Protocol for the Differentiation of Mouse Fibroblasts into Neural Stem Cells

Neural stem cells (NSCs) play a unique role in basic research and clinical application. Cell therapy based on NSC transplantation is a promising tool for treating nervous system diseases, which are primarily investigated by preclinical studies and clinical trials that are challenged by the lack of acquiring NSCs in large enough quantities. Here, we provide a protocol for differentiating mouse fibroblasts into neural stem cells.

• Add 1 mL of 0.1 g/mL gelatin in a 35 mm Petri dish, and let it stand for 1 h at 37°C, 5% CO₂ incubator.
• Discard the gelatin that coated the dish 1 h ago, and the mouse fibroblasts are digested with 0.05% trypsin for 3 min and terminated with an equal volume of fibroblast culture solution. The cells are centrifuged at 200 g for 5 min at room temperature, resuspended with fibroblast culture medium, and inoculated at a density of 2×10⁵ per 35 mm dish in dishes in which the gelatin was discarded.
• 24 hours after inoculation, polybrene is added to the fibroblast culture medium at a final concentration of 4-8 μg/mL. 10 μL of each of the nine viruses is added to every 2.5 mL of the liquid, and mixed thoroughly.
• Discard the culture liquid in the fibroblast culture dish, add 2.5 mL of virus suspension to each dish, and leave for 24 h at 37°C in a 5% CO₂ incubator.
• Discard the virus suspension and add 2 mL of cell base medium per dish.
• After 24 h, the infection is repeated once to improve the infection efficiency.
• At the end of the second infection, virus-infected cells are digested and passaged, resuspended with NSCM, and inoculated at a density of 2×10⁵ per 35 mm dish in dishes pre-coated with PDL and laminin.
• NSCM is changed daily and observed. After the neural stem cell clones appear and grow to a sufficient size, the clones are picked with a mouth pipette and a glass tube into the four-well plates pre-coated with PDL and laminin for culture expansion, and one clone is placed in each well with the culture medium of NSCM. The cells are digested and passaged at a density of 2×10⁵ per 35 mm dish and observed.

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• Stem cells should be kept sterile to avoid contamination.
• The medium is changed regularly to ensure the supply of nutrients and the elimination of wastes.

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