cells (4). Humans were the most studied species (31), and the next most studied were rabbits (9) and rats (7). There was a very high correlation between WoS and Dimension citations (p < 0.001, r = 0.937).ConclusionsFor the first time, the highest impact articles examining stem cells in the context of IDD were assessed together. The current study provides a deepened understanding of historical studies focused on stem cells in IDD and is beneficial for future studies in this field.

An international analysis of stem cell research in intervertebral disc degeneration

Stem cell therapy has been increasingly investigated as a promising strategy for intervertebral disc degeneration (IDD). However, no international analysis of stem cell research has yet been conducted. This study aimed to analyze the major characteristics of published reports of stem cell use for IDD and to present a global insight into stem cell research. The study period spanned from the inception of the Web of Science database to 2021. A search strategy using specific keywords was implemented to retrieve relevant publications. The numbers of documents, citations, countries, journals, article types, and stem cell types were evaluated. A total of 1170 papers were retrieved. The analysis showed a significant increase in the number of papers over time (p < 0.001). High-income economies accounted for the majority of papers (758, 64.79 %). China produced the most articles (378, 32.31 %), followed by the United States (259, 22.14 %), Switzerland (69, 5.90 %), United Kingdom (54, 4.62 %), and Japan (47, 4.02 %). The United States ranked first in terms of the number of citations (10,346), followed by China (9177) and Japan (3522). Japan ranked first in terms of the number of citations per paper (74.94), followed by United Kingdom (58.54) and Canada (53.74). When standardized by population, Switzerland ranked first, followed by Ireland and Sweden. When gross domestic product was considered, Switzerland ranked first, followed by Portugal and Ireland. The number of papers was positively correlated with gross domestic product (p < 0.001, r = 0.673); however, there was no significant correlation with population (p = 0.062, r = 0.294). Mesenchymal stem cells were the most investigated stem cells, followed by nucleus pulposus-derived stem cells and adipose-derived stem cells. A sharp increase in stem cell research was observed in the field of IDD. China produced the most, although several European countries were more productive relative to their populations and economies.

MCP‑1/CCR2 axis inhibits the chondrogenic differentiation of human nucleus pulposus mesenchymal stem cells.

Intervertebral disc degeneration (IDD) creates a hostile environment with high osmotic pressure, high mechanical stress, hypoxia and a low pH, where cytokines such as TNF‑α and IL‑1β are highly expressed. The degenerating intervertebral disc has high local expression of monocyte chemoattractant protein‑1(MCP‑1), which is associated with the degree of degeneration. However, there are a few reports on the influence of MCP‑1 on nucleus pulposus‑derived stem cells(NPSCs). In the present study, a significant upregulation of MCP‑1 was observed in NPSCs cultured invitro with pro‑inflammatory cytokines. MCP‑1 significantly inhibited the migration and proliferation of NPSCs in a dose‑dependent manner as detected via Cell Counting Kit‑8, wound healing and Transwell assays. Western blotting and histological analysis demonstrated that MCP‑1 significantly reduced chondrogenic NPSC differentiation. Reverse transcription‑quantitative PCR and western blotting revealed that C‑Cchemokine receptor type2(CCR2) mRNA and protein expression levels were significantly enhanced by MCP‑1. Furthermore, MCP‑1 significantly inhibited the migration, differentiation and proliferation of NPSCs, which was effectively reversed by blocking CCR2 with the inhibitor RS504393. Overall, these results demonstrated that MCP‑1 may contribute to the inhibition of chondrogenic NPSC differentiation via MCP‑1/CCR2 chemotaxis signals, providing a potential therapeutic target for IDD.

HIF1A Alleviates compression-induced apoptosis of nucleus pulposus derived stem cells via upregulating autophagy

