TITLE: Generation of Myelinating Oligodendrocytes from Pluripotent Stem Cells
AUTHORS: Melanie Gil, Vivian Gama

[DESCRIPTION]
This document contains two separate differentiation protocols, one to generate oligodendrocyte progenitor cells (OPCs) and the other to generate motor neurons. For co-culture, both protocols must be done simultaneously to have cells ready at the same time. Starting the motor neuron differentiation protocol at day 38 of the OPC protocol is recommended. Alternatively, motor neurons can be frozen at day 14 and thawed to prepare for the co-culture, which occurs on day 27 of the motor differentiation. Do not maintain OPCs in PDGF media for more than two weeks after sorting as they have the propensity to differentiate into astrocytes.

 


[BEFORE_START]
This document contains two separate differentiation protocols, one to generate oligodendrocyte progenitor cells (OPCs) and the other to generate motor neurons. For co-culture, both protocols must be done simultaneously to have cells ready at the same time. Starting the motor neuron differentiation protocol at day 38 of the OPC protocol is recommended. Alternatively, motor neurons can be frozen at day 14 and thawed to prepare for the co-culture, which occurs on day 27 of the motor differentiation. Do not maintain OPCs in PDGF media for more than two weeks after sorting as they have the propensity to differentiate into astrocytes.

[GUIDELINES]
Differentiation to OLIG2+ progenitors (Timing 12 d)
This is a step-by-step protocol that was adapted from Douvaras and Fossati, 2015(PMID: 26134954), with a few modifications. 

Generating motor neurons for co-culture (Timing 27 d)
Adapted from Ziller and Ortega, 2018 (PMID: 29551301) with a few modifications from Gabriella Robertson (Gama lab).

Setting up and maintaining co-culture (Timing 20 d)
Co-culture protocol was adapted from García-León et al., 2018 (PMID: 29337119). 


 


[STEPS]
SECTION: Day -3: Replating hESCs
1. When the cells reach 70–90% confluency, remove the medium and add 1 mL of accutase.

SECTION: Day -3: Replating hESCs
2. Incubate the plate in a 37 °C incubator for 5 min.

SECTION: Day -3: Replating hESCs
8. Plate 8 × 104 cells per well on a Matrigel-coated six-well plate with mTeSR1 supplemented with 10 micromolar (µM)Y27632.

SECTION: Day -3: Replating hESCs
7. Resuspend the cell pellet in 1 mL of mTeSR1 containing 10 micromolar (µM) Y27632 and count the cells with a hemocytometer.

SECTION: Day -3: Replating hESCs
6. Centrifuge the cells at  200 x g, 4 min.

SECTION: Day -3: Replating hESCs
5. Transfer the cells in a conical tube.

SECTION: Day -3: Replating hESCs
4. 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.

SECTION: Day -3: Replating hESCs
3. Dilute the accutase solution by adding 2 mL of DMEM/F12 medium.

SECTION: Day -2: Maintaining hESCs
9. Aspirate and add fresh mTeSR1 media to remove Y27632.

SECTION: Day -1: Maintaining hESCs
10. Add fresh mTeSR1 media.

SECTION: Day 0: Neural induction
11. By now colonies should have grown evenly and reached 80% confluency. When cells have reached this point, proceed with differentiation. 

SECTION: Day 0: Neural induction
12. Aspirate medium and add NIM to induce differentiation.

SECTION: Day 0: Neural induction
13. Change NIM daily until cells have reached day 8.

SECTION: Day 8: Oligodendrocyte lineage induction
14. Aspirate medium and add N2 medium to start directing cells to oligodendrocyte lineage commitment. Change media daily until cells reach day 12.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
15. Prepare low-attachment plate:
SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
17. Use a cell lifter and place it perpendicular to the bottom of the well.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
16. Remove the old medium from differentiation plate and add 1 mL of fresh N2B27 medium per well of a six-well plate.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
15.1. Rinse wells of a 6-well plate with anti-adherence solution (StemCell Tech Cat: 07010).

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
15.2. Add N2B27 medium to a total of 3 mL in each well.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
15.3. Put plate aside until aggregates are ready.
SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
21. Use the same cell lifter to detach the remaining adherent cells by scraping the whole well.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
20. Turn the well 45° and repeat Step 17.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
19. Turn the well 90° and repeat Step 17.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
18. 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.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
23. Feeding:

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
22. 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.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
23.1. Transfer the medium containing the cell aggregates to a 15-ml conical tube and wait for 3 min–5 min for the aggregates to sink to the bottom of the tube.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
23.2. Aspirate two-thirds of the medium and replenish it with fresh N2B27 medium.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
23.4. 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.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
24. On day 20, transfer the aggregates to a 15-ml conical tube and wait for 3 min for the aggregates to sink to the bottom of the tube.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
25. Remove two-thirds of the medium and replenish it with PDGF medium.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
26. Gently pipette five times up and down with a p1000 pipette.

