Differentiation of induced pluripotent stem cells for future olfactory repair using an indirect co-culture technique

This study was to investigate the differentiation potential of iPSCs into olfactory receptor neurons and mitral/tufted cells in vitro. We extracted mouse embryonic fibroblast and prepared feeding layer, where mouse iPSCs were inoculated on; RT-PCR were used to identify iPSCs pluripotency genes Oct4, Nanog and Sox2. Then, we separated the olfactory epithelium and olfactory bulb from mice, which contained respectively olfactory receptor neurons and mitral/tufted cells, co-cultured iPSCs with olfactory epithelium cell and olfactory bulb, and identified differentiated cells with the olfactory receptor neurons markers (OMP, GAP43, NCAM), mitral/tufted cells markers (TBX21,Iba1) after 14 days co-culturing by immunofluorescence and RT-PCR. We successfully established a stable culture system of mouse iPSCs and RT-PCR showed that pluripotency genes (Oct4, Nanog, Sox2) were expressed in mouse iPSCs. Immunocytochemical analysis or RT-PCR results indicated that the differentiated iPSCs can express olfactory receptor neurons markers (OMP, GAP43, NCAM) and mitral/tufted cells markers (TBX21, Iba1) after being co-culture with olfactory epithelium or olfactory bulb. We conclude that Mouse iPSCs can be differentiated into olfactory receptor neuron-like cells and mitral/tufted-like cells in vitro.