Generation of Induced Neural Stem Cells (iNSCs) from Peripheral Blood Mononuclear Cells (PBMNCs): A Sendai Virus-based Procedure to Reprogram PBMNCs into iNSCs

1. Reprogramming of PBMNCs to iNSCs by SeV Infection

1. Preparation of Solution and Culture Medium
   1. Prepare a poly-D-lysine (PDL) stock solution by dissolving 100 mg of PDL with 100 mL of H₂O to a concentration of 1 mg/mL. Store at -20 °C in 1 mL aliquots.
   2. Prepare an insulin stock solution by dissolving 100 mg of insulin in 20 mL of 0.01 N HCl to a concentration of 5 mg/mL. Store at -20 °C in 1 mL aliquots.
   3. To prepare 200 mL of iNSC basal medium, combine 96 mL of DMEM-F12 and 96 mL of basic medium (Table of Materials) with 2 mL of 100x glutamine stock solution, 2 mL of nonessential amino acid (NEAA), 2 mL of N2 supplement and 2 mL of B27 supplement. Add 10 ng/mL recombinant human leukemia inhibitory factor, 3 μM CHIR99021 and 2 μM SB431542 prior to use. Filter the medium and store it at 4 °C.
      NOTE: Use the medium within 2 weeks. Add recombinant human leukemia inhibitory factor, CHIR99021 and SB431542 immediately before use.
2. Reprogramming of PBMNCs to iNSCs by SeV Infection
   1. Ultraviolet-sterilize a clean bench prior to use. Sterilize all surfaces and equipment with 75% alcohol. Sterilize all tips using an autoclave.
   2. On day 0, collect the cells in MNC medium and transfer to a 15 mL conical tube. Centrifuge the cells at 200 x g for 5 min. Aspirate the supernatant and resuspend the cells with 1 mL of pre-warmed MNC medium.
   3. Count the viable cells with trypan blue. Resuspend the cells with pre-warmed (37 °C) MNC medium to a concentration of 2 x 10⁵ cells per well in 24-well plates.
   4. After removing the SeV tubes from -80 °C storage, thaw the tubes containing SeV in 37 °C water bath for 5–10 s, and then allow them to thaw at RT. Once thawed, place them on ice immediately.
   5. Add the SeV encoding human Klf4, Oct3/4, SOX2, and c-MyC to the wells, at a multiplicity of infection (MOI) of 10. Centrifuge cells with plates at 1,000 x g for 30 min to facilitate the attachment of cells. Leave the cells and supernatant in the plates. Place the plates in the incubator at 37 °C, 5% CO₂.
      CAUTION: All procedures involving SeV must be performed in a safety cabinet, and all tips and tubes should be treated with ethanol or bleach before disposal.
   6. On day 1, transfer the medium and cells to a 15 mL centrifuge tube. Rinse the well with 1 mL of MNC medium. Centrifuge the cell suspension at 200 x g for 5 min. Aspirate the supernatant and resuspend the cells with 500 μL of fresh pre-warmed MNC medium in 24-well plates.
      NOTE: Use a low attachment 24-well plate to prevent attachment of any cells before plating on PDL/laminin.
   7. On day 2, dilute 1 mL of 1 mg/mL PDL with 19 mL of D-PBS to a concentration of 50 μg/mL. Coat 6-well plates with 50 μg/mL PDL for at least 2 h at RT.
   8. Dilute 200 μL of 0.5 mg/mL laminin with 20 mL of D-PBS to a concentration of 5 µg/mL. Aspirate PDL in the 6-well plates, and dry on the vertical clean bench.
   9. Coat 6-well plates with 5 μg/mL laminin and incubate for 4–6 h at 37°C. Wash with D-PBS before use.
   10. On day 3, plate the transduced cells obtained in step 1.2.6 in iNSC medium on PDL/laminin-coated 6-well plates.   
       NOTE: Move the plates gently if needed after the cells are placed on PDL/laminin-coated plates, trying not to disturb the attachment of the cells.
   11. On day 5, add 1 mL of pre-warmed (37 °C) iNSC medium in each well in 6-well plates gently. 
       NOTE: It is expected that the cells will undergo drastic death (>60%).
   12. On day 7, add 1 mL of pre-warmed (37 °C) iNSC medium in each well in 6-well plates gently.
   13. From day 9 to day 28, replace spent medium with fresh pre-warmed (37 °C) iNSC medium every day. Monitor the emergence of iNSC colonies. Pick and transfer iNSC clones for expansion in about 2–3 weeks. Pick up colonies with appropriate morphology using burned glass pipettes, excluding any possibly contaminating cells, and aspirate the colonies with 200 μL tips.