Tomoko Nakai

Transplantation of mesenchymal stem cells embedded in Atelocollagen® gel to the intervertebral disc: a potential therapeutic model for disc degeneration

Intervertebral disc degeneration is considered to be one of the major causes of low back pain. Despite this irreversible phenomenon, attempts to decelerate disc degeneration using various techniques have been reported. However, to date there has been no proven technique effective for broad clinical application. Based on previous studies, we hypothesize that maintenance of proteoglycan content in the disc is achieved by avoiding the depletion of nucleus pulposus and preserving the structure of the annulus is a primary factor in decelerating disc degeneration. One novel approach to solve the dilemma of intervertebral disc degeneration is found at the stem cell level. Mesenchymal stem cells (MSCs) are known to possess the ability to differentiate into various kinds of cells from mesenchymal origin. Although the majority of cells that contribute to disc formation are known to obtain chondrocyte-like phenotypes, no reported study has emphasized the correlation with mesenchymal stem cells. To evaluate the possible potential of MSCs in disc cell research and treatment of degenerative disc disease, autologous MSCs embedded in Atelocollagen gel were transplanted into the discs of rabbits which had undergone a procedure proven to induce degeneration. The results suggest that MSC transplantation is effective in decelerating disc degeneration in experimental models and provided new hopes for treatment of degenerative disc disease in humans. Atelocollagen gel served as an important carrier of MSCs in transplantation, permitting proliferation, matrix synthesis and differentiation of MSCs. This study strengthens the viable efficacy of practical application of MSCs in treatment of intervertebral disc disease.

Differentiation of mesenchymal stem cells transplanted to a rabbit degenerative disc model : Potential and limitations for stem cell therapy in disc regeneration

Study Design. An in vivo study to assess the differentiation status of mesenchymal stem cells (MSCs) transplanted to the nucleus pulposus of degenerative discs in a rabbit model. Objectives. To evaluate the fate of MSCs transplanted to the nucleus pulposus of degenerative discs in a rabbit and to determine whether they are a suitable alternative for cell transplantation therapy for disc degeneration. Summary of Background Data. Although MSCs have been proposed as candidate donor cells for transplantation to treat intervertebral disc degeneration, their differentiation after transplantation has not been adequately investigated. Methods. Autologous MSCs, labeled with green fluorescent protein, were transplanted into mature rabbits. Consecutive counts of transplanted MSCs in the nucleus area were performed for 48 weeks after transplantation. Differentiation of transplanted cells was determined by immunohistochemical analysis. The proteoglycan content of discs was measured quantitatively using a dimethylmethylene blue assay, and mRNA expression of Type I and II collagen, aggrecan and versican was measured semi-quantitatively using reverse transcription polymerase chain reaction. Results. Many cells that were positive for green fluorescent protein were observed in the nucleus pulposus of cell-transplanted rabbit discs 2 weeks after transplantation. Their number increased significantly by 48 weeks. Some GFP-positive cells were positive for cell-associated matrix molecules, such as Type II collagen, keratan sulfate, chondroitin sulfate, aggrecan, and the nucleus pulposus phenotypic markers, hypoxia inducible factor 1 alpha, glutamine transporter 1, and matrix metalloproteinase 2. MSCs did not show significant expression of these molecules before transplantation. Biochemical and gene expression analyses showed significant restoration of total proteoglycan content and matrix-related genes compared with nontransplanted discs. Conclusions. MSCs transplanted to degenerative discs in rabbits proliferated and differentiated into cells expressing some of the major phenotypic characteristics of nucleus pulposus cells, suggesting that these MSCs may have undergone site-dependent differentiation. Further studies are needed to evaluate their functional role.

Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc

Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2+) and disialoganglioside 2 positive (GD2+), in the nucleus pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of nucleus pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2+ cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2+GD2+) can be induced from their precursor cells (Tie2+GD2-) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of nucleus pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard.

