Toru Iwashina

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.

Transplantation of mesenchymal stem cells in a canine disc degeneration model

Transplantation of mesenchymal stem cells (MSCs) is effective in decelerating disc degeneration in small animals; much remains unknown about this new therapy in larger animals or humans. Fas‐ligand (FasL), which is only found in tissues with isolated immune privilege, is expressed in IVDs, particularly in the nucleus pulposus (NP). Maintaining the FasL level is important for IVD function. This study evaluated whether MSC transplantation has an effect on the suppression of disc degeneration and preservation of immune privilege in a canine model of disc degeneration. Mature beagles were separated into a normal control group (NC), a MSC group, and the disc degeneration (nucleotomy‐only) group. In the MSC group, 4 weeks after nucleotomy, MSCs were transplanted into the degeneration‐induced discs. The animals were followed for 12 weeks after the initial operation. Subsequently, radiological, histological, biochemical, immunohistochemical, and RT‐PCR analyses were performed. MSC transplantation effectively led to the regeneration of degenerated discs. FACS and RT‐PCR analyses of MSCs before transplantation demonstrated that the MSCs expressed FasL at the genetic level, not at the protein level. GFP‐positive MSCs detected in the NP region 8 weeks after transplantation expressed FasL protein. The results of this study suggest that MSC transplantation may contribute to the maintenance of IVD immune privilege by the differentiation of transplanted MSCs into cells expressing FasL.

Human nucleus pulposus cells significantly enhanced biological properties in a coculture system with direct cell-to-cell contact with autologous mesenchymal stem cells

Activated nucleus pulposus (NP) cells can be reinserted into the disc to inhibit intervertebral disc degeneration. Experimental studies in animals showed that using a coculture system with direct cell‐to‐cell contact with mesenchymal stem cells (MSCs) significantly upregulated the biological activity of NP cells. The purpose of this study is to determine whether this activation of NP cells by autologous MSCs is applicable to human cells in vitro. Human NP tissue was obtained from surgical specimens and MSCs from bone marrow of 10 subjects. Six‐well culture plates and inserts were used for culture; 1.0 × 104 NP cells were seeded onto each insert and incubated alone, in standard coculture with 1.0 × 104 MSCs, or cocultured with direct cell‐to‐cell contact. NP cell proliferation, DNA synthesis, and proteoglycan (PG) synthesis were evaluated. Chromosome abnormalities in the activated NP cells and tumorigenesis of the cells were evaluated in an additional 10 patients by microscopic examination for segmented cells and histological assessment of activated cells transplanted into nude mice. Cell proliferation, DNA synthesis, and PG synthesis were significantly upregulated. The positive effects of the coculture system with direct cell‐to‐cell contact seen in animal studies were also confirmed in human cells. Chromosome abnormalities and tumorigenesis were not observed in the activated NP cells. In conclusion, a coculture system with direct cell‐to‐cell contact demonstrated a significant positive effect, enhancing the biological properties of human NP cells, as it did in animal models. These results should prove useful for conducting trials leading to the clinical use of activated NP cell transplantation.

Feasibility of using a human nucleus pulposus cell line as a cell source in cell transplantation therapy for intervertebral disc degeneration.

Study Design. Assessment of the potential use of an immortalized human nucleus pulposus cell line as an alternative cell source in cell transplantation therapy for intervertebral disc degeneration. Objectives. To evaluate the effect of transplanting the human nucleus pulposus cell line into a disc degeneration model in rabbits and to define whether it is capable of becoming an alternative cell source for cell transplantation therapy for disc degeneration. Summary of Background Data. Interest in cell transplantation therapy for disc degeneration has been growing for several years, and a range of different cell types have been examined as possible donor cells. In addition, the establishment of a novel cell line that possesses some of the major characteristics of a normal human nucleus pulposus cells has been reported. Methods. Human nucleus pulposus cell line was established, and cells were transplanted into a rabbit disc degeneration model. At 4, 8, and 24 weeks after transplantation, inhibition of intervertebral disc degeneration was assessed by examining the disc height, macroscopic appearance, histologic findings, and immunohistochemistry. In addition, aggrecan, versican, and Type II collagen gene expression in the nucleus pulposus were measured semiquantitatively at the mRNA level. Furthermore, the survival of transplanted cells was examined using immunohistochemistry for Simian Virus 40 T antigen, and the presence of graft-versus-host reaction was assessed by immunohistochemistry for CD4 and CD58. Results. The disc height was significantly greater in the transplanted group than in the degenerative group’s disc from 4 weeks’ posttransplantation. Macroscopically, the nucleus pulposus was absent and there was loss ofdisc height in the degenerative group at 24 weeks after transplantation, whereas the nucleus pulposus was preserved in the transplanted group. Histologic examination showed that the structure of the inner anulus fibrosus was significantly preserved in the transplanted group, and the boundary between the nucleus and anulus could be clearly visualized. Expression of mRNAs of the nucleus pulposus matrix, aggrecan, and Type II collagen was significantly greater in the transplanted group than in the degenerative group. This indicates that transplantation of human nucleus pulposus cell line helped to preserve the matrix of the nucleus pulposus. Thus, transplantation of a human nucleus pulposus cell line was shown to delay disc degeneration in this rabbit model. Conclusion. The human nucleus pulposus cell line may become an alternative cell source for cell transplantation therapy of intervertebral disc degeneration.

