Generating Cortical Pyramidal Neurons from Human Neural Stem Cells

In a culture plate, coat a coverslip with polyornithine, a positively charged polymer, and incubate.

Polyornithine forms a mesh-like layer that helps negatively charged cells stick to the coverslip.

Remove the solution and wash the coverslips with a buffered salt solution to remove unbound polyornithine.

Add a solution containing an extracellular matrix protein called laminin and incubate.

Laminin forms a matrix over the polyornithine-coated coverslip, enhancing cell attachment.

Discard the solution and take a small number of neural stem cells suspended in a growth factor-free medium.

Add this low-density culture over the prepared coverslip in a slow rotating movement to evenly distribute the cells.

Initially, growth factor deprivation promotes cell division.

Over time, stem cells differentiate into neurons with long projections

Eventually, neurons produce spine-like structures over their projections, like the cortical pyramidal neurons.

To prepare neuronal cultures, place glass coverslips into six-well culture dishes, and add polyornithine. Incubate overnight under a flow hood. The following day, use DPBS to wash the coverslips three times and add laminin. Incubate under a flow hood for at least 10 hours.

Prepare culture medium containing the following components. Then, use the medium to plate and dispatch neural stem cells or NSCs at a density of 50,000 cells per square centimeter, pipetting with slow rotating movements in order to reduce cell clustering.