ABSTRACT Intervertebral disc degeneration (IDD) is the primary pathological mechanism that underlies low back pain. Overloading-induced cell death, especially endogenous stem cell death, is the leading factor that undermines intrinsic repair and aggravates IDD. Previous research has separately studied the effect of oxygen concentration and mechanical loading in IDD. However, how these two factors synergistically influence endogenous repair remains unclear. Therefore, we established in vitro and in vivo models to study the mechanisms by which hypoxia interacted with overloading-induced cell death of the nucleus pulposus derived stem cells (NPSCs). We found the content of HIF1A (hypoxia inducible factor 1 subunit alpha) and the number of NPSCs decreased with disc degeneration in both rats and human discs. Hence, we isolated this subpopulation from rat discs and treated them simultaneously with hypoxia and excessive mechanical stress. Our results demonstrated that hypoxia exerted protective effect on NPSCs under compression, partially through elevating macroautophagy/autophagy. Proteomics and knockdown experiments further revealed HIF1A-BNIP3-ATG7 axis mediated the increase in autophagy flux, in which HMOX1 and SLC2A1 were also involved. Moreover, HIF1A-overexpressing NPSCs exhibited stronger resistance to over-loading induced apoptosis in vitro. They also showed higher survival rates, along with elevated autophagy after being intra-disc transplanted into over-loaded discs. Jointly, both in vivo and in vitro experiments proved the anti-apoptotic effect of HIF1A on NPSCs under the excessive mechanical loading, suggesting that restoring hypoxia and manipulating autophagy is crucial to maintain the intrinsic repair and to retard disc degeneration. Abbreviations: 3-MA: 3-methyladenine; ACAN: aggrecan; ATG7: autophagy related 7; BafA1: bafilomycin A1; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CASP3: caspase 3; CCK8: cell counting kit-8; CHT: chetomin; CMP: compression; CoCl2: cobalt chloride; COL2A1: collagen type II alpha 1 chain; Ctrl: control; DAPI: 4,6-diamidino-2-phenylindole; DEP: differentially expressed protein; DiR: 1,1-dioctadecyl-3,3,3,3-tetramethyl indotricarbocyanine; ECM: extracellular matrix; FCM: flow cytometry; GD2: disialoganglioside GD 2; GFP: green fluorescent protein; GO: gene ontology; GSEA: gene set enrichment analysis; H&E: hematoxylin-eosin; HIF1A: hypoxia inducible factor 1 subunit alpha; HK2: hexokinase 2; HMOX1: heme oxygenase 1; HX: hypoxia mimicry; IDD: intervertebral disc degeneration; IF: immunofluorescence; IHC: immunohistochemistry; IVD: intervertebral disc; KEGG: kyoto encyclopedia of genes and genomes; LBP: low back pain; Lv: lentivirus; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NC: negative control; NIR: near-infrared; NP: nucleus pulposus; NPC: nucleus pulposus cell; NPSC: nucleus pulposus derived stem cell; NX: normoxia; PPI: protein-protein interactions; RFP: red fluorescent protein; SLC2A1/GLUT1: solute carrier family 2 member 1; SQSTM1/p62: sequestosome 1; TEK/TIE2: TEK receptor tyrosine kinase; TEM: transmission electron microscopy; TUBB: tubulin beta class I.

Effects of Stromal Cell-Derived Factor-1α Secreted in Degenerative Intervertebral Disc on Activation and Recruitment of Nucleus Pulposus-Derived Stem Cells.

Stromal cell-derived factor-1α (SDF-1α) plays a significant role in mobilizing and recruiting mesenchymal stem cells (MSCs) to the sites of injury. This study investigated the potential of SDF-1α released in the degenerative intervertebral disc (IVD) to activate and recruit endogenous nucleus pulposus-derived stem cells (NPSCs) for regeneration in situ. We found SDF-1α was highly expressed and secreted by the native disc cells when cultured in the proinflammatory mediators in vitro mimicking the degenerative settings. Immunohistochemical staining also showed that the expression level of SDF-1α was significantly higher in the degenerative group compared to that in the normal group. In addition to enhancement of viability, SDF-1α significantly increased the number of NPSCs migrating into the center of the nucleotomized bovine IVD ex vivo. After the systemic delivery of exogenous PKH26-labelled NPSCs into the rats in vivo, there was a significant difference in the distribution of the migrated cells between the normal and the degenerative IVDs, which might be caused by the different expression levels of SDF-1α. However, blocking CXC chemokine receptor 4 (CXCR4) with AMD3100 effectively abrogated SDF-1α-stimulated proliferation and migration. Taken together, SDF-1α may be a key chemoattractant that is highly produced in response to the degenerative changes, which can be used to enhance the proliferation and recruitment of endogenous stem cells into the IVDs. These findings may be of importance for understanding IVD regenerative mechanisms and development of regenerative strategies in situ for IVD degeneration.