Upper cervical spinal cord tumors: review of 13 cases.

BackgroundClinical features of upper cervical spinal cord tumors are not clear because there have been too few published reports. The purpose of this study was to review the clinical features of these tumors.MethodsWe reviewed 13 patients who underwent surgery for an upper cervical spinal cord tumor. Data regarding age, sex, duration and type of symptoms, levels, topographical locations, surgical results, and histological features were investigated retrospectively.ResultsOf the 13 tumors, 5 were at C1/2, 7 at C2/3 and 1 at C1-3. Topographically, 9 of the 13 tumors were dumbbell shaped, and all 5 at C1/2 had this shape. The initial symptom was occipital pain in three patients, numbness or pain of the extremity in nine, and clumsiness of the upper extremity in one. The average duration from initial symptom to surgery was 14.9 months. There were no major surgical complications, but there were three cases of postoperative cervical kyphosis. These three patients underwent tumorectomy with total laminectomy of C2. Tumor recurrence was seen in three patients for reasons thought to be the same as tumors at other levels, being residual meningiomas lying ventral to the cord and extraforaminal neurinomas.Conclusions Spinal cord tumors in the upper cervical region tend to progress as dumbbell tumors. As all the neurinomas had this shape (Eden type 2 or 3) at C1/2, this anatomy might favor progression to the extradural and extraforaminal spaces. However, it also allows total removal of the tumor via a posterior approach. To maintain postoperative cervical alignment, the surgeon should select the least invasive approach to the paraspinal muscles attached to the spinous process of C2

Low-intensity pulsed ultrasound stimulation enhances TIMP-1 in nucleus pulposus cells and MCP-1 in macrophages in the rat.

Recent studies have reported that low‐intensity pulsed ultrasound (LIPUS) stimulates cell proliferation and proteoglycan production in rabbit intervertebral disc cells, and moreover promotes the secretion of MCP‐1 (monocyte chemotaxis protein‐1) from macrophages in a disc organ culture model. These findings suggest the possible application of LIPUS for biological repair of disc degeneration and herniation. Although the mechanisms involved are not well understood, several cytokine pathways may play a role. Therefore, in order to evaluate the effect of LIPUS stimulation on cytokine production by nucleus pulposus cells and macrophages, in vitro culture studies were designed. Nucleus pulposus cells and macrophages were collected from Sprague‐Dawley rats, cultured separately in a monolayer, and stimulated with LIPUS for 7 days. After culture, the culture medium and the cells were analyzed by cytokine array, RT‐PCR, and ELISA. Cytokine array showed that LIPUS stimulation significantly upregulated TIMP‐1 (tissue inhibitor of metalloproteinase‐1) in the nucleus pulposus and MCP‐1 in macrophages in comparison with the control. This was confirmed at the gene level by RT‐PCR in nucleus pulposus cells and macrophages after stimulation with LIPUS. Quantitative evaluation of these proteins by ELISA showed higher levels in nucleus pulposus cells and macrophages stimulated by LIPUS than in controls. These results showed that LIPUS stimulation significantly activated TIMP‐1 and MCP‐1 in nucleus pulposus cells and macrophages at both the protein and gene levels, suggesting that LIPUS may be a promising supplemental treatment for intervertebral disc herniation.

Age-Related Effects of Cocultured Rat Nucleus Pulposus Cells and Macrophages on Nitric Oxide Production and Cytokine Imbalance

Study Design. A study of age-related effects on nitric oxide (NO) and cytokine production in cocultured rat nucleus pulposus (NP) cells and macrophages. Objective. To evaluate the effects of age on NO and cytokine production in an in vitro model of cocultured NP cells and macrophages. Summary of Background Data. It is well known that the clinical characteristics of lumbar disc herniation differ with age. The relationship between age-related differences in clinical features and immuno-chemical factors, such as NO and inflammatory cytokines, has not been established. Methods. Male Sprague Dawley rats (n = 45), including 15 animals from 3 groups (3-, 12-, and 32-weeks old), were used. NP cells and exudated peritoneal macrophages were cocultured in serum-free media. NO levels were measured at 2-, 24-, 48-, and 72 hours using the Griess method. After 7 days of culture, the production of cytokines, including tissue inhibitor metalloproteinase-1, interferon-gamma (IFN-gamma), and interleukin-10 (IL-10) were evaluated. Results. NO levels of coculture increased with age. In the coculture groups, tissue inhibitor metalloproteinase-1 and IFN-gamma level of 3 weeks old were statistically higher than 12 and 32 weeks old. IL-10 level of 3 weeks old was statistically lower than 12 and 32 weeks old. Conclusion. NO levels of cocultures increased with age that suggests inflammatory reactions increase with age. This study showed an age-related cytokine imbalance, as represented by levels of IFN-gamma and IL-10. Stress and aging are thought to affect the extracellular matrix and change the immunologic response. Younger rat NP cells had higher cell-mediated immunity activity, while the older rat had higher humoral immunity activity. These results demonstrate that age affects the immunologic response attributable to NP cells. Further studies are needed to elucidate the mechanism of this newly observed occurrence and to apply these findings clinically.