TITLE: iNIL/iNIP transcription factor-induced motor neuron differentiation v2 (Mazzoni lab)
AUTHORS: Dylan E. Iannitelli, Hoa (Emma) Nguyen, Albert Tan, Esteban O. Mazzoni

[BEFORE_START]
Preparing base human motor neuron media (hMNM)
1X Neurobasal Media                500mL 
50X B27                                        10mL
100X N2                                        5mL
100X L-Glutamine                        5mL

[STEPS]
SECTION: Day 0
13. Aspirate media and gently wash iPSCs with 1X DPBS
SECTION: Day 0
14. Harvest iNIL/iNIP iPSCs

SECTION: Day 0
15. Count cells 
SECTION: Day 0
16. Seed directly into hMN media + Y-27632 (10 μM) + dox (3 μM) 
Seed at 8,000 cells/cm2 (Example: 600,000 cells/10cm dish)
 

SECTION: Day 2
17. Aspirate all media
SECTION: Day 2
18. Replenish with hMN media + Y-27632 (10 μM) + dox (3 μM), adding Retinoic Acid (RA)
iSpMNs: 1.0 μM RA
iCrMNs: 0.01 μM RA
SECTION: Day 4
19. Aspirate all media
SECTION: Day 4
20. Replenish hMN media + dox (3 μM) + RA (iSpMN 1.0 μM/iCrMN 0.01 μM), removing Y-27632
SECTION: Day 6
21. Carefully aspirate half of the total volume of media per well/dish
SECTION: Day 6
22. Replenish hMN media + dox (3 μM) + RA (iSpMN 1.0 μM/iCrMN 0.01 μM) + Adarotene (1 μM)
SECTION: Day 8
23. Carefully aspirate half of the total volume of media per well/dish
SECTION: Day 8
24. Replenish hMN media + dox (3 μM) + RA (iSpMN 1.0 μM/iCrMN 0.01 μM) + Adarotene (1 μM) + supplements:
BDNF (10 ng/mL)
GDNF (10 ng/mL)
CNTF (10 ng/mL)
L-ascorbic acid (200 ng/mL)
SECTION: Day 10 + (for a healthy culture, change media every 4-5 days)
25. Carefully aspirate half of the total volume of media per well/dish
SECTION: Day 10 + (for a healthy culture, change media every 4-5 days)
26. Replenish hMN media + dox (3 μM) + RA (iSpMN 1.0 μM/iCrMN 0.01 μM) + Adarotene (1 μM), + supplements:
BDNF (10 ng/mL)
GDNF (10 ng/mL) 
CNTF (10 ng/mL)
L-ascorbic acid (200 ng/mL)

SECTION: Day 10 + (for a healthy culture, change media every 4-5 days)
27. Optional: adding maturation cocktail (GENtonik)
GSK (1 μM)
EPZ (1 μM)
NMDA (1 μM)
BayK (1 μM)
SECTION: Day 0
14.1. Aspirate 1X PBS and dissociate iNIL/iNIP iPSCs by adding Accutase
SECTION: Day 0
14.2. Neutralize Accutase with  E8 Media + Y-27632 (10 μM) 2 times the amount of Accutase added (eg. If 2 mL of Accutase was used, resuspend in 4 mL of  E8 Media + Y-27632 (10 μM))
SECTION: Day 0
14.3. Transfer resuspended cells to a 15 mL tube
SECTION: Day 0
14.4. Spin at 900 rpm for 4 minutes

SECTION: Day 0
14.5. Aspirate media and resuspend cell pellet in 3-5 mL  of E8 Media + Y-27632 (10 μM)

SECTION: Thawing and maintaining human iPSCs prior to differentiation
1. Preparing Matrigel


SECTION: Thawing and maintaining human iPSCs prior to differentiation
1.1. Thaw an aliquot of 500 μL of Matrigel on ice in the 4 °C  fridge for a few hours

SECTION: Thawing and maintaining human iPSCs prior to differentiation
1.2. Dilute aliquot 1:100 in cold Neurobasal Media

SECTION: Thawing and maintaining human iPSCs prior to differentiation
2. Coat plates overnight with diluted Matrigel at 37 °C 

 


SECTION: Thawing and maintaining human iPSCs prior to differentiation
3. Thaw vial of cells from liquid nitrogen by placing the vial in a 37 °C  bath until partially thawed
 


SECTION: Thawing and maintaining human iPSCs prior to differentiation
4. Add  1 mL of pre-warmed E8 + Y-27632 (10 μM) media into the partially thawed vial and transfer cells to a 15 mL tube

SECTION: Thawing and maintaining human iPSCs prior to differentiation
5. Add an additional 1-2 mL of pre-warmed E8 + Y-27632 (10 μM) media into the 15 mL tube

SECTION: Thawing and maintaining human iPSCs prior to differentiation
6. Spin at 900 rpm for 4 minutes
SECTION: Thawing and maintaining human iPSCs prior to differentiation
7. While waiting for the spin to complete, prepare plate
SECTION: Thawing and maintaining human iPSCs prior to differentiation
7.1. Aspirate Matrigel from coated plate 

 

SECTION: Thawing and maintaining human iPSCs prior to differentiation
7.2. Wash plate with 1X PBS
SECTION: Thawing and maintaining human iPSCs prior to differentiation
7.3. Aspirate 1X PBS, and add the appropriate amount of pre-warmed E8 + Y-27632 (10 μM) media 
SECTION: Thawing and maintaining human iPSCs prior to differentiation
8. At the end of spin, aspirate media and resuspend cell pellet in 1 mL of E8 Media + Y-27632 (10 μM)
SECTION: Thawing and maintaining human iPSCs prior to differentiation
9. Seed directly into E8 Media + Y-27632 (10 μM)
SECTION: Thawing and maintaining human iPSCs prior to differentiation
10. Change media the next day to E8 with no Y-27632 (Rock inhibitor)

 

SECTION: Day 0
12. Coat plates with Matrigel as described above
SECTION: Thawing and maintaining human iPSCs prior to differentiation
11. Change media with E8 every other day until iPSCs are near confluent and ready for re-replating and differentiation