This study outlines a method for co-culturing human-induced neurons (iNs) with induced oligodendrocyte precursor cells (iOPCs) to investigate neuronal maturation and myelination. The process involves dissociating iNs from an extracellular matrix, followed by their integration with iOPCs to promote synaptic connections and myelin sheath formation.
Take human-induced neurons, or iNs, cultured on an extracellular matrix.
Remove the media and add digestive enzymes that disrupt the matrix, dissociating the cells.
Transfer the iNs into a tube. Centrifuge the suspension and discard the supernatant containing the enzymes.
Resuspend the iNs in a co-culture medium; transfer them onto a culture of induced oligodendrocyte precursor cells, or iOPCs.
iOPCs are precursors to oligodendrocytes that form myelin sheaths around the axons of neurons.
Upon incubation, the iOPCs form synaptic connections with the iNs. Trophic factors released by the iOPCs and growth factors in the medium promote iN maturation into neurons, exhibiting increased cellular processes that form synaptic connections with other neurons.
The electrical activity of the mature neurons and growth factors in the medium induces iOPC maturation into oligodendrocytes.
The oligodendrocytes extend cellular processes, forming myelin sheaths around the neuronal axons.
The established co-culture is ready for analysis.
On day 14, plate iOPCs at a density of 1 x 105 cells per well in a 24-well plate in OPC differentiation medium. On the next day, detach the induced human neurons with cell detachment solution. Add neurons onto the cultured OPCs, plating at the seating density of 2 x 105 cells per well. Use co-culture medium containing neurobasal A-medium, 2% B-27 Supplement, and 100 nanograms per milliliter T3 triiodothyronine. Change the medium on the next day, and then every other day. If OPCs proliferate too fast and reach confluency in less than three days, add 2 to 5 micromolar Ara-C.
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