Knockout of B2M in combination with PD-L1 overexpression protects MSC-derived new islet β cells from graft rejection in the treatment of canine diabetes mellitus

The immunogenicity of allogeneic mesenchymal stem cells (MSCs) is significantly enhanced after transplantation or differentiation, and these cells can be recognized and cleared by recipient immune cells. Graft rejection has become a major obstacle to improving the therapeutic effect of allogeneic MSCs or, after their differentiation, transplantation in the treatment of diabetes and other diseases. Solving this problem is helpful for prolonging the time that cells play a role in the recipient body and for significantly improving the clinical therapeutic effect. In this study, canine adipose-derived mesenchymal stem cells (ADSCs) were used as seed cells, and gene editing technology was used to knock out the B2M gene in these cells and cooperate with the overexpression of the PD-L1 gene. Gene-edited ADSCs (GeADSCs), whose biological characteristics and safety are not different from those of normal canine ADSCs, have been obtained. The immunogenicity of GeADSCs is reduced, the immune escape ability of GeADSCs is enhanced, and GeADSCs can remain in the body for a longer time. Using the optimized induction program, the efficiency of the differentiation of GeADSCs into new islet β-cells was increased, and the maturity of the new islet β-cells was increased. The immunogenicity of new islet β-cells decreased, and their immune escape ability was enhanced after the cells were transplanted into diabetic dogs (the graft site was prevascularized by the implantation of a scaffold to form a vascularized pouch). The number of infiltrating immune cells and the content of immune factors were decreased at the graft site. New islet β-cell transplantation, which has low immunogenicity, can reverse diabetes in dogs, and the therapeutic effect of cell transplantation is significantly enhanced. This study provides a new method for prolonging the survival and functional time of cells in transplant recipients and significantly improving the clinical therapeutic effect.