Immunomodulatory properties of mesenchymal stem cells (MSC) are key components of their successful applications in clinical setting. However, treatments based on MSC immunomodulation need understanding of cell characteristics before cell transplantation. We used live-imaging to test the suitability of the MSC motility as a parameter for quick prediction of the immunomodulatory potential of human MSC in regulating the activity of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Bone marrow MSC, from various donors and in vitro passages, were cultured with or without stimulated PBMC. After seven days, immunomodulation was assessed by measuring PBMC proliferation, IgG production and cytokine secretion in MSC and PBMC monocultures and co-cultures, and results were correlated to MSC motility. In co-culture, we observed that MSC successfully inhibited PBMC activity, reducing PBMC proliferation and IgG production compared to PBMC monoculture. MSC modulated PBMC to reduce the secretion of TNFα and IL-10, increase IL-6, G-CSF and MCP-1, while GM-CSF was not affected. By live-imaging tracking of cell trajectories, we observed that fast moving MSC were inhibiting more efficiently stimulated PBMC compared to slow ones. In co-culture, fast MSC were more effective in inhibiting IgG production (˜30% less IgG), and secreted higher levels of IL-10 (˜10% increase) and GM-CSF (˜20% increase) compared to slower cells. Furthermore, fast MSC in monocultures produced 2.3-fold more IL-6, 1.5-fold MCP-1 and 1.2-fold G-CSF in comparison to slower cells. In conclusion, live-imaging cell tracking allowed us to develop an indicative assay of the immune-regulatory potential of MSC prior to in vivo administration. Key Words: Human mesenchymal stem cells, Immunomodulatory potential, In vitro cell motility, Stem cell transplantation

Degeneration of the intervertebral disc (IVD) is a progressive and chronic process, and the high incidence of discogenic disorders calls for new therapeutic approaches, such as cell-based therapies using three dimensional cultures and mesenchymal stem cells (MSC), which can differentiate to chondrogenic-and IVD-lineages.Here, we investigated the growth and differentiation of human MSC culture on biodegradable collagen scaffolds in order to obtain an injectable suspension.Commercially available wound dressings were downsized to dimensions between 100 and 1500 μm and seeded with freshly isolated or early passages MSC.Proliferation rate and chondrogenic differentiation potential was tested at oxygenation levels of 2 %, 5 %, 10 % and 21 % in static and dynamic cultures.Evaluation methods included cell viability test, disc marker genes expression (aggrecan, collagen type I and type II), histological detection of proteoglycans and immunohistochemical analysis.On microcarriers, freshly isolated MSC had lower proliferation rate and chondrogenic differentiation potential compared with early passages MSC.Proliferation of MSC was significantly increased 1.7-fold at 5 % oxygen level and in combination with dynamic culture was further increased to 2.3-fold, with respect to normoxia.Chondrogenesis was positively affected by 2 % and 5 % hypoxia, as shown by increased transcription levels and protein expression of collagen type II and proteoglycan accumulation in static cultures, while it was inhibited in dynamic cultures.Collagen type I and aggrecan expression were not affected by hypoxia.In conclusion, collagen based microcarriers are a suitable support for in vitro MSC growth and chondrogenesis especially when cultured at 5 % oxygen level.

Degeneration of intervertebral discs (IVDs) is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP) to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and post-traumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapy-monitoring markers for degenerative disc diseases.

There is evidence that mesenchymal stem cells (MSCs) can differentiate towards an intervertebral disc (IVD)-like phenotype. We compared the standard chondrogenic protocol using transforming growth factor beta-1 (TGFß) to the effects of hypoxia, growth and differentiation factor-5 (GDF5), and coculture with bovine nucleus pulposus cells (bNPC). The efficacy of molecules recently discovered as possible nucleus pulposus (NP) markers to differentiate between chondrogenic and IVD-like differentiation was evaluated. MSCs were isolated from human bone marrow and encapsulated in alginate beads. Beads were cultured in DMEM (control) supplemented with TGFß or GDF5 or under indirect coculture with bNPC. All groups were incubated at low (2 %) or normal (20 %) oxygen tension for 28 days. Hypoxia increased aggrecan and collagen II gene expression in all groups. The hypoxic GDF5 and TGFß groups demonstrated most increased aggrecan and collagen II mRNA levels and glycosaminoglycan accumulation. Collagen I and X were most up-regulated in the TGFß groups. From the NP markers, cytokeratin-19 was expressed to highest extent in the hypoxic GDF5 groups; lowest expression was observed in the TGFß group. Levels of forkhead box F1 were down-regulated by TGFß and up-regulated by coculture with bNPC. Carbonic anhydrase 12 was also down-regulated in the TGFß group and showed highest expression in the GDF5 group cocultured with bNPC under hypoxia. Trends in gene expression regulation were confirmed on the protein level using immunohistochemistry. We conclude that hypoxia and GDF5 may be suitable for directing MSCs towards the IVD-like phenotype.