TITLE: Differentiation of iPSCs with the hNIL construct into motor neurons protocol
AUTHORS: Maria  Sckaff, Kenneth Wu, Hana  Ghanim, Aradhana  Sachdev, Gokul N Ramadoss, Carissa M. Feliciano, Luke M. Judge, Bruce  Conklin, Claire D Clelland

[DESCRIPTION]
This protocol describes the differentiation of induced pluripotent stem cells (iPSCs) into motor neurons using the hNIL transgenic factors in a CLYBL safe harbor site.

[GUIDELINES]
This protocol is based on hNIL differentiation protocol by Fernandopulle et al. from Michael Ward’s
lab1.

References

[1] Michael S Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E Ward. Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons. Curr Protoc Cell Biol. 2018 Jun;79(1): e51. doi: 10.1002/cpcb.51. Epub 2018 May 18. PMID: 29924488; PMCID: PMC6993937.

[2] Erika Lara Flores (2021). Maintenance protocol of iPSCs. Protocols.io

[3] Merissa Chen*, Nina Draeger*, Martin Kampmann*, Kun Leng*, Emmy Li*, Connor Ludwig*, Greg Mohl*, Avi Samelson*, Syd Sattler*, Ruilin Tian* (2019). Kampmann Lab iNeuron pre-differentiation & differentiation protocol. Protocols.io






[STEPS]
SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Maintaining iPSCs
1. Culture iPSCs for at least 2-3 passages after initial hNIL transfection or from frozen stocks before starting a motor neuron differentiation.
 

SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Maintaining iPSCs
2. Briefly, we maintain iPSCs on Matrigel-coated plastic culture dishes (Growth Factor Reduced Matrigel diluted in 50 mL of KnockOut DMEM to a concentration of 80 μg/ml) with mTeSR™ Plus medium.

SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Maintaining iPSCs
3. Change media every other day, and passage every 4-5 days with ReLeSR™ (see below).
SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Maintaining iPSCs
4. Add ROCK1 inhibitor at 10 micromolar (µM) in media to freshly passaged iPSCs for 1 day to limit spontaneous differentiation.

SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Passaging iPSCs
5. Passage iPSCs as clumps (for routine expansion of iPSC cultures) or single cells (for differentiation).
SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Passaging iPSCs
6. For routine expansion, wash the well with DPBS -Ca/-Mg (1-2 mL/well*) then add 1 mL per well* of ReLeSR™ for 1 min. 

SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Passaging iPSCs
7. Aspirate most of the ReLeSR™, but do not overdry, and incubate the wells at Room temperature for 3-4 minutes.

SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Passaging iPSCs
8. Add fresh mTeSR™ Plus media to the well (1 mL/well*) and pipette gently to detach the iPSCs and break colonies into small clumps.
 

SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Passaging iPSCs
9. A confluent well can be split 1:6 to 1:20 depending on the desired confluency and rate of growth of the iPSC line.
SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Passaging iPSCs
10. If desired, before replating, cells can be centrifuged at 800 rpm, 15 s to pellet clumps and remove any single cells (which remain in suspension).
 


SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Passaging iPSCs
11. For differentiation (or anytime cell counting is needed), wash the well with DPBS -Ca/-Mg (1-2 mL/well*) then add 0.5 mL* of Accutase®. 
 


SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Passaging iPSCs
12. Incubate the plate for 5 min at 37 °C.
SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Passaging iPSCs
13. After the incubation, tap the plate to fully detach the iPSCs, then add 0.5 mL* of KnockOut DMEM with 20% FBS (alternately, 2.5 mL/well* of DPBS +Ca/+Mg can be used in place of DMEM/FBS).
 

SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Passaging iPSCs
14. Transfer the cells and solution to a conical vial and centrifuge for 800 rpm, 3 min to pellet the cells.
SECTION: Maintenance and Preparation of the iPSCs for the hNIL differentiation into motor neurons: Passaging iPSCs
15. Aspirate the supernatant carefully as to not disturb the cell pellet, and resuspend the cells in mTeSR™ Plus with ROCK1 inhibitor at 10 micromolar (µM).
 


SECTION: hNIL differentiation into motor neurons: Day 0
16. Coat the receiving vessel with Matrigel at least 30 min before starting Day 0 (but no more than 24-36 hours prior to starting) and incubate at 37 °C.
SECTION: hNIL differentiation into motor neurons: Day 0
17. Bring the mTeSR™ Plus with ROCK1 inhibitor (final concentration of 10 micromolar (µM) in well, 1000X dilution from 10 millimolar (mM) stock) and the Accutase® to 37 Room temperature.

