Differentiation of WTC11 and KOLF2.1 iPSCs to dopaminergic neurons

Note: This protocol is optimized for mDA neuron differentiation in 6-well plate format. Medium change schema: Day 0-20:4mLDay 21-65:3mL Day 0-15: daily media changesDay 16-20: media changes every 2 daysDay 21-65: media changes every 2-3 days

Thaw Geltrex On iceand dilute to 100x in ice cold DMEM/F-12.

Centrifuge the cell suspension at300x g,23°C.

Discard the supernatant carefully and resuspend the cell pellet in1mLE8 Flex medium supplemented with10µMY-27632 by gently pipetting the cell suspension up and down for 3-6 times with a P1000 pipette to obtain a homogeneous cell suspension.

Adjust the volume with E8 Flex medium supplemented with10µMY-27632 to12mL. Mix well.

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 to0h 20m 0sand the number of pipetting to dissociate the cell colonies to 3-4 times.

Seed 800,000 cells per well in a total volume of4mLon 6-well plates coated with Geltrex.

Rock the plate back-forth and side-to-side for0h 0m 10sto achieve an even spread of cells in the plate well.

Keep cells intactat37°C``5%.

Coat 6-well culture plates with1mLof Geltrex solutionat37°C``5%.

Discard Geltrex solution and add2mLof E8 Flex medium supplemented with10µMY-27632 avoiding the coating to dry out.

Keep the plate at37°Cfor0h 5m 0sbefore seeding cells.

Warm PBS, Accutase and E8 Flex medium to37Room temperature.

Add1mLof Accutase to iPSCs in 6-well plate.

Dissociate cells with a P1000 pipette by pipetting the cell suspension up and down for 3-6 times.

Perform a quick microscope inspection of the cells. A single cell suspension should be obtained.

Transfer the cell suspension to a 15 mL conical centrifuge tube. A total volume of12mLhould be obtained from a full 6-well plate.

Before starting: a) Prepare enough amount of Knockout Serum Replacement (KSR) medium. For 500mLof KSR medium, add:413.5mL Knockout DMEM/F-12 medium75mL Knockout Serum Replacement5mL MEM Non-Essential Amino Acids5mL GlutaMAX 500µL 2-mercaptoethanol 1mL 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: 500nM LDN19318910µM SB431542100ng/mL SHH2µM Purmorphamine100ng/mL FGF84µM CHIR9902120ng/mL BDNF0.2mM Ascorbic acid20ng/mL GDNF0.5mM db-cAMP1ng/mL TGFβ310µMor10nM 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.

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.

Prepare differentiation medium:

Warm KSR medium at37°C.

Add:

500nM LDN193189

10µM SB431542

Mix well.

Critical step: Do not add small molecules to cold medium to avoid inadequate dissolution.

Perform full media change:

Discard old culture medium and add4mLof 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.

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 of4mL, discard 3mLof old medium and add4mLof fresh prepared differentiation medium.

Warm KSR medium at37°C.

Add:

500nM LDN193189

10µM SB431542

100ng/mL SHH

2µM Purmorphamine

100ng/mL FGF-8b

Mix well.

Perform medium change: 4mL.

Place cells back at37°C``5%.

Warm KSR medium at37°C.

Add:

500nM LDN193189

10µM SB431542

100ng/mL SHH

2µM Purmorphamine

100ng/mL FGF-8b

4µM CHIR99021

Mix well.

Perform medium change: 4mL.

Place cells back at37°C``5%.

Before starting: Prepare enough amount of N2 medium.For 500mLof N2 medium, add:479mL Neurobasal medium10mL B27 supplement without vitamin A 5mL N2 supplement 5mL GlutaMAX 1mL 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:

500nM LDN193189

10µM SB431542

100ng/mL SHH

2µM Purmorphamine

100ng/mL FGF-8b

4µM CHIR99021

Mix well.Perform medium change: 4mL.Place cells back at37°C``5%.

Warm KSR and N2 medium at37°C.

Mix:

50%

50%

Add:

500nM LDN193189

10µM SB431542

100ng/mL SHH

4µM CHIR99021

Mix well.Perform medium change: 4mL.Place cells back at37°C``5%.

Warm KSR and N2 medium at37°C.

Mix:

25%

75%

Add:

500nM LDN193189

10µM SB431542

100ng/mL SHH

4µM CHIR99021

Mix well.Perform medium change: 4mL.Place cells back at37°C``5%.

Before starting: Prepare enough amount of NB/B27 medium.For 500mLNB/B27 medium, add:484mL Neurobasal medium10mL B27 supplement without vitamin A5mL GlutaMAX1mL 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:

4µM CHIR99021

20ng/mL BDNF0.2mM Ascorbic acid20ng/mL GDNF0.5mM db-cAMP1ng/mL TGFβ310µM DAPTMix well.Perform medium change: 4mL.Place cells back at37°C``5%.

Warm NB/B27 medium at37°C.

Add:

20ng/mL BDNF0.2mM Ascorbic acid20ng/mL GDNF0.5mM db-cAMP1ng/mL TGFβ310µM DAPTMix well.Perform medium change: 4mL.Place cells back at37°C``5%.