A phenotypic comparison of intervertebral disc and articular cartilage cells in the rat

The basic molecular characteristics of intervertebral disc cells are still poorly defined. This study compared the phenotypes of nucleus pulposus (NP), annulus fibrosus (AF) and articular cartilage (AC) cells using rat coccygeal discs and AC from both young and aged animals and a combination of microarray, real-time RT-PCR and immunohistochemistry. Microarray analysis identified 63 genes with at least a fivefold difference in fluorescence intensity between the NP and AF cells and 41 genes with a fivefold or greater difference comparing NP cells and articular chondrocytes. In young rats, the relative mRNA levels, assessed by real-time RT-PCR, of annexin A3, glypican 3 (gpc3), keratin 19 (k19) and pleiotrophin (ptn) were significantly higher in NP compared to AF and AC samples. Furthermore, vimentin (vim) mRNA was higher in NP versus AC, and expression levels of cartilage oligomeric matrix protein (comp) and matrix gla protein (mgp) were lower in NP versus AC. Higher NP levels of comp and mgp mRNA and higher AF levels of gpc3, k19, mgp and ptn mRNA were found in aged compared to young tissue. However, the large differences between NP and AC expression of gpc3 and k19 were obvious even in the aged animals. Furthermore, the differences in expression levels of gpc3 and k19 were also evident at the protein level, with intense immunostaining for both proteins in NP and non-existent immunoreaction in AF and AC. Future studies using different species are required to evaluate whether the expression of these molecules can be used to characterize NP cells and distinguish them from other chondrocyte-like cells.

Upregulation of the viability of nucleus pulposus cells by bone marrow-derived stromal cells. Significance of direct cell-to-cell contact in coculture system

Study Design. Upregulation of the viability of nucleus pulposus cells by coculture with bone marrow-derived stromal cells using a novel culture system. Objectives. The objective was to apply a novel coculture system having direct cell-to-cell contact between nucleus pulposus cells and bone marrow-derived stromal cells for stimulation of nucleus pulposus cells. Summary of Background Data. Reinsertion of nucleus pulposus cells was effective for treatment of intervertebral disc degeneration. However, obtaining highly viable nucleus pulposus cells was necessary to achieve successful results. Thus, an alternative method to upregulate the biologic and metabolic viabilities of nucleus pulposus cells was desired. Methods. Nucleus pulposus cells and bone marrow-derived stromal cells were isolated from New Zealand white rabbits. A 6-well culture plate and insert with track-etched membrane having 0.4 &mgr;m pores at the bottom were used for coculture. Nucleus pulposus cells were monocultured, cocultured conventionally (having no direct cell-to-cell contact) with bone marrow-derived stromal cells, or cocultured having direct cell-to-cell contact with bone marrow-derived stromal cells. On day 4 of coculture, nucleus pulposus cells were evaluated for proliferation using WST-8 assay, deoxyribonucleic acid synthesis by measuring [3H]-thymidine uptake, and proteoglycan synthesis by measuring [35S]-sulfate uptake. We also quantified cytokines in supernatants from the culture system. Results. Cell proliferation, deoxyribonucleic acid synthesis, and proteoglycan synthesis of nucleus pulposus cells were significantly upregulated in samples cocultured having direct cell-to-cell contact. Moreover, evaluations of supernatants revealed that growth factors associated with proliferation and cellular metabolism of nucleus pulposus cells were increased. Conclusions. Direct cell-to-cell contact in coculture system between nucleus pulposus cells and bone marrow-derived stromal cells accomplished significant upregulation in viability of nucleus pulposus cells.

Differential phenotype of intervertebral disc cells: microarray and immunohistochemical analysis of canine nucleus pulposus and anulus fibrosus.