 

SECTION: OPC differentiation in adherent cultures (Timing 35 d)
31. Every other day, carefully replenish two-thirds of the medium with fresh PDGF medium until day 65 of differentiation.

SECTION: OPC differentiation in adherent cultures (Timing 35 d)
31.1. 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.

SECTION: Preparing Cells for MACS
32. Remove media from cells and add with 1 mL of accutase per well of a 6-wellplate. Incubate for 20 min at 37 °C.

SECTION: Preparing Cells for MACS
33. Check under the scope to see if cells have lifted off plate. Incubate for another 5 min-10 min at 37 °C if needed.

SECTION: Preparing Cells for MACS
35. Spin down at 200 x g, 4 min .

SECTION: Preparing Cells for MACS
34. Dilute Accutase with 2 mL of DMEM with 10 micromolar (µM) Y27632 and place in a 15mL conical.

SECTION: Preparing Cells for MACS
39. Gentle pipette up and down

SECTION: Preparing Cells for MACS
38. Incubate for an additional 5 min.

SECTION: Preparing Cells for MACS
37. Gentle pipette up and down with a P1000 to break up the clumps.

SECTION: Preparing Cells for MACS
36. Aspirate media and resuspend in 4 mL of DMEM with 10 micromolar (µM) Y27632 and 100 1527 DNase and incubate for 5 min at Room temperature .

SECTION: Preparing Cells for MACS
41. Spin down at 200 x g, 4 min .

SECTION: Preparing Cells for MACS
40. Run cells through a cell strainer.

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

SECTION: Preparing Cells for MACS
42. Aspirate media and resuspend in 60 µL of cold 0.5% BSA.

SECTION: Magnetic labeling
43. 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.

SECTION: Magnetic labeling
47. Aspirate supernatant completely and resuspend in 1 mL of 0.5% BSA.

SECTION: Magnetic labeling
46. Wash cells by adding 2 mL of 0.5% BSA and spin down at 200 x g, 4 min.

SECTION: Magnetic labeling
45. Add 40 µL of the Anti-A2B5 MicroBeads per 10⁷ total cells and incubate for15 min in the refrigerator.

SECTION: Magnetic separation
48. Place column in the magnetic field of a suitable MACS® Separator.

SECTION: Magnetic separation
49. Prepare column by rinsing with 500 µL of 0.5% BSA.

SECTION: Magnetic separation
54. Pipette 1 mL of 0.5% BSA with 10 micromolar (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.

SECTION: Magnetic separation
53. Remove column from the separator and place it on a suitable collection tube.

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

SECTION: Magnetic separation
51. Collect flow-through containing unlabeled cells in a 2mL or 5mL tube.

SECTION: Magnetic separation
50. Apply cell suspension onto the column. 

SECTION: Magnetic separation
57. Plate labeled and unlabeled cells and in imaging dish to check if any cells of interest remain.

SECTION: Magnetic separation
56. Alternatively, to maintain OPCs, count cells and plate accordingly on poly-l-ornithine and laminin plates. Keep cells in 10 micromolar (µM)Y27632 for 960 min. Continue to maintain OPCs in PDGF media.

 

SECTION: Magnetic separation
55. 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"
SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
27. 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.

SECTION: Day 30: Selection and plating of OLIG-2-enriched aggregates (Timing 1–4 h)
28. Have a poly-l-ornithine and laminin 6-well plate ready to go. 

 

SECTION: Day 30: Selection and plating of OLIG-2-enriched aggregates (Timing 1–4 h)
29. Add 3 mL of PDGF medium per well.

SECTION: Day 30: Selection and plating of OLIG-2-enriched aggregates (Timing 1–4 h)
30. 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.

SECTION: Magnetic labeling
44. Mix the cell suspension well and incubate for 10 min in the refrigerator.

SECTION: Day 12: Cell detachment and formation of OLIG2-enriched aggregates (Timing 18 d)
23.3. Gently pipette five times up and down with a p1000 pipette.

 

SECTION: Day -2: Replating hiPCS
58. When the cells reach 70–90% confluency, remove the medium and add 1 mL of accutase.
SECTION: Day -2: Replating hiPCS
59. Incubate the plate in a 37 °C incubator for 5 min.
SECTION: Day -2: Replating hiPCS
63. Centrifuge the cells at 200 x g, 4 min.
SECTION: Day -2: Replating hiPCS
62. Transfer the cells in a conical tube.
SECTION: Day -2: Replating hiPCS
61. 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.

