A protocol for the differentiation of human embryonic stem cells into midbrain dopaminergic neurons

Summary Here, we present a protocol for the generation of functional midbrain dopaminergic (mDA) neurons from human embryonic stem cells (hESCs), which mimics the development of the human ventral midbrain. We describe steps for hESC proliferation, induction of mDA progenitors, freezing stocks of mDA progenitors as an intermediate starting point to reduce the time to make mDA neurons, and maturation of mDA neurons. The entire protocol is feeder-free and uses chemically defined materials. For complete details on the use and execution of this protocol, please refer to Nishimura et al. (2023).1


Highlights
Step-by-step protocol for the induction of mDA neurons from hESCs The protocol recapitulates key aspects of in vivo human midbrain development Frozen mDA progenitor stocks can be used for faster production of mDA neurons

MATERIALS AND EQUIPMENT
Optional: Penicillin/streptomycin can be supplemented in the media to prevent bacterial contamination.
Note: Media can be aliquoted and stored at À20 C. Avoid repeated freezing and thawing.

Reagent
Final concentration Amount

STEP-BY-STEP METHOD DETAILS
Expansion and preparation of hESCs before differentiation of mDA progenitors Timing: 2-3 weeks (for all steps in this section) Timing: 1 h (for step 1) Timing: 6-7 days (for step 3) Timing: 1 h (for steps 3a to 3i) hESCs are cultured under xeno-and feeder-free conditions using laminin-521 (LN521), to recapitulate the embryonic stem cell niche, and are passaged at least twice before starting differentiation.
Here we describe the procedure for a 6-well plate.If you use other plates, please calculate the concentration of LN521 according to the surface area (See Table 1).
Note: The culture medium, PBS, TrypLE Select and DTI should be warmed in a water bath at 37 C before use.

Optional:
The plate can be coated with LN521 at 4 C for overnight (15-22 h).
b. Slowly thaw a cryovial containing approximately 500,000 cells using a water bath at 37 C until the cells are half-thawed, then immediately collect them in a 15-mL tube with 10 mL of NutriStem hPSC XF medium.
CRITICAL: Do not completely thaw the cells in the water bath to avoid cell death.
c. Centrifuge the cells at 180 G for 3-5 min at room temperature (20 C-25 C). d.In the meantime, wash the coated well with PBS(+Ca 2+ /+Mg 2+ ) twice and add 2 mL of NutriStem hPSC XF medium supplemented with 10 mM Y-27632 to prevent cell death.
Note: A ROCK inhibitor Y-27632 drastically improves cell viability of single-cell dissociated cells. 4After centrifugation, discard the supernatant and resuspend the cells in 1 mL of NutriStem hPSC XF medium supplemented with 10 mM Y-27632.f.Seed the cells in a final volume of 3 mL of NutriStem hPSC XF medium supplemented with 10 mM Y-27632 in one well of 6-well plate.g.The next day, change the culture medium to 3 mL of NutriStem hPSC XF medium without Y-27632.

Maintenance of hESCs
Maintain the hESCs in NutriStem hPSC XF medium until they reach 70-80% confluency, changing the medium daily.The volume of NutriStem hPSC XF medium should be increased from 3 mL to 8 mL according to the growth of the cells to avoid the medium turning yellow.

Passage of hESCs.
a. Mix 100 mL of LN521 and 1,400 mL of PBS(+Ca 2+ /+Mg 2+ ) and coat a well of a 6-well plate at 37 C for at least 2 h.b.Discard the supernatant of the cells in culture and wash the cells with PBS(-Ca 2+ /-Mg 2+ ) twice.c.Add 1 mL of TrypLE Select to the well and incubate at 37 C for 4 min.d.Gently pipette to yield a single-cell suspension, add 1 mL of defined trypsin inhibitor (DTI) to inactivate TrypLE Select and then collect all cells in a 15-mL tube.e. Centrifuge the cells at 180 G for 3-5 min at room temperature (20 C-25 C). f.In the meantime, wash the coated well with PBS(-Ca 2+ /-Mg 2+ ) twice and add 1 mL of NutriStem hPSC XF medium supplemented with 10 mM Y-27632.g.After centrifugation, discard the supernatant and resuspend the cells in 1 mL of NutriStem hPSC XF medium supplemented with 10 mM Y-27632.h.Count the cells using a hemocytometer.i. Seed 50,000 cells in a final volume of 2 mL of NutriStem hPSC XF medium supplemented with 10 mM Y-27632.j.The next day, change the culture medium to 2 mL of NutriStem hPSC XF medium without Y-27632.k.Change the medium daily until they reach 70-80% confluency (usually in 6-7 days).l.Repeat the entire process at least one more time.

