Take human induced pluripotent stem cell, or iPSC, colonies cultured on an extracellular matrix-coated plate.
The iPSCs are genetically modified to express transcription factors that induce motor neuron differentiation.
Remove the media and add enzymes to detach the colonies.
Mechanically dissociate the cells into a single-cell suspension and collect them.
Centrifuge and remove the supernatant.
Resuspend the cells in media containing an inhibitor that prevents cellular apoptosis.
Plate the cells on a matrix-coated dish.
Add fresh differentiation media containing the antibiotic doxycycline and small molecule inhibitors.
Doxycycline induces transcription factor expression, while the inhibitors block specific signaling pathways, promoting iPSC differentiation into motor neuron progenitors.
Add enzymes to detach the cells.
Collect the cells and mechanically dissociate them.
Centrifuge and discard the supernatant. Resuspend the cells in neuronal media.
Plate the cells on adhesion protein-coated microslides to promote cell adherence.
Growth factors in the media induce progenitor maturation into motor neurons.
