Generation of Monkey Induced Pluripotent Stem Cell-Derived Cartilage Lacking Major Histocompatibility Complex Class I Molecules on the Cell Surface.

Due to the poor capacity for articular cartilage to regenerate, its damage tends to result in progressively degenerating conditions such as osteoarthritis. To repair the damage, the transplantation of allogeneic human induced pluripotent stem cell (iPSC)-derived cartilage is being considered. However, although allogeneic cartilage transplantation is effective, immunological reactions can occur. One hypothetical solution is to delete the expression of major histocompatibility complex (MHC) class I molecules to reduce the immunological reactions. For this purpose, we deleted the β2 microglobulin (B2M) gene in a cynomolgus monkey (crab-eating monkey [Macaca fascicularis]) iPS cells (cyiPSCs) to obtain cyiPSCs using the CRISPR/Cas9 system. Western blot analysis confirmed cyiPSCs lacked B2M protein, which is necessary for MHC class I molecules to be transported to and expressed on the cell surface by forming multimers with B2M. Flow cytometry analysis revealed no cyiPSCs expressed MHC class I molecules on their surface. The transplantation of cyiPSCs in immunodeficient mice resulted in teratoma that contained cartilage, indicating that the lack of MHC class I molecules on the cell surface affects neither the pluripotency nor the chondrogenic differentiation capacity of cyiPSCs. By modifying the chondrogenic differentiation protocol for human iPSCs, we succeeded at differentiating and cyiPSCs toward chondrocytes followed by cartilage formation , as indicated by histological analysis showing that and cyiPSC-derived cartilage were positively stained with safranin O and expressed type II collagen. Flow cytometry analysis confirmed that MHC class I molecules were not expressed on the cell surface of chondrocytes isolated from cyiPSC-derived cartilage. An mixed lymphocyte reaction assay showed that neither nor cyiPSC-derived cartilage cells stimulated the proliferation of allogeneic peripheral blood mononuclear cells. On the contrary, osteochondral defects in monkey knee joints that received allogeneic transplantations of cyiPSC-derived cartilage showed an accumulation of leukocytes with more natural killer cells around cyiPSC-derived cartilage than cartilage, suggesting complex mechanisms in the immune reaction of allogeneic cartilage transplanted in osteochondral defects . Impact statement The transplantation of allogeneic induced pluripotent stem cell (iPSC)-derived cartilage is expected to treat articular cartilage damage, although the effects of major histocompatibility complex (MHC) in immunological reactions have not been well studied. We succeeded at creating cynomolgus monkey (cy)iPSCs and cyiPSC-derived cartilage that lack MHC class I molecules on the cell surface. cyiPSC-derived cartilage cells did not stimulate the proliferation of allogeneic peripheral blood mononuclear cells . On the contrary, the transplantation of cyiPSC-derived cartilage into osteochondral defects in monkey knee joints resulted in survival of transplants and accumulation of leukocytes, including natural killer cells, suggesting complex mechanisms for the immune reaction.