Autophagy mediates serum starvation-induced quiescence in nucleus pulposus stem cells by the regulation of P27

BackgroundAdult 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.MethodsFirst, 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.ResultsNPSC 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.ConclusionsSerum 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.

Stromal cell-derived factor-1α promotes recruitment and differentiation of nucleus pulposus-derived stem cells.

BACKGROUNDIntervertebral disc (IVD) degeneration is a condition characterized by a reduction in the water and extracellular matrix content of the nucleus pulposus (NP) and is considered as one of the dominating contributing factors to low back pain. Recent evidence suggests that stromal cell-derived factor 1α (SDF-1α) and its receptor C-X-C chemokine receptor type 4 (CXCR4) direct the migration of stem cells associated with injury repair in different musculoskeletal tissues.AIMTo investigate the effects of SDF-1α on recruitment and chondrogenic differentiation of nucleus pulposus-derived stem cells (NPSCs).METHODSWe performed real-time RT-PCR and enzyme-linked immunosorbent assay to examine the expression of SDF-1α in nucleus pulposus cells after treatment with pro-inflammatory cytokines in vitro. An animal model of IVD degeneration was established using annular fibrosus puncture in rat coccygeal discs. Tissue samples were collected from normal control and degeneration groups. Differences in the expression of SDF-1α between the normal and degenerative IVDs were analyzed by immunohistochemistry. The migration capacity of NPSCs induced by SDF-1α was evaluated using wound healing and transwell migration assays. To determine the effect of SDF-1α on chondrogenic differentiation of NPSCs, we conducted cell micromass culture and examined the expression levels of Sox-9, aggrecan, and collagen II. Moreover, the roles of SDF-1/CXCR4 axis in the migration and chondrogenesis differentiation of NPSCs were analyzed by immunofluorescence, immunoblotting, and real-time RT-PCR.RESULTSSDF-1α was significantly upregulated in the native IVD cells cultured in vitro with pro-inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, mimicking the degenerative settings. Immunohistochemical staining showed that the level of SDF-1α was also significantly higher in the degenerative group than in the normal group. SDF-1α enhanced the migration capacity of NPSCs in a dose-dependent manner. In addition, SDF-1α induced chondrogenic differentiation of NPSCs, as evidenced by the increased expression of chondrogenic markers using histological and immunoblotting analyses. Real-time RT-PCR, immunoblotting, and immunofluorescence showed that SDF-1α not only increased CXCR4 expression but also stimulated translocation of CXCR4 from the cytoplasm to membrane, accompanied by cytoskeletal rearrangement. Furthermore, blocking CXCR4 with AMD3100 effectively suppressed the SDF-1α-induced migration and differentiation capacities of NPSCs.CONCLUSIONThese findings demonstrate that SDF-1α has the potential to enhance recruitment and chondrogenic differentiation of NPSCs via SDF-1/CXCR4 chemotaxis signals that contribute to IVD regeneration.

Is exclusion of leukocytes from platelet-rich plasma (PRP) a better choice for early intervertebral disc regeneration?

BackgroundPlatelet-rich plasma (PRP) is becoming a promising strategy to treat early intervertebral disc degeneration (IDD) in clinics. Pure PRP without leukocytes (P-PRP) may decrease the catabolic and inflammatory changes in the early degenerated intervertebral discs. The aim of this study was to investigate the effects of P-PRP on nucleus pulposus-derived stem cells (NPSCs) isolated from early degenerated intervertebral discs in vitro.MethodsNPSCs isolated from early degenerated discs of rabbits were treated with P-PRP or leukocyte-platelet-rich PRP (L-PRP) in vitro, followed by measuring cell proliferation, stem cell marker expression, inflammatory gene expression, and anabolic and catabolic protein expression by immunostaining, quantitative real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay.ResultsCell proliferation was induced by P-PRP in a dose-dependent manner with maximum proliferation at 10% P-PRP dose. P-PRP induced differentiation of NPSCs into active nucleus pulposus cells. P-PRP mainly increased the expression of anabolic genes and relative proteins, aggrecan (AGC), collagen types II (Col II), while L-PRP predominantly increased the expression of catabolic and inflammatory genes, matrix metalloproteinase-1 (MMP-1), MMP-13, interleukin-1 beta (IL-1β), IL-6, tumor necrosis factor alpha (TNF-α), and protein production of IL-1β and TNF-α.ConclusionsLeukocytes in PRP activate inflammatory and catabolic effects on NPSCs from early degenerated intervertebral discs. Hence, P-PRP may be a more suitable therapeutic strategy for early IDD.