Study Design. Microarray gene expression profiling, quantitative gene expression analysis, and immunohistochemistry was used to investigate molecular variations between nucleus pulposus (NP) and anulus fibrosus (AF) of the dog intervertebral disc (IVD). Objective. To identify specific molecules with differing expression patterns in NP and AF and compare their profile with articular cartilage (AC). Summary of Background Data. Although experimental and animal studies have demonstrated the potential of cell based approaches for NP regeneration, there is still a deficiency of basic knowledge about the phenotype of IVD cells. Methods. Comparative microarray analysis of beagle lumbar NP and AF was performed. Molecules of interest were evaluated by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, comparing lumbar and coccygeal NP and AF and AC. To assess interspecies variations, genes that had been found differentially expressed in rat tissues were also investigated. Results. Forty-five genes with NP/AF signal log ratio ≥1 were identified. &agr;-2-Macroglobulin, cytokeratin-18, and neural cell adhesion molecule (CD56) mRNA were higher in NP compared to AF and AC, and desmocollin-2 mRNA was higher in NP than AF. The expression profiles were similar in lumbar and coccygeal discs, although certain variations were noticed. Interspecies differences between rat and dog were evident in the expression of several genes. Immunohistochemistry confirmed differences in gene expression at the protein level. Conclusion. This study reports on the expression of molecules that have not been described previously in IVD, in non-notochordal discs comparable with human. Interspecies differences were noted between rat and dog tissues, whereas variations between caudal and lumbar discs were less prominent. The NP of the beagle as a chondrodystrophoid dog breed is potentially more similar to the human than the NP of species whose discs do not naturally degenerate. Therefore, studies on appropriate species may contribute to a better understanding of the cell types residing in the IVD.

Synergistic role of c-Myc and ERK1/2 in the mitogenic response to TGFβ-1 in cultured rat nucleus pulposus cells

IntroductionAlthough transforming growth factor β1 (TGFβ1) is known to be a potent inhibitor of proliferation in most cell types, it accelerates proliferation in certain mesenchymal cells, such as articular chondrocytes and nucleus pulposus cells. The low ability for self-renewal of nucleus pulposus cells is one obstacle in developing new therapeutic options for intervertebral disc diseases, and utilizing cytokines is one of the strategies to regulate nucleus pulposus cell proliferation. However, the precise cell cycle progression and molecular mechanisms by which TGFβ1 stimulates cell growth remain unclear. The aim of this study was to elucidate a mechanism that enables cell proliferation with TGFβ1 stimulation.MethodsWe tested cultured rat nucleus pulposus cells for proliferation and cell cycle distribution under exogenous TGFβ1 stimulation with and without putative pharmaceutical inhibitors. To understand the molecular mechanism, we evaluated the expression levels of key regulatory G1 phase proteins, c-Myc and the cyclin-dependent kinase inhibitors.ResultsWe found that TGFβ1 promoted proliferation and cell cycle progression while reducing expression of the cyclin-dependent kinase inhibitors p21 and p27, which are downregulators of the cell cycle. Robust c-Myc expression for 2 h and immediate phosphorylation of extra cellular signal regulated kinase (ERK1/2) were detected in cultures when TGFβ1 was added. However, pretreatment with 10058-F4 (an inhibitor of c-Myc transcriptional activity) or PD98059 (an inhibitor of ERK1/2) suppressed c-Myc expression and ERK1/2 phosphorylation, and inhibited cell cycle promotion by TGFβ1.ConclusionsOur experimental results indicate that TGFβ1 promotes cell proliferation and cell cycle progression in rat nucleus pulposus cells and that c-Myc and phosphorylated ERK1/2 play important roles in this mechanism. While the difference between rat and human disc tissues requires future studies using different species, investigation of distinct response in the rat model provides fundamental information to elucidate a specific regulatory pathway of TGFβ1.

CD146 defines commitment of cultured annulus fibrosus cells to express a contractile phenotype

