This article describes a protocol for studying the maturation of oligodendrocyte precursor cells (OPCs) into oligodendrocytes and their ability to form myelin sheaths around dorsal root ganglion (DRG) neurons. The method involves the use of lentiviral vectors to express signaling proteins that regulate myelination.
Take adherent cultures of primary oligodendrocyte precursor cells, or OPCs, in a growth medium.
Add control lentiviral vectors to the control well and test vectors, encoding signaling proteins that regulate myelination, to the test well, then incubate.
The cells internalize the vectors, and the viral RNA is reverse-transcribed into DNA and incorporated into the host genome, expressing the signaling proteins in the test cells.
Introduce enzymes to detach the OPCs from the substrate.
Neutralize the enzymes and centrifuge to remove the enzymes.
Add the growth medium and transfer the OPCs onto coverslips inside a microplate with adhered dorsal root ganglion or DRG neurons. Allow the OPCs to settle.
Fill the wells with the growth medium and incubate. Interaction with the DRG neurons triggers OPC maturation into oligodendrocytes.
The expressed signaling proteins induce oligodendrocytes to form myelin sheaths around the DRG neurons.
Assess the signaling protein-induced myelin sheath formation in the test cells compared to the control vector-transduced cells.
For this protocol, have primary oligodendrocyte precursor cells, OPCs, cultured in 10-centimeter plates with 10 milliliters of SATO media containing the following growth factors — CNTF, PDGF, NT3, and Forskolin. The cells must have been cultured for 24 hours under 8% carbon dioxide.
Begin with aspirating the media and remove it completely. Then, replace it with 10 milliliters of the same media with the growth factors. Infect your OPCs with lentivirus, expressing the protein of interest by adding the required volume of virus dropwise into the media.
Also, in a separate OPC plate, mix the control lentivirus that expresses only GFP into the media. Culture the cells for another 48 hours, then collect the OPCs from the plates for seeding onto the cultured DRG neurons.
First, rinse each plate with 8 milliliters of EBSS twice. Make up 1 milliliter of trypsin-EDTA 0.05% with 9 milliliters of warm EBSS. Add 5 milliliters of the diluted trypsin to each plate. Incubate at 37 degrees Celsius for up to two minutes. Neutralize the trypsin by adding 5 milliliters of 30% FBS in EBSS.
Then, using a 10-milliliter pipette, wash the OPCs from the plate's surface. Turn the plate by a quarter-turn each time to collect all the cells. Transfer the cell suspension to a 15-milliliter tube. Centrifuge the cells at room temperature for 15 minutes at 180 g's.
Now, aspirate the supernatant and resuspend the cell pellet in a milliliter of warm SATO media without growth factors, and count the cells. Next, prepare the DRG neurons that are cultured on the 22-millimeter coverslips by completely aspirating the media from their plate.
Then, gently seed 200,000 OPCs, dropwise, to each coverslip. The seeding volume must be less than 200 microliters per coverslip. Let the cells settle without moving the plate. After 10 minutes, top each well with a milliliter of warm SATO media. Now replate the remaining sister OPCs with SATO media containing growth factors. They can be used later to verify expression of the protein of interest.
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