Identification Of Bone Marrow-Derived Mesenchymal Stem Cells In Systemic Sclerosis

Objective:To establish a method for in vitro isolation and expansion of bone marrow-derived mesenchymal stem cells in systemic sclerosis, based on which to evaluate the biological properties and immunomodulation activity of SSc derived MSCs.Methods:MSCs were isolated and expanded from SSc and control bone marrow by density gradient centrifugation plus adhesive culture and were identified by morphology, phenotype, function and growth properties. Mixed lymphocyte reaction and lymphocyte transformation test were performed and expression of immune cytokines was determined with RT-PCR for evaluation of immunomodulation function of MSCs.Results:1. MSCs were successfully isolated and expanded from both SSc and control bone marrow, with an aging process significant after6passages, indicating a limit expansion potential.2. SSc and control MSCs displayed similar phenotype profile which was affected by culture conditions. MSCs expressed CD166, CD73, CD105, CD90, HLA-ABC but not CD34, CD45, CD31, CD83, CD80and HLA-DR. Phenotype assay confirmed a high purity of MSCs.3. Cell cycle assay demonstrated that over90%of SSc and control MSCs remained in G0/G1phase without significant difference between passages. 4. SSc derived MSCs can be differentiated into adipocytes and osteoblasts, with some patient inclined to osteogenic differentiation.5. Both SSc derived and control MSCs conditionally acted as stimuli to allogenic T cells, but inhibited mitogen-induced T cell proliferation in a dose-dependent manner.6. SSc derived MSCs presented an immune cytokines expression profile, with an observation of reduced expression of M-CSF and TGF-β1in some patients.Conclusions:1. MSCs were isolated and expanded from bone marrow of SSc patients and cultured over6passages with sternness unchanged.2. SSc derived MSCs possessed multiple differentiation potential while some cases inclined to osteogenic differentiation.3. SSc derived MSCs conditionally inhibited T cell proliferation in a dose-dependent manner.4. SSc derived MSCs showed an immune cytokines expression profile.