Generation of Myelinating Oligodendrocytes from Pluripotent Stem Cells

When the cells reach 70–90% confluency, remove the medium and add 1mL of accutase.

Incubate the plate in a 37°C incubator for 0h 5m 0s.

Dilute the accutase solution by adding 2mL of DMEM/F12 medium.

Use a cell lifter to remove cells from well and gently pipette the mixture 2–5 times with the p1000 pipette to fully dissociate the hESC cell colonies to single cells.

Transfer the cells in a conical tube.

Centrifuge the cells at 200x g.

Resuspend the cell pellet in 1mL of mTeSR1 containing 10micromolar (µM) Y27632 and count the cells with a hemocytometer.

Plate 8 × 10⁴ cells per well on a Matrigel-coated six-well plate with mTeSR1 supplemented with 10micromolar (µM)Y27632.

Aspirate and add fresh mTeSR1 media to remove Y27632.

Add fresh mTeSR1 media.

By now colonies should have grown evenly and reached 80% confluency. When cells have reached this point, proceed with differentiation.

Aspirate medium and add NIM to induce differentiation.

Change NIM daily until cells have reached day 8.

Aspirate medium and add N2 medium to start directing cells to oligodendrocyte lineage commitment. Change media daily until cells reach day 12.

Prepare low-attachment plate:

Rinse wells of a 6-well plate with anti-adherence solution (StemCell Tech Cat: 07010).

Add N2B27 medium to a total of 3mL in each well.

Put plate aside until aggregates are ready.

Remove the old medium from differentiation plate and add 1mL of fresh N2B27 medium per well of a six-well plate.

Use a cell lifter and place it perpendicular to the bottom of the well.

Press the cell lifter against the bottom of the plate and create a cut to the cell layer. Create at least 20 such lines parallel to each other, to cover the whole well.

Turn the well 90° and repeat Step 17.

Turn the well 45° and repeat Step 17.

Use the same cell lifter to detach the remaining adherent cells by scraping the whole well.

With a p1000 pipette, gently pipette the clumps of cells 3–5 times, and transfer the contents of one well into two wells of an ultra-low-attachment six-well plate. Feed cells every 2 days.

Feeding:

Transfer the medium containing the cell aggregates to a 15-ml conical tube and wait for 0h 3m 0s–0h 5m 0s for the aggregates to sink to the bottom of the tube.

Aspirate two-thirds of the medium and replenish it with fresh N2B27 medium.

Gently pipette five times up and down with a p1000 pipette.

Return the aggregates to the same ultra-low-attachment plate and redistribute an approximately equal number of aggregates in each well with a p1000 pipette.

On day 20, transfer the aggregates to a 15-ml conical tube and wait for 0h 3m 0s for the aggregates to sink to the bottom of the tube.

Remove two-thirds of the medium and replenish it with PDGF medium.

Gently pipette five times up and down with a p1000 pipette.

Return the aggregates to the same ultra-low-attachment plate and redistribute an approximately equal number of aggregates in each well with the p1000 pipette. Feed every other day until day 30.

Have a poly-l-ornithine and laminin 6-well plate ready to go.

Add 3mL of PDGF medium per well.

Use a microscope under sterile conditions, and with a p200 pipette pick the aggregates that are round, that have a diameter of 300 - 800 µm, and that appear golden or brown with a dark center. Plate 20 spheres in a well of a six-well plate.

Every other day, carefully replenish two-thirds of the medium with fresh PDGF medium until day 65 of differentiation.

Be extremely gentle during medium changes. Do not tilt the plate to aspirate the old medium. The cell aggregates should be covered with liquid at all times.

• Gently aspirate the old medium with a p1000 pipette, by placing the tip close to the wall, without disturbing the cells.
• Add fresh medium very slowly by aiming at the wall of the well.
• Avoid rough and sudden movements even when you transfer the plate from the incubator, especially after day 40, because the cells will detach from the plate, typically in the form of a sheet.

Remove media from cells and add with 1mL of accutase per well of a 6-wellplate. Incubate for 0h 20m 0s at 37°C.

Check under the scope to see if cells have lifted off plate. Incubate for another 0h 5m 0s-0h 10m 0s at 37°C if needed.

Dilute Accutase with 2mL of DMEM with 10micromolar (µM) Y27632 and place in a 15mL conical.

Spin down at 200x g .

Aspirate media and resuspend in 4mL of DMEM with 10micromolar (µM) Y27632 and 100 DNase and incubate for 0h 5m 0s at Room temperature .

Gentle pipette up and down with a P1000 to break up the clumps.

Incubate for an additional 0h 5m 0s.

Gentle pipette up and down

Run cells through a cell strainer.

• Make sure to rinse strainer with DMEM right before placing cells on it.

Spin down at 200x g .

Aspirate media and resuspend in 60µL of cold 0.5% BSA.

Work fast, use pre-cooled solutions and keep cells cold, at all times. This will prevent capping of antibodies on the cell surface and non-specific cell labeling.

