Effect of cell number on mesenchymal stem cell transplantation in a canine disc degeneration model.

Transplantation of mesenchymal stem cells (MSCs) inhibits the progression of disc degeneration in animal models. We know of no study to determine the optimal number of cells to transplant into the degenerated intervertebral disc (IVD). To determine the optimal donor cell number for maximum benefit, we conducted an in vivo study using a canine disc degeneration model. Autologous MSCs were transplanted into degenerative discs at 10(5), 10(6), or 10(7) cells per disc. The MSC-transplanted discs were evaluated for 12 weeks using plain radiography, magnetic resonance imaging, and gross and microscopic evaluation. Preservation of the disc height, annular structure was seen in MSC-transplantation groups compared to the operated control group with no MSC transplantation. Result of the number of remaining transplanted MSCs, the survival rate of NP cells, and apoptosis of NP cells in transplanted discs showed both structural microenvironment and abundant extracellular matrix maintained in 10(6) MSCs transplanted disc, while less viable cells were detected in 10(5) MSCs transplanted and more apoptotic cells in 10(7) MSCs transplanted discs. The results of this study demonstrate that the number of cells transplanted affects the regenerative capability of MSC transplants in experimentally induced degenerating canine discs. It is suggested that maintenance of extracellular matrix by its production from transplanted cells and/or resident cells is important for checking the progression of structural disruption that leads to disc degeneration.

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; 1.0x10(4) NP cells were seeded onto each insert and incubated alone, in standard coculture with 1.0x10(4) 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.

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.