The isolation, characterization and application of a population of highly immunosuppressive bone marrow-derived mesenchymal stromal cells in autoimmune disease

Abstract

Autoimmune disease is a debilitating disease caused by immune responses towards particular self-antigens whereby inflammation in the target organs leads to tissue damage, which is associated with serious morbidity and disability. An example of autoimmune disease is multiple sclerosis (MS), a chronic inflammatory disorder of the human central nervous system (CNS) in which lymphocytic infiltration into the CNS damages myelin and axons. MS patients generally have poor quality of life with patients suffering from recurrent acute neurological episodes with persistent symptoms or progressive development of the disease. To date, there is no cure for MS, with management of the disease dependent on immunomodulatory interventions to minimize disease relapses and retard disease progression. However, available therapies have poor efficacy and many are associated with serious side-effects. Recently, cell therapy has emerged as a promising treatment regime for various autoimmune diseases including MS. Mesenchymal stem/stromal cells (MSC), plastic adherent fibroblastic-like cells, have emerged as the promising cell therapy for the treatment of MS. In pre-clinical studies, MSC ameliorate disease in experimental autoimmune encephalomyelitis (EAE) with associated decreased in lymphocyte infiltration and demyelination in the CNS. Due to the lack of a consensus method for effective isolation of well-characterized mouse MSC, different MSC preparations have been used across different studies, making comparison of the data difficult. Moreover, the majority of these studies use poorly characterized MSC preparation lacking in tri-lineage differentiation capacity. Therefore, the true therapeutic effect of MSC for the treatment of MS has not been established. In this study, an improved methodology aimed at isolating a well-characterized MSC population from mouse BM has been developed. By preconditioning the donor mice with 5-FU, a cytotoxic drug that kills proliferating cells, to remove hematopoietic cells and combining flushing of the medullary cavity with scraping of the endosteum to remove additional BM cells, the isolation efficacy was significantly increased. Using MACS® separation column, CD11b-CD45- MSC was efficiently and consistently isolated from multiple mouse strains. Isolated MSC from 5-FU-treated mice (5-FU-MSC) displayed enhanced functional characteristics such as higher proliferation potential, tri-lineage differentiation, immunosuppression capacity and CD49d (α4 integrin) expression compared to untreated MSC. Importantly, the administration of 5-FU-MSC in an EAE model of MS completely remitted disease, associated with the reduction in lymphocytes infiltration into the CNS, demyelination, and reduction in immune response such as lower splenocyte proliferation to MOG and serum anti-MOG antibodies. The complete remission was accompanied by elevated anti-inflammatory Th2 cytokines (IL-4, IL-5 and IL-6) and decreased proinflammatory cytokine, IL-17, in MOG-stimulated splenocytes. Soluble factors, IL-1ra, IL-10 and PGE2, were found to be the mediators of 5-FU-MSC-induced immunosuppression. Together, these findings suggest that immunosuppression by 5-FU-MSC is mediated by a combination of elevated IL-1ra, IL-10, PGE2 and anti-inflammatory Th2 cytokines and decreased IL-17. These findings suggest that the improved isolation method developed in this study can firstly, be adopted by other researchers to isolate a well-characterised MSC from multiple mouse strains and secondly, the method can be modified, for example changing 5-FU treatment from in vivo to in vitro, to isolate a similar population of highly immunosuppressive human MSC for exploitation to remit autoimmune diseases.