Mix the cell suspension well and incubate for 0h 10m 0s in the refrigerator.

Add 40µL of the Anti-A2B5 MicroBeads per 10^⁷ total cells and incubate for0h 15m 0s in the refrigerator.

Wash cells by adding 2mL of 0.5% BSA and spin down at 200x g.

Aspirate supernatant completely and resuspend in 1mL of 0.5% BSA.

Place column in the magnetic field of a suitable MACS® Separator.

Prepare column by rinsing with 500µL of 0.5% BSA.

Apply cell suspension onto the column.

Collect flow-through containing unlabeled cells in a 2mL or 5mL tube.

Wash column with 1mL of 0.5% BSA 3 times. Collect unlabeled cells that pass through and combine with the flow-through from step 55.

Remove column from the separator and place it on a suitable collection tube.

Pipette 1mL of 0.5% BSA with 10micromolar (µM) Y27632 onto the column. Immediately flush out the magnetically labeled cells by firmly and SLOWLY pushing the plunger into the column. These are sorted OPCs.

At this stage OPCs are ready for co-culture. Perform cell count. Please see the accompanying protocol section: "Day 0 of co-culture: Introducing OPCs to motor neurons"

Alternatively, to maintain OPCs, count cells and plate accordingly on poly-l-ornithine and laminin plates. Keep cells in 10micromolar (µM)Y27632 for 16h 0m 0s. Continue to maintain OPCs in PDGF media.

Plate labeled and unlabeled cells and in imaging dish to check if any cells of interest remain.

When the cells reach 70–90% confluency, remove the medium and add 1mL of accutase.

Incubate the plate in a 37°C incubator for 0h 5m 0s.

Dilute the accutase solution by adding 2mL of DMEM/F12 medium.

Use a cell lifter to remove cells from well and gently pipette the mixture 2–5 times with the p1000 pipette to fully dissociate the hiPCS cell colonies to single cells.

Transfer the cells in a conical tube.

Centrifuge the cells at 200x g.

Resuspend the cell pellet in 1mL of E8 containing 10micromolar (µM) Y27632 and count the cells with a hemocytometer.

Plate 7×10⁵ cells per well on a Matrigel-coated six-well plate with E8 supplemented with 10micromolar (µM) Y27632.

Aspirate and add fresh E8 media to remove Y27632.

By now colonies should have grown evenly and reached 80-90% confluency. When cells have reached this point, proceed with differentiation.

Aspirate medium and add MNIM + SB4, LDN, RA, SAG to induce differentiation.

Change MNIM + SB4, LDN, RA, SAG daily until cells have reached day 5.

Aspirate medium and add MNIM + SU, DAPT, RA, SAG to direct cells to motor neuron lineage.

Change MNIM + SU, DAPT, RA, SAG daily until cells have reached day 13.

At this stage, cells can be frozen for later use.

To replate cells, remove the medium and add 1mL of accutase.

Incubate the plate in a 37°C incubator for 0h 10m 0s.

Dilute the accutase solution by adding 2mLof DMEM/F12 medium.

Use a cell lifter to remove cells from well and gently pipette the mixture 2–5 times with the p1000 pipette to fully dissociate the motor neurons.

Transfer the cells in a conical tube.

Centrifuge the cells at 200x g.

Resuspend the cell pellet in 1mL of MNMM containing 10micromolar (µM)Y27632 and count the cells with a hemocytometer.

Plate 1 × 10⁶cells per well on a poly-l-ornithine and laminin six-well plate with MNMM supplemented with 10micromolar (µM) Y27632.

Remove and add fresh MNMM to remove Y27632. Continue to feed cells with MNMM every two days until day 22.

To replate cells, remove the medium and add 1mLof accutase.

Incubate the plate in a 37°C incubator for 0h 10m 0s.

Dilute the accutase solution by adding 2mL of DMEM/F12 medium.

Use a cell lifter to remove cells from well and gently pipette the mixture 2–5 times with the p1000 pipette to fully dissociate the motor neurons.

Transfer the cells in a conical tube.

Centrifuge the cells at 200x g.

Resuspend the cell pellet in 1mL of MNMM containing 10micromolar (µM) Y27632 and count the cells with a hemocytometer.

Plate 5 × 10⁴ cells per well on a poly-l-ornithine and laminin 24-well glass bottom plate with MNMM supplemented with 10micromolar (µM) Y27632.

Remove and add fresh MNMM to remove Y27632.

Remove and add fresh media mixture composed of 50% MNMM and 50% co-culture media. The following day, the cell will be ready for co-culture.

Use the OPC cell count obtained after sorting.

Prepare a cell solution in co-culture media with 10micromolar (µM) Y27632 at a density of 1× 10⁵cells per mL.

Remove media from motor neurons and replenish with cell suspension (500µL for a 24-well plate) from step 36.

Remove and add fresh co-culture media to remove Y27632.

Continue to feed cells every other day with co-culture media until day 20 when they are ready for fixation.