The Effects Of Human Wharton’s Jelly-derived Mesenchymal Stem Cells Transplantation On The Degenerative Intervertebral Disc

Background and Purpose:Nowadays, the degenerative disc disease (DDD) is considered to be a leading cause of low back pain, which is one of the largest medical burdens for the health care system in the world. Current treatments of DDD, both conservative and invasive that are symptom relieving instead of addressing underlying problems-biological and structural deterioration of the NP, often have short-term solutions and lead to variety of complications. So, it is of great therapeutic significance to assess biological repair therapies by studying the disc regeneration based on tissue engineering or cell therapy. However, the lack of ideal seed cells remained a serious problem among these methods. Recently many researchers have focused on the Wharton’s jelly-derived mesenchymal stem cells (WJMSCs), which express mesenchymal stem cell surface markers, self-renew, and are multipotent in virto. WJMSC have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, nerve, tendon, muscle, and fat. WJSMCs were immunosuppressive and could be tolerated in allogeneic and xenogeneic transplantation. Furthermore, the harvest of WJMSCs was not an invasive procedure. WJMSCs had been considered as an ideal seed cells used in tissue engineering or cell-based therapies approaches. So, the objectives of this study were to the effects of the Wharton’s jelly-derived mesenchymal stem cells transplantation on the intervertebral disc using a canine disc degeneration model.Methods:Human umbilical cord was collected from full-term births after normal birth with informed consent from the patient and approval from the hospital for the use of the sample for research purpose. WJMSCs were taken for flow cytometric analysis (CD90/CD73/CD105/CD29/HAL-ABC/CD34/CD45and HLA-DR). The WJMSCs were then infected with recombinant adeno-associated virus vector2(rAAV2), which expressing the enhanced green fluorescent protein (EGFP) gene. The cells were infected with rAAV2-EGFP at multiplicity of infection (MOI)1×104、1×105、1×106v.g/cell. No infected cells were served as a negative control. After3days, GFP expression was confirmed using fluorescence microscope. The ability of proliferation was observed by CCK-8. Twenty skeletal mature beagle dogs (12months old,10kg) in total were used in this study. To induce disc degeneration, a needle was inserted at the center of the nucleus pulposus (NP). The NP was then aspirated using a10mL syringe. Four weeks after the operation, evaluation of radiologic of all animals and histologic analysis of two animals were performed. And the other eighteen animals were used to investigate the effects of Wharton’s jelly-derived mesenchymal stem cells transplantation on the degenerative intervertebral disc. L4-5of each canine was used as degenerative segments (group B), L5-6was injected with0.9%saline4weeks after puncture injury (group C), and L6-7was injected with EGFP-labbled WJMSCs4weeks after puncture injury (group D). And the L3-4intervertebral disc was as the control group (group A). The animals were followed for24weeks after the initial operation. Evaluation of radiologic, histologic, biomechanics and gene expression analysis were performed. Immunohistochemistry for aggrecan, types Ⅱ collagen, SOX-9together with hematoxylin and eosin and Safranin O staining was employed to investigate the matrix formation in the NP.Results:WJMSCs gradually spread out and were shown as fibroblast-like morphology after isolated from human umbilical cord. Flow cytometric analysis showed that the cells expressed CD90/CD73/CD105/CD29and HAL-ABC, but did not express CD34/CD455and HLA-DR. After3days of infected by rAAV2-EGFP, GFP expression was confirmed using fluorescence microscope. And the ratio of vector incorporation was more following the time of infection. CCK-8results showed that the ability of proliferation of WJMSCs didn’t decline in MOI of1×104、1×105(v.g/cell), but decline in MOI of1×106(v.g/cell). Four weeks after the first operation, MRI assays showed a significant decrease in the T2-weighted signal intensity in L4-5/L5-6/L6-7. And Safranin O staining showed a low aggrecan content in L4-5/L5-6/L6-7.20weeks after cell transplantation operation, GFP-positive WJMSCs were detected in the nucleus pulposus of group D. Disc height and T2-weighted signal intensity were well preserved in group D (P<0.05). The biomechanical testing showed that the cell transplantation restored better spinal segmental stability than the group B and group C (P<0.05). Safranin O staining showed a high aggrecan content in the group D. Group D displayed more immunopositive staining for SOX-9, type Ⅱ collagen and aggrecan in matrix than that in group B and C with the immunohistochemical analyses. Disc regeneration was also confirmed at the gene expression level in group D using real-time polymerase chain reaction. The relative gene expressions of matrix-related genes (aggrecan, type Ⅱ collagens and SOX-9) were significantly increased in group D compared with group B and C (P<0.05).Conclusions:We succeed in isolation mesenchymal stem cells from human umbilical cord Wharton’s jelly. WJMSCs could be infected by rAAV2-EGFP, and the best ratio of vector incorporation was1×103(v.g/cell). Puncture and aspirating could induce disc degeneration after4weeks. The transplanted human WJMSCs could survive in the canine degenerated intervertebral discs. The transplantation of WJMSCs into intervertebral discs increased the disc height, enhanced T2-weighted signal intensity, and promoted the disc matrix formation. Thus, the human WJMSCs may have value in the treatment of degenerated discs and serve as a new valuable resource in cell transplantation therapy for degenerative disc disease.