Characteristics and potentials of stem cells derived from human degenerated nucleus pulposus: potential for regeneration of the intervertebral disc

BackgroundEliminating the symptoms during treatment of intervertebral disc degeneration (IVDD) is only a temporary solution that does not cure the underlying cause. A biological method to treat this disorder may be possible by the newly discovered nucleus pulposus derived stem cells (NPDCs). However, the uncertain characteristics and potential of NPDCs calls for a comprehensive study.MethodsIn the present study, nucleus pulposus samples were obtained from 5 patients with IVDD undergoing discectomy procedure and NPDCs were harvested using fluorescence activated cell sorting (FACS) by the co-expression of GD2+ and Tie2+. After in vitro expansion, the properties of NPDCs were compared with those of bone marrow mesenchyme stem cells (BMSCs) from the same subjects.ResultsNPDCs performed similar properties in cell colony-forming ability, cell proliferation rate, cell cycle and stem cell gene expression similar to those of BMSCs. In addition, NPDCs could be differentiated into osteoblasts, adipocytes, and chondrocytes, and are found to be superior in chondrogenesis but inferior in adipocyte differentiation.ConclusionsNPDCs derived from the degenerated intervertebral disc still keep the regeneration ability similar to BMSCs. Besides, the superior capacity in chondrogenesis may provide a promising cell candidate for cell-based regenerative medicine and tissue engineering in IVDD.

The characteristics of stem cells in human degenerative intervertebral disc.

Background:The aim of this study is to identify which possessed the best stem-cell-like characteristics in 3 kinds of cell in human degenerative intervertebral disc: NPSCs (nucleus pulposus-derived stem cells), AFSCs (annulus fibrosus-derived stem cells), or CESCs (cartilage endplate-derived stem cells).Methods:We separated the disc samples obtained from 15 surgically treated patients with disc degenerative diseases into nucleus pulposus, annulus fibrosus, and cartilage endplate. After cultivating, we used the cell counting kit-8 to analysis the cell activity of 3 kinds of disc tissue-derived stem cell separately; different stem cells were defined with multilineage (osteogenic, chondrogenic, and adipogenic) differentiation. We extracted the total RNA and detected the expression of different lineage differentiation-related genes with the real-time polymerase chain reaction (RT-PCR).Results:Cell morphology of NPSCs, AFSCs, and CESCs did not show significant difference. Cell proliferation capacity of NPSCs and AFSCs was stronger than that of CESCs. The differentiation outcomes showed that osteocyte-like cells were stained red by Alizarin red S, chondrocyte-like cells blue by toluidine blue, and adipocyte-like red by oil red O. The RT-PCR reflected that the expression of different lineage differentiation-related genes of AFSCs was stronger than NPSCs and CESCs.Conclusion:In conclusion, we found that the cell morphology was not significantly different among NPSCs, AFSCs, and CESCs. Both differentiation and RT-PCR tests demonstrated that AFSCs had the best stem-cell-like characteristics in the human degenerative intervertebral disc.

The comparison of biological characteristics of three kinds of stem cells in human degenerative intervertebral disc

