TITLE: Differentiation of WTC11 and KOLF2.1 iPSCs to dopaminergic neurons
AUTHORS: Nisha Mohd Rafiq, Pietro De Camilli

[DESCRIPTION]
This protocol describes the differentiation of iPSCs (WTC11 and KOLF2.1) to dopaminergic neurons according to Bressan et al 2021.                                                            

[STEPS]
SECTION: Differentiation of iPSCs into midbrain dopaminergic (mDA) neurons
1. Note: This protocol is optimized for mDA neuron differentiation in 6-well plate format.

Medium change schema:
Day 0-20:4 mL
Day 21-65:3 mL 
Day 0-15: daily media changes
Day 16-20: media changes every 2 days
Day 21-65: media changes every 2-3 days
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.3. 
Discard Geltrex solution and add2 mLof E8 Flex medium supplemented with10 µMY-27632 avoiding the coating to dry out.
SECTION: Day -2: Coating of 6-well plates with Geltrex
1.2. Coat 6-well culture plates with1 mLof Geltrex solution at37 °C5 %.
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.4. Keep the plate at37 °Cfor5 minbefore seeding cells.
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.5. Warm PBS, Accutase and E8 Flex medium to37 Room temperature.
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.6. Add1 mLof Accutase to iPSCs in 6-well plate.
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.7. Dissociate cells with a P1000 pipette by pipetting the cell suspension up and down for 3-6 times. 
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.8. Perform a quick microscope inspection of the cells. A single cell suspension should be obtained.

SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.9. Transfer the cell suspension to a 15 mL conical centrifuge tube. A total volume of12 mLhould be obtained from a full 6-well plate.

SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.10. Centrifuge the cell suspension at300 x g, 5 min, 23 °C.
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.11. Discard the supernatant carefully and resuspend the cell pellet in1 mLE8 Flex medium supplemented with10 µMY-27632 by gently pipetting the cell suspension up and down for 3-6 times with a P1000 pipette to obtain a homogeneous cell suspension. 
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.12. Adjust the volume with E8 Flex medium supplemented with10 µMY-27632  to12 mL. Mix well.
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.13. Perform two separate live-cell counts using a hemocytometer or an automated cell counter. 
Critical step: Adjust the cell suspension volume accordingly to obtain an accurate counting. 
Calculate the mean achieved from the two counts and determine the concentration of live cells per milliliter. 
 
Note: Working with a different brand of Accutase might impact the cell viability when following the dissociation protocol described above. If cell viability is lower than expected, reduce the incubation time with Accutase to20 minand the number of pipetting to dissociate the cell colonies to 3-4 times.
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.14. Seed 800,000 cells per well in a total volume of4 mLon 6-well plates coated with Geltrex.
SECTION: Day 0: Start differentiation
2. Before starting: 

a) Prepare enough amount of Knockout Serum Replacement (KSR) medium. 

For 500 mLof KSR medium, add:
413.5 mL Knockout DMEM/F-12 medium
75 mL Knockout Serum Replacement
5 mL MEM Non-Essential Amino Acids
5 mL GlutaMAX 
500 µL 2-mercaptoethanol 
1 mL Penicillin-Streptomycin  
Storage: KSR medium can be stored for 5 days at4 °Cor for up to one month at-20 °C. 

b) Reconstitute lyophilized reagents following the instructions and stock concentrations indicated in Materials (1.1.4 Reconstitution of reagents). 

Use the following final concentrations: 
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
2 µM Purmorphamine
100 ng/mL FGF8
4 µM CHIR99021
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 µMor10 nM DAPT
Storage: Once thawed, the stocks of small molecules and growth factors can be stored for up to 5 days at4 °C. 

Notes: Small molecules and growth factors must be freshly added immediately before each medium change. It is strongly advised to avoid mixing different lots of reagents in the same differentiation.
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.16. Keep cells intact at37 °C5 %.
SECTION: Day 0: Start differentiation
2.1. Perform a quick microscope inspection to the cells to check confluency.

Critical step: Start the differentiation with a 100% confluent cell culture, meaning that the culture area of the plate should be completely covered with a cell monolayer. Not confluent cell cultures might affect differentiation efficiency. If cell lines did not achieve 100% confluency on day 0, the number of cells seeded per cm2 on day -1 should be adjusted accordingly. 
 
