A Co-culture Technique for Differentiating Human Neural Progenitor Cells into Neurons
Transkript
After generating induced pluripotent stem cells or iPSCs, induce and maintain the cells as a human neural progenitor cell or NPC culture according to the text protocol. In this example, the NPCs are labeled using the lentiviral vector Synapsin1-tdTomato. The day after adding primary neurons to an astrocyte culture, use 1X PBS to wash human NPCs once. Then, add cell detachment solution and incubate for five minutes at 37 degrees Celsius.
Ensure that all the cells have detached, then, transfer them into a 15-milliliter tube containing twice the amount of DMEM/F12 and centrifuge at 200 g for five minutes. Remove the supernatant and use neuronal differentiation medium to gently resuspend the pellet into single cells. Use a hemocytometer to count the cells.
Next, carefully aspirate the medium from the astrocyte cortical neuron cultures. Plate the human NPCs at 5,000 cells per square centimeter per well in neuronal differentiation medium. Incubate at 37 degrees Celsius and 5% CO2 and replace the medium every two or three days for at least 28 days.
To confirm whether human iPSCs behave like mature neurons, use a confocal microscope equipped with a 60x water immersion lens. To find cells differentiated from human iPSCs by visualizing tdTomato.
