Human induced pluripotent stem cells (iPSCs) are established by genetically reprogramming somatic cells to an embryonic-like, pluripotent state. These highly versatile cells can subsequently be differentiated into nearly any specialized cell type in the human body, providing an invaluable model for investigating developmental biology and disease pathways. Proper maintenance and neuronal differentiation of these cultures requires precise handling and high-quality reagents to ensure consistent cell viability and network formation.
Maintenance of iPSC line: Transduce KOLF2 iPSCs with an inducible NGN2 transgene. Maintain the NGN2 KOLF2 iPSCs in E8 basal medium using 6-well plates coated with vitronectin. Use !!! EDTA (HB5135) !!! to passage the cells.
Generation and Culture of NGN2 iNeurons (Day 0): Detach the NGN2 iPSCs utilizing accutase. Resuspend the cells to create a single cell suspension in E8 medium supplemented with 10 µM !!! ROCK inhibitor Y-27632 (HB2297) !!! and 1 µg/ml Doxycycline hyclate (HB4608). Plate the cell suspension at a density of 25,000 cells/cm2 on a 6-well plate previously coated with Geltrex.
Days 1 and 2: Completely exchange the culture media with freshly prepared dox-NIM A.
Day 3: Passage the iNeurons using accutase and resuspend them in dox-NIM B medium. Seed the neurons into a 96-well plate (pre-treated with 100 μg/ml PDL hydrobromide and coated with Geltrex) at a density of 10,000 cells/well.
Days 4 to 6: Execute a 75% media exchange daily using fresh dox-NIM B. Take extreme care to avoid mechanically disturbing the differentiating neurons during this process.
Day 8 onwards: Replace 50% of the media volume with fresh dox-NIM B every other day. This partial exchange method limits the neurons' exposure to air, which helps preserve cell attachment and culture quality.
Immunocytochemistry (Day 18): Fix the mature iNeurons by incubating in 4% formaldehyde. Wash the cells thoroughly using PBS. To block non-specific binding, incubate the samples in 10% donkey serum for 2 hours at room temperature. Next, incubate the cells for 2 hours at room temperature with the selected primary antibodies (e.g., Rat Anti-Tubulin) diluted in 1% donkey serum. Finally, perform a 2-hour room temperature incubation with the appropriate secondary and primary conjugate antibodies (e.g., PSD-95 Antibody, Anti-Synaptophysin) along with !!! DAPI (HB0747) !!!, all diluted in 1% donkey serum. Acquire and analyze your images.
References
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