SECTION: Day 0: Start differentiation
2.2. Prepare differentiation medium: 

Warm KSR medium at37 °C.
Add:
500 nM LDN193189
10 µM SB431542
Mix well.

Critical step: Do not add small molecules to cold medium to avoid inadequate dissolution. 
SECTION: Day 0: Start differentiation
2.3. Perform full media change:
Discard old culture medium and add4 mLof differentiation medium very carefully avoiding touching the bottom of the well. 

Critical step: To prevent cells from drying out during full media changes, change the medium of one 6-well plate at each time. Add differentiation medium very gently (dropwise) to avoid perturbation of the cell layer. Cell detachments might affect the differentiation efficiency. In case of cell detachment, a confluency above 95% is desired to continue the differentiation.
SECTION: Media changes during differentiation
3. From day 1, change 75% of the differentiation medium daily until day 15, and then, every 2 days until day 20.
Note: to perform 75% medium change of a working volume of4 mL, discard 3 mLof old medium and add4 mLof fresh prepared differentiation medium.
SECTION: Day 1 and 2
3.1. Warm KSR medium at37 °C.
Add:
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
2 µM Purmorphamine
100 ng/mL FGF-8b
Mix well.
Perform medium change: 4 mL.
Place cells back at37 °C5 %.
SECTION: Day 3 and 4
3.2. Warm KSR medium at37 °C.
Add:
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
2 µM Purmorphamine
100 ng/mL FGF-8b
4 µM CHIR99021
Mix well.
Perform medium change: 4 mL.
Place cells back at37 °C5 %.
SECTION: Day 5 and 6
3.3. Before starting:

Prepare enough amount of N2 medium.

For 500 mLof N2 medium, add:
479 mL Neurobasal medium
10 mL B27 supplement without vitamin A 
5 mL N2 supplement 
5 mL GlutaMAX 
1 mL Penicillin-Streptomycin  
Storage: N2 medium can be stored for 5 days at4 °Cor for up to one month at-20 °C. 

Warm KSR and N2 medium at37 °C.
Mix:
75 % 
25 % 
Add:
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
2 µM Purmorphamine
100 ng/mL FGF-8b
4 µM CHIR99021
Mix well.
Perform medium change: 4 mL.
Place cells back at37 °C5 %.
SECTION: Day 7 and 8
3.4. Warm KSR and N2 medium at37 °C.
Mix:
50 % 
50 % 
Add:
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
4 µM CHIR99021
Mix well.
Perform medium change: 4 mL.
Place cells back at37 °C5 %.
SECTION: Day 9 and 10
3.5. Warm KSR and N2 medium at37 °C.
Mix:
25 % 
75 % 
Add:
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
4 µM CHIR99021
Mix well.
Perform medium change: 4 mL.
Place cells back at37 °C5 %.
SECTION: Day 11 and 12
3.6. Before starting:

Prepare enough amount of NB/B27 medium.

For 500 mLNB/B27 medium, add:
484 mL Neurobasal medium
10 mL B27 supplement without vitamin A
5 mL GlutaMAX
1 mL Penicillin-Streptomycin
Storage: NB/B27 medium can be stored for 5 days at4 °Cor for up to one month at-20 °C. 

Warm NB/B27 medium at37 °C.
Add:
4 µM CHIR99021
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 µM DAPT
Mix well.
Perform medium change: 4 mL.
Place cells back at37 °C5 %.
SECTION: Day 13 -15, 17 and 19
3.7. Warm NB/B27 medium at37 °C.
Add:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 µM DAPT
Mix well.
Perform medium change: 4 mL.
Place cells back at37 °C5 %.
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4. Note: At day 20 of differentiation, mDA neuron precursors can be replated as describe below or cryopreserved.

Before starting:

Coating of 6-well plates step 1
Coat 6-well culture plates with1 mL0.1 mg/mLPoly-L-Ornithine (PLO) in PBS. Incubate plates at37 °C. Wash plates three times with PBS. Discard PBS and proceed to coating step 2.

