This article describes a protocol for differentiating human neural progenitor cells (NPCs) into mature neurons using a co-culture system with mouse astrocytes and rat cortical neurons. The NPCs are engineered to express a fluorescent protein for visualization under a confocal microscope.
Take a culture of human neural progenitor cells, or NPCs, supported on an extracellular matrix.
The NPCs are engineered to express a fluorescent protein.
Discard the medium and wash the cells to remove any residual medium.
Incubate the culture with enzymes that disrupt the matrix, dissociating the cells.
Transfer the detached cells into a tube and add a medium to neutralize the enzymatic action.
Centrifuge to pellet the cells, discard the supernatant, and resuspend the NPCs in a neuronal differentiation medium.
Transfer the NPCs onto an established co-culture of mouse astrocytes and rat cortical neurons.
Growth factors released by the established co-culture, along with the cell-cell contacts of the NPCs with the cortical neurons and astrocytes, promote differentiation of the NPCs into mature neurons.
The mature neurons exhibit extended cellular processes and form synaptic connections.
Under a confocal microscope, use the signal from the fluorescent proteins to visualize the human NPC-derived neurons.
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.
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