Note:
We usually obtain 2-3 million cells from a well of 6-well plate when the cells reach 70%-80% confluency.But it depends on cell lines and culture conditions.
Note: Handle carefully the plate when it contains large volumes of media to avoid spillage and contamination.
Optional: Excess cells can be frozen and stored in liquid nitrogen.Differentiation of hESCs into mDA progenitors is performed in feeder-free culture conditions (Figure 1A) using LN511 to support midbrain development. 5Please, ensure the quality of undifferentiated hESCs before starting the differentiation.hESCs should be 70-80% confluent at the start of the differentiation and should be a homogeneous population, without any sign of differentiation such as morphological changes (fibroblast-like morphology).Here we describe the procedure for a 6-well plate.

Protocol
Note: Media must be changed daily using the volumes indicated as follows.If the color of the culture media turns too yellow or orange, increase the volume of media.
4. Seeding hESCs for differentiation (Day -2).a. Mix 100 mL of LN511 and 1,400 mL of PBS(+Ca 2+ /+Mg 2+ ) and coat a well of a 6-well plate at 37 C for at least 2 h.b.Remove the NutriStem hPSC XF medium from the cells and wash the cells twice with PBS(-Ca 2+ /-Mg 2+ ).c. Add 1 mL of TrypLE Select to the well and incubate at 37 C for 4 min.d.Gently pipette into a single cell suspension, then add 1 mL of DTI and then collect all cells in a 15-mL tube with 5 mL of NutriStem hPSC XF medium.e. Centrifuge the cells at 180 G for 3-5 min at room temperature (20 C-25 C). f.In the meantime, wash the coated well twice with PBS(-Ca 2+ /-Mg 2+ ) and add 1 mL of NutriStem hPSC XF medium supplemented with 10 mM Y-27632.g.After centrifugation, discard the supernatant and resuspend the cells in 1 mL of NutriStem hPSC XF medium supplemented with 10 mM Y-27632.h.Count the cells using a hemocytometer.i. Seed 5 million cells in a final volume of 5 mL of NutriStem hPSC XF medium supplemented with 10 mM Y-27632, to achieve a final seeding density of 500,000 cells/cm 2 .j.The next day (day -1), change the culture medium: 5 mL of NutriStem hPSC XF medium without Y-27632.
CRITICAL: Initial cell density is particularly important for a successful differentiation outcome.A seeding density of 500,000 cells/cm 2 should be achieved.5. On day 0, discard the culture medium and add 5 mL of neural induction medium supplemented with 200 nM LDN193189 and 10 mM SB431542.
Note: LDN193189 and SB431542 inhibit SMAD signaling to induce neuroectoderm. 6On day 1 and 2, discard the culture medium and add 5 mL of the neural induction medium supplemented with 200 nM LDN193189, 10 mM SB431542 and 2 mM purmorphamine.
Note: Purmorphamine is a potent agonist of the Smoothened receptor, activates Sonic Hedgehog signaling and is used to induce floorplate identity. 7 On day 3 and 4, discard the culture medium and add 5 mL of the neural induction medium supplemented with 200 nM LDN193189, 10 mM SB431542, 2 mM purmorphamine and 1.5 mM CHIR99021.
Note: CHIR99021 is a GSK3b inhibitor that activates Wnt/b-catenin signaling and was used to induce caudal ventral midbrain progenitors. 8 From day 5 to day11, media was gradually changed from neural induction medium to neural differentiation medium.On day 5 and 6, discard the culture medium and add a total volume 6 mL (4.5 mL of the neural induction medium + 1.5 mL) of neural differentiation medium supplemented with 200 nM LDN193189, 10 mM SB431542, 2 mM purmorphamine and 1.5 mM CHIR99021.9. On day 7 and 8, discard the medium and add a total volume 6 mL (3 mL of neural induction medium + 3 mL of neural differentiation medium) supplemented with 200 nM LDN193189, 2 mM purmorphamine, 1.5 mM CHIR99021 and 100 ng/mL WNT5A.
Note: WNT5A is a signaling molecule which activates the Wnt/Rac1 pathway and is used to promote mDA differentiation. 9.On day 9 and 10, discard the culture medium and add a total volume 8 mL (2 mL of neural induction medium + 6 mL of the neural differentiation medium) supplemented with 200 nM LDN193189, 2 mM purmorphamine, 1.5 mM CHIR99021, 100 ng/mL WNT5A and 100 ng/mL FGF8B.