Objective To compare the biological characteristics of nucleus pulposus-derived stem cells (NPSCs), annulus fibrosus-derived stem cells (AFSCs) and cartilage endplate-derived stem cells (CESCs) from the same patient who had degenerative disc. Methods The human discs isolated from 7 patients with intervertebral disc herniation were separated carefully and digested and filtered before getting the original NPSCs, AFSCs and CESCs. When cultivated to third passage, the cell activity of stem cells was analyzed by using cell counting kit-8 (CCK-8); multilineage (adipogenic, osteogenic and chondrogenic) differentiation potential was analyzed to define different stem cells. The total RNA of third-passage-cells was extracted, and then reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the relative expression of adipogenic, osteogenic and chondrogenic differentiation-related genes. Results Cell morphology of NPSCs, AFSCs and CESCs from the same patient showed no significant difference. The growth curve showed that proliferative ability of CESCs (A value at day 1, 3, 5, 7, 9, 11, 13, 15 was 0.35±0.01, 0.52±0.02, 0.64±0.08, 0.80±0.19, 0.79±0.2, 1.06±0.11, 1.37±0.09, 1.31±0.41) and AFSCs (0.38±0.01, 0.50±0.03, 0.72±0.06, 0.82±0.13, 0.87±0.08, 0.87±0.09, 1.20±0.05, 1.43±0.05)was more active than that of NPSCs (0.32±0.02, 0.42±0.04, 0.59±0.01, 0.64±0.17, 0.65±0.19, 0.68±0.21, 0.67±0.12, 0.59±0.12). Multilineage differentiation results showed adipocyte-like red by oil red O, osteocyte-like cells were stained red by Alizarin red, and chondrocyte-like cells blue by toluidine blue. The RT-PCR results showed that AFSCs was better than NPSCs and CESCs in the aspect of the expression of adipogenic [the relative expression of peroxisome proliferator-activated receptor gamma (PPARγ) gene was 6.85±0.32 vs. 6.63±0.50 and 7.21±0.21; the relative expression of lipoprotein lipase (LPL) gene was 13.72±0.37 vs. 8.41±0.42 and 10.62±0.37] and chondrogenic [the relative expression of type 2 collagen (COL-2) gene was 10.45±1.10 vs. 3.53±0.62 and 8.26±0.49; the relative expression of sex determining region Y-box 9(SOX-9) gene was 14.39±1.22 vs. 7.66±0.68 and 13.70±1.10] differentiation-related genes, whereas the expression of osteogenic differentiation-related genes was best in CESCs[the relative expression of runt related transcription factor-2(RUNX-2) gene was 7.30±1.1 vs. 2.61±0.06 and 6.20±0.92; the relative expression of COL-2 gene was 8.22±0.84 vs. 7.05±0.82 and 4.94±0.90] and the expression of adipogentic differentiation-related genes, particularly LPL gene was worse in NPSCs. Conclusion The cell morphology was similar among NPSCs, AFSCs and CESCs in vitro, but biological characteristics of those were different. Induced differentiation and RT-PCR results both showed that AFSCs had more advantages than NPSCs and CESCs in the field of intervertebral disc tissue engineering. Key words: Intervertebral disc degeneration; Nucleus pulposus stem cells; Annulus fibrosus stem cells; Cartilaginous endplate stem cells; Biological characteristics

BMP7-Based Functionalized Self-Assembling Peptides Protect Nucleus Pulposus-Derived Stem Cells From Apoptosis In Vitro.

Tissue engineering has shown great success in the treatment of intervertebral disk degeneration (IVDD) in the past decade. However, the adverse and harsh microenvironment associated in the intervertebral disks remains a great obstacle for the survival of transplanted cells. Although increasing numbers of new materials have been created or modified to overcome this hurdle, a new effective strategy of biological therapy is still required. In this study, bone morphogenic protein 7 (BMP7)-based functionalized self-assembling peptides were developed by conjugating a bioactive motif from BMP-7 (RKPS) onto the C-terminal of the peptide RADARADARADARADA (RADA16-I) at a ratio of 1:1 to form a new RADARKPS peptide. Human nucleus pulposus-derived stem cells (NPDCs) were cultured in the presence of RADA-RKPS or RADA16-I in an apoptosis-promoting environment that was induced by tumor necrosis factor-alpha, and cells were cultured with RADA16-I in normal medium that served as the control group. After 48 h of apoptosis induction, the viability, proliferation, apoptosis rate, and expression of apoptosis-related genes of NPDCs in the different groups were evaluated, and the differentiation of NPDCs toward nucleus pulposus-like cells was tested. The results showed that the RADA-RKPS peptide could significantly protect the survival and proliferation of NPDCs. In addition, the application of RADA-RKPS decreased the rate of cell apoptosis, as detected by TUNEL-positive staining. Furthermore, our in vitro study confirmed the apoptosis-protecting effects of RADA-RKPS peptides, which significantly reduced the BAX/BCL-2 ratio of NPDCs and upregulated the gene expression of collagen II a1, aggrecan, and Sox-9 after 48 h of apoptosis induction. Collectively, these lines of evidence suggest that RADA-RKPS peptides confer a protective effect to NPDCs in an apoptosis environment, suggesting their potential application in the development of new biological treatment strategies for IVDD.

