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

To reprogram somatic cells into induced neural stem cells or iNSCs - stem cells possessing high self-renewal and differentiation capability, begin by culturing peripheral blood mononuclear cells in a serum-free medium to expand erythroblasts. 

Next, pipette the erythroblast suspension into a culture plate. Treat the cells with recombinant Sendai virus or SeV. These viruses encode reprogramming transcription factors. Centrifuge to sediment the cells and incubate.

Following SeV fusion to the host cell membrane, the virus genome undergoes transcription followed by translation in the cytoplasm. The ectopic expression of transcription factors triggers the cell reprogramming process without integrating the genes into the host genome. 

Now, centrifuge the culture to remove the viruses. Resuspend the transduced cells in a suitable media, followed by plating. Further, centrifuge to remove cellular debris. Resuspend the cells in iNSC media.

Subsequently, seed the cells in a protein-coated plate. The cells attach to the protein coating in the plate. Supplement the adhered cells with iNSC media on alternate days. After a week of culture, replace the spent media with fresh iNSC media daily. 

The growth factors and chemical constituents in the media maintain self-renewal and support pluripotency of a subset of transduced cells, reprogramming them completely to iNSCs. Over time, the cells begin to form iNSC colonies. Select the desired colonies for culturing. 

On day 0, collect the cells in MNC medium and transfer to a 15-milliliter conical tube. After centrifuging the cells at 200 x g for 5 minutes, aspirate the supernatant and resuspend the cells with 1 milliliter of pre-warmed MNC medium. Count the viable cells with Trypan Blue and then resuspend them with pre-warmed MNC medium to a concentration of 200,000 cells per well in 24-well plates.

Thaw the tubes removed from minus 80 degrees Celsius storage containing Sendai virus in a 37 degrees Celsius water bath for 5 to 10 seconds and then leave them to thaw at room temperature. Once thawed, place them immediately on ice. Add the thawed Sendai virus to the wells of the 24-well plate with MNCs at a multiplicity of infection of 10. To facilitate the attachment of cells, centrifuge the plates at 1,000 x g for 30 minutes, and after that, place the plates in the incubator at 37 degrees Celsius, 5% carbon dioxide.

On day 1, transfer the cells with the medium to a 15-milliliter centrifuge tube. To further detach the remaining cells, rinse the wells with 1 milliliter of MNC medium per well and add the cells with medium to the tube. After centrifuging this cell suspension at 200 x g for 5 minutes, aspirate the supernatant, resuspend the cells with 500 microliters of fresh pre-warmed MNC medium and add to a well of a 24-well plate.

On day 2, use 1 milliliter per well of previously diluted 50 micrograms per microliter poly-D-lysine and D-PBS to coat 6-well plates for at least 2 hours at room temperature. Aspirate poly-D-lysine from the 6-well plates and dry on the vertical clean bench for about 10 minutes. Then, add 1 milliliter per well of a previously diluted 5 micrograms per milliliter laminin to the 6-well plates and incubate for 4 to 6 hours at 37 degrees Celsius to coat. Wash the plates with D-PBS before use.

On day 3, plate all Sendai virus transduced MNCs in induced neural stem cell medium on the poly-D-lysine laminin coated 6-well plates. On days 5 and 7, gently add 1 milliliter of 37 degrees Celsius pre-warmed iNSC medium to each well of the 6-well plates.

From day 9 to day 28, replace the medium with fresh pre-warmed iNSC medium daily. Monitor the emergence of iNSC colonies, and in about 2 to 3 weeks, pick colonies with appropriate morphology using burned glass pipettes. Then, transfer each colony to a separate well of a 6-well plate for expansion.