Generation of induced pluripotent stem cells from rat fibroblasts and optimization of its differentiation into mature functional neurons

Background: Induced pluripotent stem cells (iPSCs) derived neural stem cells (NSCs) provide a potential for autologous neural transplantation therapy following neurological insults. Thus far, in preclinical studies the donor iPSCs-NSCs are mostly of human or mouse origin with concerns centering around graft rejection when applied to rat brain injury models. For better survival and integration of transplanted cells in the injured brain in rat models, use of rat-iPSC-NSCs and in combination with biomaterials is of advantageous. Herein, we report a detailed method in generating rat iPSCs with improved reprogramming efficiency and differentiation into neurons. New method: Rat fibroblasts were reprogrammed into iPSCs with polybrene and EF1 alpha-STEMCCA-LoxP lentivirus vector. Pluripotency characterization, differentiation into neuronal linage cells were assessed with RT-qPCR, Western blotting, immunostaining and patch-clamp methods. Cells were cultured in a custom-designed integrin array system as well as in a hydrogel-based 3D condition. Results: We describe a thorough method for the generation of rat-iPSC-NSCs, and identify integrin alpha vl38 as a substrate for the optimal growth of rat-iPSC-NSCs. Furthermore, with hydrogel as the supporting biomaterial in the 3-D culture, when combined with integrin alpha vl38 binding peptide, it forms a conducive environment for optimal growth and differentiation of iPSC-NSCs into mature neurons. Comparison with existing methods: Published studies about rat-iPSC-NSCs are rare. This study provides a detailed protocol for the generation of rat iPSC-NSCs and optimal growth conditions for neuronal differentiation. Our method is useable for studies to assess the utility of rat iPSC-NSCs for neural transplantation in rat brain injury models.