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 with1mL``0.1mg/mLPoly-L-Ornithine (PLO) in PBS. Incubate platesat37°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 with1mL``10µg/mLLaminin plus 2µg/mLFibronectin, both diluted in PBS. Incubate platesat37°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:

20ng/mL BDNF0.2mM Ascorbic acid20ng/mL GDNF0.5mM db-cAMP1ng/mL TGFβ310nM DAPT10µM Y-27632Discard coating reagents and add2mLof NB/B27 complete medium.

Keep the plate at37°Cfor0h 15m 0sbefore seeding cells.

Warm PBS, Accutase and NB/B27 medium to37Room temperature.

Discard the supernatant carefully and add1mLNB/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.

Complete the volume to12mLwith NB/B27 complete medium and mix well.

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.

Seed desired density (300,000 to 500, 000 cells) on MatTek dishes or 6-well plate coated with PLO/Laminin.

Rock the plate back-forth and side-to-side for0h 0m 10sto achieve an even spread of cells in the plate well.

Keep cells intact at37°C``5%.

Discard old culture media and wash cells once with1mLof PBS.

Discard PBS and add1mLof Accutase.

Incubate cells at37°Cfor0h 15m 0s.

After incubation, block Accutase with1mLNB/B27 medium supplemented with10µMY-27632.

Dissociate cells with a P1,000 pipette by pipetting the cell suspension up and down for 3-6 times.

Perform a quick microscope inspection of the cells. A single cell suspension should be obtained.

Transfer the cell suspension to a conical tube. A total volume of12mLshould be obtained from a full 6-well plate.

Centrifuge the cell suspension at300x g,23°C.

Before starting:

Warm NB/B27 medium at37°C.

Make NB/B27 complete medium by adding:

20ng/mL BDNF0.2mM Ascorbic acid20ng/mL GDNF0.5mM db-cAMP1ng/mL TGFβ310nM DAPTPrepare enough amount of NB/B27 complete medium supplemented with1Parts per Million (PPM)Supplement K (BrainXell). Mix well.

Discard old culture medium and add2mLof NB/B27 complete medium supplemented with Supplement K.

Place cells back at37°C``5%.

Warm NB/B27 medium at37°C.

Add:

20ng/mL BDNF

0.2mM Ascorbic acid20ng/mL GDNF0.5mM db-cAMP1ng/mL TGFβ310nM DAPT

Mix well by 20x full inversions of the conical tube or flask. Perform medium change: 2mL.

Place cells back at37°C``5%.

Warm NB/B27 medium at37°C.

Make NB/B27 complete medium by adding:

20ng/mL BDNF0.2mM Ascorbic acid20ng/mL GDNF0.5mM db-cAMP1ng/mL TGFβ310nM DAPTCool down NB/B27 complete medium toRoom temperature.Add:10µg/mL LamininMix well.

Discard old culture medium. Perform medium change as following:3mL in 6-well plates200µL in 96-well plates

Place cells back at3°C``5%.

Before starting:

Thaw Synth-a-Freeze cryopreservation medium from -20C.

Perform cells dissociation and previously described.

After determining the number of live cells, centrifuge the cell suspension at400x g,23°C.

Resuspend the cell pellet very gently in Synth-a-Freeze medium to 3-5x10^6 cells/mL.

Distribute500µLof the cell suspension in cryogenic vials.

Transfer cryogenic vials in a container at-80°C.

Transfer cells to the vapor phase of a liquid nitrogen storage facility.

One day before thawing:

Coat 6-well culture plates with Geltrex diluted in ice cold DMEM/F-12 mediumat37°C.

Before starting

Prepare:

Wash medium

Warm NB/B27 medium at37°C.

Add: 10µMY-27632.

Recovery medium

Warm NB/B27 medium at37°C.

Make NB/B27 complete medium by adding:

20ng/mL BDNF0.2mM Ascorbic acid20ng/mL GDNF0.5mM db-cAMP1ng/mL TGFβ310nM DAPT

10µM Y-27632

Cool down NB/B27 complete medium toRoom temperature.

6-well plates

Discard coating reagent and add2mLof recovery medium.

Keep plates at37°Cfor0h 15m 0sbefore seeding cells.

Conical centrifuge tubes

Label 15-mL conical tubes and fill with5mLwash medium. Keep atRoom temperature .

Keep cells intact at37°C``5%.

For long-term culture of mDA neurons, change differentiation medium every 5 days as described previously.

Thaw cryopreserved mDA neurons by placing the cryogenic vial containing cells in a37°Cwater bath for approximately0h 1m 0sor until no ice is visible but the liquid is still cold.

Fill a 5-mL serological pipette with4mLwash medium and collect the thawed cell suspension very carefully.

Transfer the cell suspension dropwise to the 15-mL conical tube containing5mLof wash medium.

Centrifuge the cell suspension at400x g,23°C.

Discard the supernatant carefully and resuspend the cell pellet with1mLrecovery medium by gently pipetting up and down 3-6 times to obtain a homogeneous cell suspension.

Seed cells in a total volume of2mLon 6-well plates coated with Geltrex.

Treat cells with Supplement K following the same procedure described on day 23 of differentiation.

Rock the plate back-forth and side-to-side for0h 0m 10sto achieve an even spread of cells in the plate well.

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 .

Seed cells at desired cell density and plate format.