SECTION: hNIL differentiation into motor neurons: Day 0
18. Aspirate the spent media and wash the wells with cells once with 1X DPBS -Ca/-Mg.
SECTION: hNIL differentiation into motor neurons: Day 0
19. Add appropriate volume of Accutase® per well.
SECTION: hNIL differentiation into motor neurons: Day 0
20. Incubate the plate for 5 min at 37 °C.
SECTION: hNIL differentiation into motor neurons: Day 0
21. Remove plate from the incubator and tap the plate to release the cells.
SECTION: hNIL differentiation into motor neurons: Day 0
22. Quench the Accutase® using five times the volume DPBS +Ca/+Mg (if you used 0.5 mL of Accutase®, quench with 2.5 mL of DPBS).

SECTION: hNIL differentiation into motor neurons: Day 0
23. Transfer the cells to a conical vial.
SECTION: hNIL differentiation into motor neurons: Day 0
24. Centrifuge the vial at 800 rpm, 3 min.

SECTION: hNIL differentiation into motor neurons: Day 0
25. Aspirate the Accutase® with DPBS, being careful not to disturb the cell pellet at the bottom of the vial.
SECTION: hNIL differentiation into motor neurons: Day 0
26. Resuspend the cells with an appropriate volume of mTeSR™ Plus with ROCK1 inhibitor (final concentration of 10 micromolar (µM) in well, 1000X dilution from 10 millimolar (mM) stock). Aim for roughly 1 mL of mTeSR™ Plus with ROCK1 inhibitor per 1 million cells.

SECTION: hNIL differentiation into motor neurons: Day 0
27. Pipette up and down to mix well and produce an evenly distributed solution.
SECTION: hNIL differentiation into motor neurons: Day 0
28. Count the number of cells using a Countess II (any preferred equivalent way of counting cells is also appropriate).

 

SECTION: hNIL differentiation into motor neurons: Day 0
29. Aspirate the Matrigel from the receiving vessel.
SECTION: hNIL differentiation into motor neurons: Day 0
30. Add appropriate volume of media (the mTeSR™ Plus with ROCK1 inhibitor at 10 millimolar (mM)) to the receiving vessel.

SECTION: hNIL differentiation into motor neurons: Day 0
31. Add volume of cell mixture appropriate for the number of cells desired in the well. For a 10cm dish, we have found 1-2 million iPSCs at day 0 produces 3-5 million cells at day 3.
SECTION: hNIL differentiation into motor neurons: Day 0
32. Place the receiving vessels (which now has your cells) back in the incubator at 37 °C.

SECTION: hNIL differentiation into motor neurons: Day 0
33. Shake the plate in all four directions to ensure the cells are evenly distributed in the well.
SECTION: hNIL differentiation into motor neurons: Day 1
34. Prepare the neural induction medium (NIM) as follows:
 
      DMEM/F12  97 mL    N-2  1 mL    NEAA  1 mL    GlutaMAX  1 mL  
 
SECTION: hNIL differentiation into motor neurons: Day 1
35. Calculate the volume of freshly prepared NIM needed to perform a full media change (see Table 2) and aliquot in a vial of appropriate size. 
 
     Cell   culture vessel      Surface area (cm2)      Media volume per  well      Day 3   Seeding density for ICC applications      Day 3   Seeding density for protein, RNA,  DNA extraction        96-well plate      0.32      150 µL      20-25k cells      -        24-well plate      1.9      500 µL      80-100k cells      -        12-well plate      3.5      1 mL      2 x 105 cells      -        6-well plate      9.6      2 mL      0.5 x 106 cells      1 x 106 cells        10-cm dish      56.7      10 mL      -      5 x 106 cells        15-cm dish      145      30 mL      -      15 x 106 cells   
 
SECTION: hNIL differentiation into motor neurons: Day 1
36. Add the appropriate factors for day 1 to the NIM aliquot you aliquoted in step 35:
 
      Factor  Final Concentration  Stock  Dilution    ROCK1 inhibitor  10 μM  10 mM  1:1,000    Doxycycline  2 μg/mL  2 mg/mL  1:1,000    Compound E  0.2 μM  2 mM  1:10,000  
 
SECTION: hNIL differentiation into motor neurons: Day 1
37. Aspirate the spent media from the wells with cells.
SECTION: hNIL differentiation into motor neurons: Day 1
38. Add the appropriate volume of media to each well.
SECTION: hNIL differentiation into motor neurons: Day 1
39. Place the plate with cells in fresh media back in the incubator at 37 °C.
SECTION: hNIL differentiation into motor neurons: Day 2
40. Select the receiving cell-culture vessel. This is the plate on which your neurons will grow permanently (see recommendations below).
 

SECTION: hNIL differentiation into motor neurons: Day 2
41. Coat the wells which will be receiving cells on day 3 with the appropriate volume of 1X PDL at 0.1 mg/mL (see Table 1).
 
