A Co-culture Technique for Differentiating Human Neural Progenitor Cells into Neurons

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.