SECTION: Day -2: Replating hiPCS
60. Dilute the accutase solution by adding 2 mL of DMEM/F12 medium.
SECTION: Day -2: Replating hiPCS
64. Resuspend the cell pellet in 1 mL of E8 containing 10 micromolar (µM) Y27632 and count the cells with a hemocytometer.

SECTION: Day -2: Replating hiPCS
65. Plate 7×105 cells per well on a Matrigel-coated six-well plate with E8 supplemented with 10 micromolar (µM) Y27632.

SECTION: Day -1: Maintaining hiPCS
66. Aspirate and add fresh E8 media to remove Y27632.

SECTION: Day 0: Neural induction
67. By now colonies should have grown evenly and reached 80-90% confluency. When cells have reached this point, proceed with differentiation. 

SECTION: Day 0: Neural induction
68. Aspirate medium and add MNIM + SB4, LDN, RA, SAG to induce differentiation.

SECTION: Day 0: Neural induction
69. Change MNIM + SB4, LDN, RA, SAG daily until cells have reached day 5.

SECTION: Day 6: Directed differentiation
70. Aspirate medium and add MNIM + SU, DAPT, RA, SAG to direct cells to motor neuron lineage.

SECTION: Day 6: Directed differentiation
71. Change MNIM + SU, DAPT, RA, SAG daily until cells have reached day 13.

SECTION: Day 14: Motor neuron maintenance
72. At this stage, cells can be frozen for later use. 

SECTION: Day 14: Motor neuron maintenance
73. To replate cells, remove the medium and add 1 mL of accutase.

SECTION: Day 14: Motor neuron maintenance
74. Incubate the plate in a 37 °C incubator for 10 min.

SECTION: Day 14: Motor neuron maintenance
75. Dilute the accutase solution by adding 2 mLof DMEM/F12 medium.

SECTION: Day 14: Motor neuron maintenance
76. 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.

SECTION: Day 14: Motor neuron maintenance
77. Transfer the cells in a conical tube.

SECTION: Day 14: Motor neuron maintenance
78. Centrifuge the cells at 200 x g, 4 min.

SECTION: Day 14: Motor neuron maintenance
79. Resuspend the cell pellet in 1 mL of MNMM containing 10 micromolar (µM)Y27632 and count the cells with a hemocytometer.

SECTION: Day 14: Motor neuron maintenance
80. Plate 1 × 106 cells per well on a poly-l-ornithine and laminin six-well plate with MNMM supplemented with 10 micromolar (µM) Y27632.

 

SECTION: Day 15: Motor neuron maintenance
81. Remove and add fresh MNMM to remove Y27632. Continue to feed cells with MNMM every two days until day 22.

SECTION: Day 23: Replate motor neurons for co-culture
82. To replate cells, remove the medium and add 1 mLof accutase.

SECTION: Day 23: Replate motor neurons for co-culture
83. Incubate the plate in a 37 °C incubator for 10 min.

SECTION: Day 23: Replate motor neurons for co-culture
84. Dilute the accutase solution by adding 2 mL of DMEM/F12 medium.

SECTION: Day 23: Replate motor neurons for co-culture
85. 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.

SECTION: Day 23: Replate motor neurons for co-culture
86. Transfer the cells in a conical tube.

SECTION: Day 23: Replate motor neurons for co-culture
87. Centrifuge the cells at 200 x g, 4 min.

SECTION: Day 23: Replate motor neurons for co-culture
88. Resuspend the cell pellet in 1 mL of MNMM containing 10 micromolar (µM) Y27632 and count the cells with a hemocytometer.

SECTION: Day 23: Replate motor neurons for co-culture
89. Plate 5 × 104 cells per well on a poly-l-ornithine and laminin 24-well glass bottom plate with MNMM supplemented with  10 micromolar (µM) Y27632.

 

SECTION: Day 24: Motor neuron maintenance
90. Remove and add fresh MNMM to remove Y27632.

SECTION: Day 26: Introducing co-culture media to motor neurons
91. 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.

SECTION: Day 0 of co-culture: Introducing OPCs to motor neurons
92. Use the OPC cell count obtained after sorting. 

SECTION: Day 0 of co-culture: Introducing OPCs to motor neurons
93. Prepare a cell solution in co-culture media with 10 micromolar (µM) Y27632 at a density of 1× 105 cells per mL. 

 

SECTION: Day 0 of co-culture: Introducing OPCs to motor neurons
94. Remove media from motor neurons and replenish with cell suspension (500 µL for a 24-well plate) from step 36.

SECTION: Day 1 of co-culture: Maintaining co-culture
95. Remove and add fresh co-culture media to remove Y27632. 

SECTION: Day 1 of co-culture: Maintaining co-culture
96. Continue to feed cells every other day with co-culture media until day 20 when they are ready for fixation.