Note: FGF8B is a growth factor that is used to induce caudal midbrain identity. 10.Replate the cells on day 11.a. Mix 100 mL of LN511 and 1,400 mL of PBS(+Ca 2+ /+Mg 2+ ) and coat a well of a 6-well plate at 37 C for at least 2 h.b.Remove the culture medium from the cells and wash the cells twice with PBS(-Ca 2+ /-Mg 2+ ).c. Add 1 mL of TrypLE Select with 100 mg/mL of DNase I to the well and incubate at 37 C for 10 min.d.Gently pipette into a single-cell suspension, add 1 mL of DTI with 100 mg/mL of DNase I for effective cell dissociation and then collect all cells in a 15-mL tube with 5 mL of neural differentiation medium.e. Count the cells using a hemocytometer.f.Centrifuge the cells at 180 G for 3-5 min at room temperature (20 C-25 C). g.In the meantime, wash the coated well twice with PBS(-Ca 2+ /-Mg 2+ ) and add 2 mL of the neural differentiation medium supplemented with 10 mM Y-27632, 2 mM purmorphamine, 7.5 mM CHIR99021 and 100 ng/mL FGF8B.h.After centrifugation, discard the supernatant and resuspend the cells in 1 mL of the neural differentiation medium supplemented with the factors in step 11g.
i. Seed 5 million cells in a final volume of 5 mL of the neural differentiation medium supplemented with the factors in step 11g, to achieve a final seeding density of 500,000 cells/cm 2 .12. From day 12 to day 15, discard the culture medium daily and add 5 mL of the neural differentiation medium supplemented with 2 mM purmorphamine, 7.5 mM CHIR99021 and 100 ng/mL FGF8B.Figure 1C shows an example of cell morphologies at this stage.13.Replate the cells on day 16.a. Mix 100 mL of LN511 and 1,400 mL of PBS(+Ca 2+ /+Mg 2+ ) and coat a well of a 6-well plate at 37 C for at least 2 h.b.Remove the culture medium from the cells and wash the cells twice with PBS(-Ca 2+ /-Mg 2+ ).c. Add 1 mL of TrypLE Select with 100 mg/mL of DNase I to the well and incubate at 37 C for 15 min.d.Gently pipette into a single-cell suspension, add 1 mL of DTI with 100 mg/mL of DNase I and then collect all cells in a 15-mL tube with 5 mL of neural differentiation medium.e. Count the cells using a hemocytometer.f.Centrifuge the cells at 180 G for 3-5 min at room temperature (20 C-25 C). g.In meantime, wash the coated well twice with PBS(-Ca 2+ /-Mg 2+ ) and add 2 mL of the neural differentiation medium supplemented with 10 mM Y-27632, 10 mM GW3965, 10 mM DAPT, 20 ng/mL BDNF and 200 mM ascorbic acid.
Note: GW3965 is a synthetic liver X receptor ligand that is used to promote mDA neurogenesis. 11,12DAPT is a g-secretase inhibitor used to promote neuronal maturation. 13After centrifugation, discard the supernatant and resuspend the cells in 1 mL of the neural differentiation medium with the factors in step 13g.i. Seed 7 million cells in a final volume of 5 mL of the neural differentiation medium supplemented with the factors in step 13g, to achieve a seeding density of 700,000 cells/cm 2 .
Note: It can be hard to completely dissociate the cells into a single-cell suspension at this time.It is normal to observe some cell clumps.We do not recommend persisted pipetting to avoid damaging the cells.
14. Quality check of the cells by immunostaining for LMX1, FOXA2, OTX2 and EN1 (Figure 2).a. Seed 200,000 cells dissociated in step 13 in a well of 96-well black flat bottom plate coated with LN511.b.The next day, wash the cells with PBS(-Ca 2+ /-Mg 2+ ) and fix with 4% PFA for 20 min at 4 C. c. Remove the PFA and wash the cells three times with PBS.d.Remove the PBS and block the cells for 1 h at room temperature (20 C-25 C) in blocking solution (PBS with 0.1% Triton-X and 5% normal donkey serum).e. Dilute the primary antibodies in the blocking solution and incubate at 4 C for overnight (15-22 h).See key resources table for antibody dilution factors.f.Remove the antibody solution and wash the cells three times with PBS.g.Dilute the secondary antibodies (1:500) in the blocking solution and incubate for 1-2 h at room temperature (20 C-25 C) in the dark.h.Remove the secondary antibody solution and incubate with DAPI (diluted 1:5,000 in blocking solution) for 15 min at room temperature (20 C-25 C) in the dark.i. Remove the DAPI solution and wash the cells three times with PBS.j.Image the cells or store them in PBS at 4 C in the dark until imaging.

