ABSTRACT: Human clinical trials in type 1 diabetes (T1D) patients are underway using mesenchymal stem cells (MSC) without prior validation in a mouse model for the disease. In response to this void, we characterized bone marrow-derived murine MSC for their ability to modulate immune responses in the context of T1D, as represented in non-obese diabetic (NOD) mice. In comparison to NOD-, BALB/c-MSC express higher levels of the negative costimulatory molecule PD-L1 and promote a shift toward Th2-like responses in treated NOD mice. In addition, transfer of MSC from resistant strains (i.e. NOR or BALB/c), but not from NOD mice, conferred disease protection when administered to prediabetic NOD mice. The number of BALB/c-MSC trafficking to the pancreatic lymph nodes of NOD mice was higher than in NOD mice provided autologous NOD-MSC. Administration of BALB/c-MSC resulted in reversal of hyperglycemia in 90% of NOD mice (p=0.002). Transfer of autologous NOD-MSC imparted no such therapeutic benefit, and in fact soft tissue and visceral tumors were uniquely observed in this setting (i.e. no tumors were present with BALB/c- or NOR-MSC transfer). These data provide important preclinical data supporting the basis for further development of allogeneic MSC-based therapies for T1D and potentially, other autoimmune disorders. To generate MSC, BM mononuclear cells were isolated from the femurs and tibiae of at least 5 mice in order to minimize cell variability. Cells are seeded in flasks at a concentration of 10x106/25 cm2 in M10 medium (DMEM medium [Cambrex, East Rutherford, New Jersey] containing 10% fetal calf serum [HyClone, Logan, Utah], 1% penicillin-streptomycin, and 1% L-glutamine [both from Cambrex]). To examine MSC in an inflammatory setting, 7.5x105 NOD- or BALB/c-MSC/well were cultured for 48h in 6-well plates in M10 medium containing 10 ng/ml recombinant murine IL-1beta (Peprotech, Rocky Hill, NJ). To ensure that our cultured cells had multipotent potential, we tested MSC P4 cultures for their ability to undergo differentiation into chondrocytes, osteocytes, and adipocytes as previously published.10 Chondrogenic differentiation was induced by 50 ug/ml ascorbic acid and 1 ng/ml TGF?-1 (Peprotech), osteogenic differentiation was induced by 50 ug/ml ascorbic acid, 10 mM sodium alpha-glycerophosphate, and 10-8 M dexamethasone, and adipogenic differentiation was induced by 10-7 M dexamethasone and 6 ng/ml insulin. MSC were analyzed for expression at passage 4 (P4). We examined the gene expression profile of NOD- (samples #1 to 4) and BALB/c-MSC (samples #5 to 8), obtained from normoglycemic NOD mice and age-matched BALB/c mice. A striking concordance in the pattern of gene regulation across the entire screen was observed when BALB/c-MSC genes were compared to the NOD-MSC 4 different culture of NOD derived MSC were compared to 4 differente cell culture of Balb/c derived MSC, Balb/c cell coltures were considered reference samples.