Distinguishing characteristics of stem cells derived from different anatomical regions of human degenerated intervertebral discs.

Several types of stem cells have been successfully demonstrated to exist in the human degenerated intervertebral disc (IVD), which is composed of annulus fibrosus (AF), nucleus pulposus (NP) and cartilage endplate (CEP). However, the differences in the biological characteristics among these and bone marrow derived mesenchymal stem cells (BM-MSCs) remain unclear. To investigate this issue, cells were harvested from human AF, NP, CEP, and bone marrow, respectively; passage 2 cells were selected using the agarose suspension culture system to obtain stem cell clones. Following expansion in vitro, stem cells from different anatomical regions were compared regarding the morphology, proliferation ability, immunophenotypic expression, and multi-lineage differentiation capacity. In addition, stem cell-alginate bead compositions were constructed for the comparison of DNA and sGAG content. There were subtle differences regarding cell morphology, but no significant differences in proliferation ability among the four types of stem cells. For the immunophenotypic analysis, all stem cells basically fulfilled the criteria for mesenchymal stem cells (MSCs), which have been published by the International Society for Cellular Therapy (ISCT), with a significant difference in CD105 expression. A comparison of the osteogenic capacities indicated: cartilage endplate-derived stem cells (CESCs)>annulus fibrosus-derived stem cells (AFSCs)>BM-MSCs>nucleus pulposus-derived stem cells (NPSCs). The chondrogenesis difference was similar to osteogenesis. For adipogenesis: BM-MSCs>NPSCs>CESCs>AFSCs. In the stem cell/alginate composition, the CESCs consistently showed the superior chondrogenic potential among all those cell types. Our data indicated that all the four types of stem cells shared some similar biological properties (regarding shape, proliferation ability and immunophenotypic expression). CESCs, which had the strongest osteogenic and chondrogenic potentials, may serve as excellent seed cells for NP/cartilage or bone tissue engineering.

A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc degeneration in a rabbit model

IntroductionIn recent years, nucleus pulposus cell (NPC) transplantation has been used to treat intervertebral disc degeneration (IDD); however, the degenerative nature of NPCs influences its effectiveness. Nucleus pulposus-derived stem cells (NPSCs), which are self-renewing, have high expansion potential and can adapt to the intervertebral disc (IVD) microenvironment and may have a better regenerative capacity, which is favourable for treating IDD. The aim of this study was to compare the effectiveness of transplantation with NPSCs and NPCs on the regeneration of the IVD in rabbit models. MethodsNPSCs and NPCs were isolated from human degenerate nucleus pulposus tissue by differential adhesion method, and stem cell surface markers were detected by flow cytometry. Degenerative discs in rabbits were randomly distributed into three groups: NPSCs, NPCs and vehicle control group; the normal discs served as the normal control group. Cells of the P3 generation were prepared for transplantation. About 20 μl of cell suspension (NPSCs or NPCs) or DMEM was injected into corresponding discs, while the normal discs were left untreated. After 8 weeks, disc height was evaluated using X-ray, water content was evaluated by MRI, and gene and protein expression levels of collagen II and aggrecan in the nucleus were determined by real-time PCR and ELISA. ResultsNPCs and NPSCs from the P3 generation were polygonal and spindle-shaped, respectively. Both NPSCs and NPCs strongly expressed surface markers CD73, CD90, and CD105 and weakly expressed CD34 and CD45. The relative rates of expression of CD73, CD90, and CD105 were higher in NPSCs than in NPCs. After 8 weeks, X-ray results showed no significant difference in disc height index among the groups (p > 0.05). MRI revealed that the intensity of the nucleus pulposus signal was increased in NPSCs (p < 0.05). The results from PCR and ELISA demonstrated that NPSCs promoted gene and protein expression of aggrecan instead of collagen II (p < 0.05). ConclusionCompared to NPCs, NPSCs harvested by differential adhesion method displayed a higher positive rate of stem cell surface markers and showed superior regenerative effectiveness for treating IDD in rabbit models. Therefore, NPSCs are potential candidates for cell therapy for the regeneration of the IVD.