CRITICAL:
The concentration of CHIR99021 is essential for the cells to adopt a midbrain fate.The optimal concentration to specify mDA progenitors depends on the hESC line, the medium and the duration of CHIR99021 treatment.Therefore, the optimal concentration of CHIR99021 for midbrain patterning should be examined and adjusted for each individual cell line and culture condition before starting a full differentiation experiment.See also troubleshooting.Optional: The cells can be frozen and stored at this stage (day 16) in liquid nitrogen.k.Making an intermediate frozen stock at day 16.i. Resuspend the cells dissociated in step 13 at a density of 5 million cells/mL in STEM-CELLBANKER GMP-Grade.ii.Distribute 0.5 mL of the cell suspension per cryovial (2.5 million cells/vial) and store at À80 C in a cold Cryo1 C freezing container.Transfer the cryovials to a liquid nitrogen tank the next day for long-term storage.l.Thawing the frozen stock.
i. Mix 100 mL of LN511 and 1,400 mL of PBS(+Ca 2+ /+Mg 2+ ) and coat a well of a 6-well plate at 37 C for at least 2 h.ii.Slowly thaw three vials (7.5 million cells in total) in a 37 C water bath, and immediately collect into a 15-mL tube with 10 mL of the neural differentiation medium.iii.Centrifuge the pooled cells at 180 G for 3-5 min at room temperature (20 C-25 C). iv.In the meantime, wash the coated well twice with PBS(-Ca 2+ /-Mg 2+ ) and add 1 mL of the neural differentiation medium supplemented with 10 mM Y-27632, 10 mM GW3965, 10 mM DAPT, 20 ng/mL BDNF and 200 mM ascorbic acid.v.After centrifugation, discard the supernatant and resuspend in 4 mL of the neural differentiation medium supplemented with the factors in point iv.vi.For neuronal differentiation, seed all cells in a final volume of 5 mL of the neural differentiation medium supplemented with the factors in point iv.

Maturation of progenitors into mDA neurons
Timing: > 6 weeks (for all steps in this section) Timing: 4 days (for step 15) Timing: 6 days (for step 16) Timing: > 30 days (for step 17) Timing: 2 days (for step 18) This step is to terminally differentiate mDA progenitors into mDA neurons.Here we describe the procedure for a 6-well plate.Please choose the appropriate well for maturation culture according to the experiments you analyze.
15. From day 17 to day 20, discard the culture medium daily and add 5 mL of neural differentiation medium supplemented with 10 mM GW3965, 10 mM DAPT, 20 ng/mL BDNF and 200 mM ascorbic acid.
Optional: Replate the cells on day 21 to avoid clumping if the cells will be cultured beyond day 28.
a. Mix 100 mL of LN511 and 1,400 mL of PBS(+Ca 2+ /+Mg 2+ ) and coat a well of a 6-well plate at 37 C for at least 2 h.b.Remove the culture medium from the cells and wash the cells twice with PBS(-Ca 2+ /-Mg 2+ ).c. Add 1 mL of TrypLE Select with 100 mg/mL of DNase I to the well and incubate at 37 C for 20 min.d.Gently pipette into a single-cell suspension, add 1 mL of DTI with 100 mg/mL of DNase I and then collect all cells in a 15-mL tube with 5 mL of neural differentiation medium.e. Count the cells using a hemocytometer.f.Centrifuge the cells at 180 G for 3-5 min at room temperature (20 C-25 C). g.In the meantime, wash the coated well twice with PBS(-Ca 2+ /-Mg 2+ ) and add 2 mL of the neural differentiation medium supplemented with 10 mM Y-27632, 10 mM DAPT, 20 ng/mL BDNF, 10 ng/mL GDNF, 200 mM ascorbic acid, 500 mM dbcAMP, 1 mM PD0325901, 5 mM SU5402 and 1 ng/mL TGFb3.h.After centrifugation, discard the supernatant and resuspend the cells in 1 mL of the neural differentiation medium supplemented with the factors in step 15g.i. Seed 5 million cells in a final volume of 5 mL of the neural differentiation medium supplemented with the factors in step 15g.
Note: Do not replate the cells after day 22 since mature neurons are vulnerable to mechanical stress.
Note: Media should be carefully changed (slow pipetting) after day 28 to avoid cell detachment.
Note: The cells formed aggregates and neurite extensions during long-term culture (Figure 1).Action potentials and dopamine release were observed at day 56.