Coating of 6-well plates step 2
Coat 6-well culture plates with1 mL10 µg/mLLaminin plus 2 µg/mLFibronectin, both diluted in PBS. Incubate plates at37 °C. Do not store coated plates. Proceed with preparation of plates for seeding cells.

Preparation of 6-well plates for seeding cells
Warm NB/B27 medium at37 °C.
Make NB/B27 complete medium by adding:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 nM DAPT
10 µM Y-27632
Discard coating reagents and add2 mLof NB/B27 complete medium.
Keep the plate at37 °Cfor15 minbefore seeding cells.
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.1. Warm PBS, Accutase and NB/B27 medium to37 Room temperature.
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.2. Discard old culture media and wash cells once with1 mLof PBS. 
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.3. Discard PBS and add1 mLof Accutase.
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.5. After incubation, block Accutase with1 mLNB/B27 medium supplemented with10 µMY-27632. 

SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.7. Perform a quick microscope inspection of the cells. A single cell suspension should be obtained.
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.8. Transfer the cell suspension to a conical tube. A total volume of12 mLshould be obtained from a full 6-well plate.
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.9. Centrifuge the cell suspension at300 x g, 5 min, 23 °C.
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.10. Discard the supernatant carefully and add1 mLNB/B27 complete medium. Resuspend the cell pellet very gently with a P1,000 pipette by pipetting the cell suspension up and down for 3-6 times.

SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.11. Complete the volume to12 mLwith NB/B27 complete medium and mix well.
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.12. Perform two separate live-cell counts using a hemocytometer or an automated cell counter. 
Critical step: Adjust the cell suspension volume accordingly to obtain an accurate counting. 
Calculate the mean achieved from the two counts and determine the concentration of live cells per milliliter. 
 

SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.13. Seed desired density (300,000 to 500, 000 cells) on MatTek dishes or 6-well plate coated with PLO/Laminin.
SECTION: Day 23: Treatment with antimitotic inhibitor (Supplement K)
5. Before starting: 

Warm NB/B27 medium at37 °C.
Make NB/B27 complete medium by adding:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 nM DAPT
Prepare enough amount of NB/B27 complete medium supplemented with1 Parts per Million (PPM)Supplement K (BrainXell). Mix well.
SECTION: Day 23: Treatment with antimitotic inhibitor (Supplement K)
5.1. Discard old culture medium and add2 mLof NB/B27 complete medium supplemented with Supplement K.
SECTION: Day 23: Treatment with antimitotic inhibitor (Supplement K)
5.2. Place cells back at37 °C5 %.
SECTION: Every 5 days: Change medium for terminal differentiation of mDA neurons
6. Warm NB/B27 medium at37 °C.
Add:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 nM DAPT
Mix well by 20x full inversions of the conical tube or flask. 
Perform medium change: 2 mL.
Place cells back at37 °C5 %.
SECTION: Every 10 days: Supplement differentiation medium with Laminin
6.1. Warm NB/B27 medium at37 °C.
Make NB/B27 complete medium by adding:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 nM DAPT
Cool down NB/B27 complete medium toRoom temperature.
Add:
10 µg/mL Laminin

Mix well.
SECTION: Every 10 days: Supplement differentiation medium with Laminin
6.2. Discard old culture medium. Perform medium change as following:
3 mL in 6-well plates
200 µL in 96-well plates
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.15. Keep cells intact  at37 °C5 %.
SECTION: Cryopreservation of mDA neurons on day 20
7. Before starting:
Thaw Synth-a-Freeze cryopreservation medium from -20C.

SECTION: Cryopreservation of mDA neurons on day 20
7.1. Perform cells dissociation and previously described .
SECTION: Cryopreservation of mDA neurons on day 20
7.2. After determining the number of live cells, centrifuge the cell suspension at400 x g, 5 min, 23 °C.
SECTION: Cryopreservation of mDA neurons on day 20
7.3. Resuspend the cell pellet very gently in Synth-a-Freeze medium to 3-5x10^6 cells/mL.
SECTION: Cryopreservation of mDA neurons on day 20
7.4. Distribute500 µLof the cell suspension in cryogenic vials.
SECTION: Cryopreservation of mDA neurons on day 20
7.6. Transfer cells to the vapor phase of a liquid nitrogen storage facility.
SECTION: Thawing cryopreserved mDA neurons
8. One day before thawing:

Coat 6-well culture plates with Geltrex diluted in ice cold DMEM/F-12 medium at37 °C.
SECTION: Day of thawing
8.1. Before starting

Prepare: 

Wash medium
Warm NB/B27 medium at37 °C.
Add: 10 µMY-27632.