Matrix stiffness determines the fate of nucleus pulposus–derived stem cells

Intervertebral disc (IVD) degeneration and consequent low-back pain present a major medical challenge. Nucleus pulposus–derived stem cells (NP–SCs) may lead to a novel therapy for this severe disease. It was recently shown that survival and function of mature NP cells are regulated in part by tissue stiffness. We hypothesized that modification of matrix stiffness will influence the ability of cultured NP-SCs to proliferate, survive, and differentiate into mature NP cells. NP-SCs were subcultured in three-dimensional matrices of varying degrees of stiffness as measured by the material's shear storage modulus. Cell survival, activity, and rate of differentiation toward the chondrogenic or osteogenic lineage were analyzed. NP-SCs were found to proliferate and differentiate in all matrices, irrespective of matrix stiffness. However, matrices with a low shear storage modulus (G′ = 1 kPa) promoted significantly more proliferation and chondrogenic differentiation, whereas matrices with a high modulus (G′ = 2 kPa) promoted osteogenic differentiation. Imaging performed via confocal and scanning electron microscopes validated cell survival and highlighted stiffness-dependent cell-matrix interactions. These results underscore the effect of the matrix modulus on the fate of NP-SCs. This research may facilitate elucidation of the complex cross-talk between NP-SCs and their surrounding matrix in healthy as well as pathological conditions.

Utilization of Stem Cells in Alginate for Nucleus Pulposus Tissue Engineering

Introduction In a general view of anatomy, intervertebral disc is composed of three parts: annulus fibrosus, nucleus pulposus (NP), and cartilage endplate. Recently, several types of stem cells were successfully isolated from these corresponding regions, but up to now, no research was performed about which kind of stem cells is the most efficient candidate for NP tissue engineering or for stem cell-based disc regeneration therapy. Materials and Methods In this study, we compared the regenerative potentials of the above-mentioned three kinds of disc-derived stem cells with that of the classic bone marrow (BM)-mesenchymal stem cells (MSCs) in a rabbit disc degeneration model. By magnetic resonance imaging (MRI), X-ray, histology, and so on. Results We found that cartilage endplate-derived stem cells (CESCs) showed superior capacity compared with the annulus fibrosus-derived stem cells (AFSCs), nucleus pulposus-derived stem cells (NPSCs), and BM-MSCs ( p&lt; 0.05); in addition, when comparing the CESC group with the normal control group, there existed no statistical difference in X-ray ( p&gt;0.05). Those results demonstrated that the CESC-seeded alginate construct performed the most powerful ability for NP regeneration, while AFSCs showed the most inferior potency, NPSCs and BM-MSCs had similar regenerative capacity and located in the middle. Conclusion Our study showed that CESCs might act as an efficient seed cell source for NP tissue engineering, which paved a new way for the biological solution of disc degeneration diseases. Disclosure of Interest None declared

Utilization of Stem Cells in Alginate for Nucleus Pulposus Tissue Engineering

In a general view of anatomy, intervertebral disc is composed of three parts: annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP). Recently, several types of stem cells were successfully isolated from these corresponding regions, but up to now, no research was performed about which kind of stem cells is the most efficient candidate for NP tissue engineering or for stem cell-based disc regeneration therapy. In this study, we compared the regenerative potentials of the above-mentioned three kinds of disc-derived stem cells with that of the classic bone marrow (BM)-mesenchymal stem cells (MSCs) in a rabbit disc degeneration model. By magnetic resonance imaging (MRI), X-ray, histology, etc. evaluations, we found that cartilage endplate-derived stem cells (CESCs) showed superior capacity compared with the annulus fibrosus-derived stem cells (AFSCs), nucleus pulposus-derived stem cells (NPSCs), and BM-MSCs (p<0.05); additionally, when comparing the CESC group with the normal control group, there existed no statistical difference in X-ray (p>0.05). Those results demonstrated that the CESC-seeded alginate construct performed the most powerful ability for NP regeneration, while AFSCs showed the most inferior potency, NPSCs and BM-MSCs had similar regenerative capacity and located in the middle. All in all, our study showed that CESCs might act as an efficient seed cell source for NP tissue engineering, which paved a new way for the biological solution of disc degeneration diseases.