EXPECTED OUTCOMES
This protocol leads to the generation of mDA progenitors of high quality, which can give rise to functional mDA neurons in the four hESC lines we tested.We investigated and characterized mDA progenitors and neurons by immunostaining and qPCR.The functionality of the mDA neurons was examined by HPLC, to detect DA release, and by electrophysiology.We also characterized the cell types generated by differentiation and maturation of hESCs by single-cell RNA-sequencing.We found that the in vitro differentiation process correctly captured human ventral midbrain development as previously defined by single-cell RNA-sequencing by La Manno et al. (2016). 18For more information, please refer to Nishimura et al. ( 2023). 1

LIMITATIONS
The proliferation and differentiation efficiency can vary depending on the cell line.Each cell line needs to be examined in small-scale experiments to identify any possible differentiation bias and optimize the concentration of CHIR99021 for appropriate midbrain patterning.After day 16, cells exit from cell cycle and need to be carefully manipulated (slow media changes) until the end of the experiment to avoid detachment and/or cell loss.To ensure that high quality mDA neurons are generated, the expression of midbrain transcription factors and markers must be examined as indicated above at different intermediate time points during the differentiation process.This protocol requires previous experience in working with hESCs, is time-consuming and expensive, but results in high-quality mDA neurons as defined by in vitro functionality and single-cell RNAsequencing compared to the endogenous human ventral midbrain.

Potential solution
Increase the volume of the culture medium.This protocol starts with high cell density (500,000 cells/ cm 2 ).Cells require media changes every day.Proliferation is promoted by adding CHIR99021 and/or FGF8B.This can be noticed by the fast change in color of the culture media from red towards yellow.At this stage, the culture media needs to be changed daily and increasing volumes should be used to avoid it becoming yellow.
You need to check the cells by immunostaining for LMX1, FOXA2 and OTX2 on day16 (step 14).If you observe LMX1 + and FOXA2 + cells, and many OTX2 -cells, the cell identity is biased towards a caudal cell fate.Conversely, if you observe few LMX1 + and FOXA2 + and many OTX2 + cells, the cell identity is biased towards other rostral cell fates.

Potential solution
The concentration of CHIR99021 needs to be adjusted.Correct CHIR99021 concentration is essential to induce ventral midbrain identity.CHIR99021 needs to be adjusted to your cell line and culture media/conditions.We recommend purchasing a large volume of CHIR99021 and making aliquots to avoid variability.A successful differentiation shows R 80% of LMX1 and FOXA2 cells, R 80% of LMX1 and OTX2 cells, and z60% of LMX1 and EN1 cells on day 16.

Problem 3
Cells detach from the edge of well during maturation into mDA neurons (steps 15-17).

Potential solution
Make sure that you change the culture media slowly and carefully, you have used an appropriate well and that coating is optimal (coating can be extended for overnight (15-22 h), the laminin solution may contain sodium azide, which needs to be properly washed).

Problem 4
The number of differentiated mDA neurons is low (step 18).
m. Resuspend cells dissociated in step 3 at 1 million cells/mL in STEM-CELLBANKER GMP-Grade.n.Distribute 0.5 mL per cryovial (500,000 cells/vial) and store at À80 C in a cold Cryo1 C freezing container.Transfer the cryovials to a liquid nitrogen tank the next day for long-term storage.Differentiation of hESCs into mDA progenitorsTiming: 3 weeks (for all steps in this section) Timing: 2 days (for step 4)Timing: 1 h (for steps 4a to 4i) Timing: 11 days (for steps 5 to 10)Timing: 1 h (for step 11) Timing: 4 days (for step 12)Timing: 1 h (for step 13) Timing: 2 days (for step14)

Figure 1 .
Figure 1.Overview of mDA neuron induction protocol from hESCs (A) A Schematic procedure for induction of mDA neurons from hESCs.(B-I) Phase contrast images during mDA differentiation of hESCs.(B) Undifferentiated hESCs, (C, D) mDA progenitor at days 16 and 21, and (E-I) mDA neuron maturation from day 28 to 56.Scale bars, 100 mm.
Prepare aliquots and store at À20 C. Thawed aliquots can be stored up to 4 weeks at 4 C. Recombinant proteins can be stored up to a week at 4 C. Avoid repeated freezing and thawing of the aliquots.All factors should be supplemented in the media just before use.All factors should be supplemented in the media just before use.
All factors should be supplemented in the media just before use.All factors should be supplemented in the media just before use.

Table 1 .
Coating the well