Characterization of cells is important for facilitating cell‐based therapies for degenerative diseases of intervertebral discs. For this purpose, we analyzed mouse annulus fibrosus cells by flowcytometory to detect phenotypic change in their primary cultures. After examination of sixteen cell surface proteins, we focused on CD146 that solely increased during culture expansion. CD146 is known to be a marker for mesenchymal stem cells and for their vascular smooth muscle commitment with expression of contractile phenotype enhanced by SM22α. We sorted CD146+ cells to elucidate their characteristics and the key factors that play a role in this change. Whole cell cultures showed the ability for tripotent differentiation toward mesenchymal lineages, whereas sorted CD146+ cells did not. Expression of CD146 was elevated by addition of transforming growth factor β1, and sorted CD146+ cells expressed higher levels of mRNA for SM22α and Elastin than did CD146− cells. Morphologically, CD146+ cells more broadly deposited extracellular type I collagen than CD146− cells and showed filamentous actin bundles traversing their cytoplasm and cell–cell junctions. Moreover, CD146+ cells demonstrated significantly higher gel contraction properties than CD146− cells when they were embedded in collagen gels. Human annulus fibrosus CD146+ cells also showed higher contractility. Immunohistochemistry determined CD146+ cells localized to the outermost annulus layers of mouse intervertebral disc tissue with co‐expression of SM22α. These results suggest that increment of CD146 expression indicates gradual change of cultured annulus fibrosus cells to express a contractile phenotype and that transforming growth factor β1 enhances this cellular commitment.

Sciatic nerve regeneration by transplantation of in vitro differentiated nucleus pulposus progenitor cells

AIM To assess the applicability of mouse intervertebral disc-derived nucleus pulposus (NP) progenitor cells as a cell source for sciatic nerve regeneration. MATERIALS & METHODS P0-Cre/Floxed-EGFP-transgenic mouse-derived NP progenitor cells were differentiated to Schwann-like cells in conventional induction medium. Schwann-like cells were subsequently transplanted into a mouse model of sciatic nerve transection, and nerve regeneration assessed by immunohistochemistry, electron microscopy and functional walking track analysis and heat stimulus reflex. RESULTS & CONCLUSION NP progenitor cells differentiated into Schwann-like cells. Transplantation of these cells promoted myelinated axon formation, morphology restoration and nerve function improvement. NP progenitor cells have the capacity to differentiate into neuronal cells and are candidates for peripheral nerve regeneration therapy.

Upregulation of glycosaminoglycan synthesis by Neurotropin in nucleus pulposus cells via stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1: A new approach to attenuation of intervertebral disc degeneration

It is suggested that most cases of low back pain are related to degeneration of intervertebral discs. Disc degeneration is a chronic and progressive disease and the search for effective medical treatments continues. Neurotropin is widely used in Japan and China to treat low back pain and neck–shoulder–arm syndrome. The present study aimed to investigate the effect of Neurotropin on glycosaminoglycan synthesis in nucleus pulposus cells. Cultured human nucleus pulposus cells were treated with Neurotropin every second day for two weeks. Production of glycosaminoglycan was assessed using a dimethyl-methylene blue assay and PicoGreen was used to measure DNA content. Microarray analysis, real-time PCR, and western blotting were performed to assess the biological processes related to Neurotropin-stimulated glycosaminoglycan synthesis. The results showed that the level of glycosaminoglycan normalized to DNA content was significantly upregulated by the addition of Neurotropin. Gene expression profiling showed over two-fold upregulation of 697 genes in response to Neurotropin treatment. Among these genes, ontological analysis suggested significant implication of phosphatidylinositol 3-kinase signaling, and analysis focused on this pathway demonstrated marked upregulation of angiopoietin 1 and insulin-like growth factor 1. Activation of phosphorylation of the signal transducer protein AKT was detected by western blotting. Of the genes related to sulfated glycosaminoglycan synthesis, the greatest increase in mRNA levels was observed for chondroitin sulfate N-acetylgalactosaminyltransferase 1, an enzyme initiating synthesis of chondroitin sulfate side chains attached to a core protein of aggrecan, which is a predominant disc matrix component. These findings suggest that Neurotropin may activate the phosphatidylinositol 3-kinase–AKT pathway and stimulate glycosaminoglycan synthesis through upregulation of expression of mRNA for chondroitin sulfate N-acetylgalactosaminyltransferase 1. Because there was no cytotoxic cellular growth inhibition, Neurotropin treatment might offer an accessible therapeutic strategy for intervertebral disc degeneration.