In-vitro generation of interleukin-10 secreting B-regulatory cells from donor adipose tissue derived mesenchymal stem cells and recipient peripheral blood mononuclear cells for potential cell therapy

Background: Interleukin-10 secreting B-cells are a major subset of B-regulatory cells (B-regs), commonly recognized as CD19(+)/38(hi)/24(hi)/IL10(+). They carry out immunomodulation by release of specific cytokines and/or cell-to-cell contact. We have generated B-regs in-vitro from donor adipose tissue derived mesenchymal stem cells (AD-MSC) and renal allograft recipient (RAR) peripheral blood mononuclear cells (PBMC) for potential cell therapy. Material and methods: Mononuclear cells separated by density gradient centrifugation from 50 ml anti-coagulated blood of 15-RAR and respective donors were analysed for baseline B-regs using appropriate antibodies. Equal amount (20 x 10(6) cells/ml) of stimulator (irradiated at 7.45 Gy/min for 10 min) and responder (non-irradiated) cells were co-cultured with in-vitro generated AD-MSC (1 x 10(6) cells/ml) in proliferation medium containing lipopolysaccharide from E. coli K12 strain at 37 degrees C with 5% CO2. Cells were harvested on day-7 and analyzed for viability, sterility, quantity, morphology and phenotyping. In-vitro generated B-reg levels were compared with baseline B-regs. Results: In-vitro generated B-reg count increased to 16.75% from baseline count of 3.35%. Conclusion: B-regs can be successfully generated in-vitro from donor AD-MSC and RAR PBMC for potential cell therapy.