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