Recovery medium
Warm NB/B27 medium at37 °C.
Make NB/B27 complete medium by adding:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 nM DAPT
10 µM Y-27632
Cool down NB/B27 complete medium toRoom temperature.


6-well plates
Discard coating reagent and add2 mLof recovery medium.
Keep plates at37 °Cfor15 minbefore seeding cells.

Conical centrifuge tubes 
Label 15-mL conical tubes and fill with5 mLwash medium. Keep atRoom temperature .

SECTION: Day of thawing
8.2. Thaw cryopreserved mDA neurons by placing the cryogenic vial containing cells in a37 °Cwater bath for approximately1 minor until no ice is visible but the liquid is still cold.
SECTION: Day of thawing
8.3. Fill a 5-mL serological pipette with4 mLwash medium and collect the thawed cell suspension very carefully. 
SECTION: Day of thawing
8.4. Transfer the cell suspension dropwise to the 15-mL conical tube containing5 mLof wash medium. 
SECTION: Day of thawing
8.5. Centrifuge the cell suspension at400 x g, 5 min, 23 °C.
SECTION: Day of thawing
8.6. Discard the supernatant carefully and resuspend the cell pellet with1 mLrecovery medium by gently pipetting up and down 3-6 times to obtain a homogeneous cell suspension.
SECTION: Day of thawing
8.7. Seed cells  in a total volume of2 mLon 6-well plates coated with Geltrex.

Treat cells with Supplement K following the same procedure described on day 23 of differentiation .
SECTION: Day of thawing
8.10. Keep cells intact  at37 °C5 %.
SECTION: Passaging of iPSCs with Gentle Differentiation Reagent (GDR)
None. Discard coating and add2 mLof E8 medium. Keep the plate at37 °Cfor15 minbefore seeding cells.

SECTION: Day -2: Coating of 6-well plates with Geltrex
1.1. Thaw Geltrex  on iceand dilute to 100x in ice cold DMEM/F-12.
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.6. Dissociate cells with a P1,000 pipette by pipetting the cell suspension up and down for 3-6 times.
SECTION: Day -1: Single cell dissociation and seeding for differentiation
1.15.  Rock the plate back-forth and side-to-side for10 sto achieve an even spread of cells in the plate well.
SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.14. Rock the plate back-forth and side-to-side for10 sto achieve an even spread of cells in the plate well.
SECTION: Day 32: Replating of mDA neurons at low cell density (optional)
None. Rock the plate back-forth and side-to-side for10 sto achieve an even spread of cells in the plate well.
SECTION: Day of thawing
8.8. Rock the plate back-forth and side-to-side for10 sto achieve an even spread of cells in the plate well.
SECTION: Day 7 after thawing: Replate mDA neurons
8.9. Replate mDA neurons 7 days after thawing on MatTek or 6-well plates at appropriate cell densities following the procedure described for replating mDA neurons on day 20  .
SECTION: Day 7 after thawing: Replate mDA neurons
8.9. Seed cells at desired cell density and plate format.

SECTION: Day 20: Replating of mDA neuron precursors at high cell density
4.4. Incubate cells at37 °Cfor15 min. 
SECTION: Day 7 after thawing: Replate mDA neurons
8.10. For long-term culture of mDA neurons, change differentiation medium every 5 days as described previously .
SECTION: Every 10 days: Supplement differentiation medium with Laminin
6.3. Place cells back at3 °C5 %.   
SECTION: Passaging of iPSCs with Gentle Differentiation Reagent (GDR)
None. Incubate cells at37 °Cfor3 min.
SECTION: Cryopreservation of mDA neurons on day 20
7.5. Transfer cryogenic vials in a container at-80 °C .