     Cell culture vessel      Matrigel,   PDL, and laminin in KO DMEM volume per well      DPBS volume   for washes per well      Accutase®   volume per well        96-well plate      120 µL      150 µL      50 µL        24-well plate      250 µL      400 µL      125 µL        12-well plate      500 µL      1 mL      250 µL        6-well plate      1 mL      2 mL      500 µL        10-cm dish      4 mL      6 mL      3 mL        15-cm dish      12 mL      18 mL      9 mL   
 

SECTION: hNIL differentiation into motor neurons: Day 2
42. Place the coated plate back in the incubator at 37 °C.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 1
43. 
 
Aspirate out the 1X PDL from the receiving cell-culture vessel.

SECTION: hNIL differentiation into motor neurons: Day 3; Part 1
44. Wash the receiving wells.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 1
45. Let receiving wells dry in the laminar flow hood for 20-30 minutes.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 1
46. Coat receiving cell-culture vessel with the appropriate volume of NIM with laminin mouse protein (at a concentration of 15 μg/ml) for 2-5 hours at 37 °C (see Table 1 for volume)
 
     Cell culture vessel      Matrigel,   PDL, and laminin in KO DMEM volume per well      DPBS volume   for washes per well      Accutase®   volume per well        96-well plate      120 µL      150 µL      50 µL        24-well plate      250 µL      400 µL      125 µL        12-well plate      500 µL      1 mL      250 µL        6-well plate      1 mL      2 mL      500 µL        10-cm dish      4 mL      6 mL      3 mL        15-cm dish      12 mL      18 mL      9 mL   
 

SECTION: hNIL differentiation into motor neurons: Day 3; Part 1
44.1. Wash the receiving wells with 1X DPBS. (1/2)
SECTION: hNIL differentiation into motor neurons: Day 3; Part 1
44.2. Wash the receiving wells with 1X DPBS. (2/2)
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
47. Prepare quenching media DMEM/F12 with 20% FBS at an appropriate volume. Per well in a 6- well plate, 0.5 mL of quenching media will be needed.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
48. Calculating the volume of day 3 NIM media needed

 

 
     Cell   culture vessel      Surface area (cm2)      Media volume per  well      Day 3   Seeding density for ICC applications      Day 3   Seeding density for protein, RNA,  DNA extraction        96-well plate      0.32      150 µL      20-25k cells      -        24-well plate      1.9      500 µL      80-100k cells      -        12-well plate      3.5      1 mL      2 x 105 cells      -        6-well plate      9.6      2 mL      0.5 x 106 cells      1 x 106 cells        10-cm dish      56.7      10 mL      -      5 x 106 cells        15-cm dish      145      30 mL      -      15 x 106 cells   
 
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
49. Preparing day 3 NIM media:
 
      Factor  Final Concentration  Stock  Dilution    ROCK1 inhibitor  10 μM  10 mM  1:1,000    Doxycycline  2 μg/mL  2 mg/mL  1:1,000    Compound E  0.2 μM  2 mM  1:10,000    Laminin  1 μg/mL  1 mg/mL  1:1,000    BrdU  40 μM  40 mM  1:1,000  
 
 

SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
50. Aspirate the spent media and wash the wells with cells once with 1X DPBS -Ca/-Mg.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
51. Add appropriate volume of Accutase® per well (0.5 mL/well in a 6-well plate).

SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
52. Incubate the plate for 5 min at 37 °C.

SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
53. Remove plate from the incubator and tap the plate to release the cells.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
54. Quench the Accutase® with DMEM/F12 + 20% FBS, at a volume equal to the volume of Accutase® per well (if you used 0.5 mL of Accutase®, quench Accutase® with 0.5 mL of DMEM/F12 + 20% FBS). DPBS +Ca/+Mg is also suitable.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
55. Pipette the solution gently around the well thoroughly to ensure cells have dissociated from the well bottom.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
56. Move the cells from their wells to a 15-mL vial (or vial of appropriate volume depending on how much volume of cells you have).
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
57. Centrifuge the vial at 800 rpm, 3 min.

SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
58. Aspirate the Accutase® with DMEM/F12 + 20% FBS, being careful not to disturb the cell pellet at the bottom of the vial.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
59. Resuspend the cells with an appropriate volume of day 3 NIM media (about 1 mL per 1 million cells in the vial).
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
60. Count the cells using a Countess™ (or preferred counting method) using the same parameters from Day 0.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
61. Aspirate the NIM with 15 μg/ml laminin from each receiving well.

SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
62. Plate appropriate number of cells per well using the appropriate media volume per well depending on your receiving cell culture vessel.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
63. Sample calculation and coating advice for uniform seeding of cells onto receiving vessel:
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
63.1. Calculate the volume of day 3 NIM media needed per well.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
63.2. Multiply that volume by the desired concentration of cells per well (e.g., 20k cells/well or 1 x 106 cells/well).
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
63.3. Divide that number by the concentration found by counting using the Countess™ (e.g. 2 x 106 cells/well).
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
63.4. The resulting number is the volume you need from the cell solution you resuspended in day 3 NIM media in step 59.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
63.5. Add that volume to your final desired volume of day 3 NIM media (e.g., 4 mL if seeding onto 2 wells of a 6-well plate).

SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
63.6. Mix well to ensure a uniform distribution of cells.
SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
63.7. Seed appropriate media volume (which now has cells as well) onto the receiving cell culture vessel.

 

SECTION: hNIL differentiation into motor neurons: Day 3; Part 2
64. Carefully place the plate with cells back in the incubator at 37 °C, rocking the plate briefly after placing the plate back, such that the cells are uniformly distributed onto the well.

SECTION: hNIL differentiation into motor neurons: Day 4
65. Prepare the day 4 NIM media for performing a full-media change
 
      Factor  Final Concentration  Stock  Dilution    B-27   Supplement  50x  01:50    Culture One   Supplement  100x 1:100    Laminin  1 μg/mL  1 mg/mL  1:1,000    BDNF  20 ng/mL  20 μg/mL  1:1,000    GDNF  20 ng/mL  20 μg/mL  1:1,000    NT3  20 ng/mL  20 μg/mL  1:1,000  
 
 

SECTION: hNIL differentiation into motor neurons: Day 4
66. Aspirate the media slowly from the wells, using a pipette, discarding into a reservoir.
SECTION: hNIL differentiation into motor neurons: Day 4
67. Add the fresh media slowly at a direction normal to the well side wall. 
 

SECTION: hNIL differentiation into motor neurons: Day 4
68. Place the plate back in the incubator at 37 °C.

SECTION: hNIL differentiation into motor neurons: Day 7
69. The risk of lifting only increases as the motor neurons age and mature on the well bottom.
 

SECTION: hNIL differentiation into motor neurons: Day 7
70. Prepare the day 7 NIM media for performing a half-media change.
 
 
      Factor  Final Concentration  Stock  Dilution    B-27   Supplement  50x  01:50    Culture One   Supplement  100x  0.111111    Laminin  500 ng/mL  1 mg/mL  1:1,000    BDNF  20 ng/mL  20 μg/mL  0.388889    GDNF  20 ng/mL  20 μg/mL  0.388889    NT3  20 ng/mL  20 μg/mL  0.388889  
 
 

SECTION: hNIL differentiation into motor neurons: Day 7
71. Aspirate the media slowly from the wells, using a pipette, discarding into a reservoir.
SECTION: hNIL differentiation into motor neurons: Day 7
72. Add the fresh media slowly in at a direction normal to the well side wall (which is easier by tilting the plate about 60-degrees towards you). 
 

SECTION: hNIL differentiation into motor neurons: Day 7
73. Place the plate of motor neurons back in the incubator at 37 °C.

SECTION: hNIL differentiation into motor neurons: Day 10 on
74. Prepare Neurobasal Medium (NMM) as follows:
 
      Neurobasal  97 mL    N-2  1 mL    NEAA  1 mL    GlutaMAX  1 mL  
 
SECTION: hNIL differentiation into motor neurons: Day 10 on
75. Prepare the day 10 NMM media for performing a half-media change.
 
      Factor  Final Concentration  Stock  Dilution    B-27   Supplement  50x  01:50    Culture One   Supplement  100x  0.111111    Laminin  500 ng/mL  1 mg/mL  1:1,000    BDNF  20 ng/mL  20 μg/mL  0.388889    GDNF  20 ng/mL  20 μg/mL  0.388889    NT3  20 ng/mL  20 μg/mL  0.388889  
 
 

SECTION: hNIL differentiation into motor neurons: Day 10 on
76. Aspirate the media slowly from the wells, using a pipette, discarding into a reservoir.
SECTION: hNIL differentiation into motor neurons: Day 10 on
77. Add the fresh media slowly in at a direction normal to the well side wall (which is easier by tilting the plate about 60-degrees towards you).
SECTION: hNIL differentiation into motor neurons: Day 10 on
78. Place the plate of motor neurons back in the incubator at 37 °C.

SECTION: hNIL differentiation into motor neurons: Day 10 on
79. Perform a half-media change with NMM and factors every 7 days for up to 9 weeks or more (if using the plate for imaging) or every 4 days (if using the plate for protein, RNA, or DNA extraction).
 

SECTION: hNIL differentiation into motor neurons: Day 10 on
80.