TITLE: An Optimized Protocol for the Generation of Midbrain Dopamine Neurons from human iPSCs under Defined Conditions
AUTHORS: Carlos  W. Gantner, Agustín Cota-Coronado, Lachlan H. Thompson, Clare L Parish

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center"><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;"> </span></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/d2df7vcw.jpg" /></div><div class = "text-block">Here, we describe a xeno-free, feeder-free, and chemically defined protocol for the generation of ventral midbrain dopaminergic (vmDA) progenitors from human pluripotent stem cells (hPSCs). This simple-to-follow protocol results in high yields of cryopreservable dopamine neurons across multiple hPSC lines. Wnt signaling is the critical component of the differentiation and can be finely adjusted in a line-dependent manner to enhance production of dopamine neurons for the purposes of transplantation, studying development and homeostasis, disease modeling, drug discovery, and drug development. </div><div class = "text-block">For complete details on the use and execution of this protocol, please refer to Gantner et al. (2020) and Niclis et al. (2017a).</div><br/><br/><div class = "text-block"><span style = "font-weight:bold;">HIGHLIGHTS</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Reproducible differentiation of human dopamine neurons from multiple hPSC lines</li><li style = "counter-reset:ol0;list-style-type:disc;">Dopamine progenitors can be cryopreserved for downstream applications</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Dopamine neurons mature </span><span style = "font-style:italic;">in vitro</span><span>, enabling screening or developmental studies</span></li><li style = "counter-reset:ol0;list-style-type:disc;">Transplanted dopamine progenitors are capable of restoring motor function</li></ul></div></div>

[STEPS]
?. [BEFORE YOU BEGIN: Pluripotent Stem Cell Thawing ]
Coat a T25 flask with – and leave in the incubator for ≥ (see Tables 1 and 2 for laminin-521 dilution).
[Laminin-521]
Note:   is sufficient for cell lines that have been adapted to Laminin-521. For non-adapted lines (i.e. hPSC lines maintained on substrates other than laminin-521)   may be required to ensure attachment during initial passages. Laminin-521 can also be added for  –  at  , although ≥  at   is preferable.
CRITICAL: Laminin-521 must be diluted in PBS+/+. Diluting in PBS-/- will result in poor attachment and survival. Ensure that enough laminin solution is made to coat the entire flask throughout the coating period. Do not use flasks which have dried as cells will not attach in these areas.
?. [BEFORE YOU BEGIN: Pluripotent Stem Cell Thawing ]
Equilibrate  in a tightly closed 15 mL tube in the incubator for ≥.
[pre-made mTeSR1]
?. [BEFORE YOU BEGIN: Pluripotent Stem Cell Thawing ]
Rapidly thaw a cryovial of hPSC in a  water bath and transfer the contents to  using a P1000 pipette.
37 °C
[pre-warmed mTeSR1 solution]
?. [BEFORE YOU BEGIN: Pluripotent Stem Cell Thawing ]
Centrifuge cells at , aspirate supernatant, and resuspend the cell pellet in , supplemented with .
Centrifuge: 300 34, 3 min, 21 10
[mTeSR1 media]
[Y27632]
CRITICAL: Addition of the ROCK inhibitor Y27632 is essential for hPSC survival, particularly if the cells have been cryopreserved as single cells rather than aggregates.
?. [BEFORE YOU BEGIN: Pluripotent Stem Cell Thawing ]
Aspirate laminin from the T25 flask and immediately plate the cell suspension, gently rocking the flask multiple times up-and-down and then side-to-side to evenly distribute the cells.
?. [BEFORE YOU BEGIN: Pluripotent Stem Cell Culture ]
One day after thawing, wash once with PBS-/- to remove debris, and replace with fresh media.
?. [BEFORE YOU BEGIN: Pluripotent Stem Cell Culture ]
Continue to culture hPSC until they reach 70%–80% confluence, changing media every day.
Note: Generally, cells will recover quickly after thawing and should reach confluency within 5 days. However, if cells are growing slowly it is best to passage at a lower density (50%– 60% confluence) or after 5–6 days.
?. [BEFORE YOU BEGIN: Pluripotent Stem Cell Culture ]
To passage hPSC, wash once in PBS-/- and add . Leave at  and monitor cell morphology under the microscope until colonies are loosely attached (–).
[ReLeSR]
37 Room temperature
?. [BEFORE YOU BEGIN: Pluripotent Stem Cell Culture ]
Remove ReLeSR carefully and rapidly tap the flask hard 3–4 times on each long edge to detach colonies.
?. [BEFORE YOU BEGIN: Pluripotent Stem Cell Culture ]
Resuspend by dissociating colonies in  using a P1000 pipette. Dilute cells (1:10–1:20) and transfer using a 5 mL serological pipette to a Laminin-521 coated T25 flask.
[equilibrated mTeSR1]
Note: Morphology during passage can vary between cell lines and the optimal timing of ReLeSR should be determined accordingly. The majority of cells should remain as 10–50 cell aggregates, removing the need for Y27632. Add Y27632 if passaged as single cells. Likewise, the growth rate of each line will differ, and passage ratios should be adjusted to compensate. We recommend passaging every 3–5 days.
Optional: Cryopreserve stem cells at low passage number by adding  ,   and   to isolated cell pellets (after ReLeSR dissociation) and slow-freeze at . 3–4 cryovials can be generated from a single, confluent T25 flask. Transfer to liquid nitrogen for long-term storage.
Pause Point: hPSC can be maintained by passaging every 3–5 days until the desired experimental start time. Thinking ahead and knowing the growth characteristics of the cell line are helpful in aligning cells at 70%–80% confluence with the desired start date.
CRITICAL: Always passage thawed cells ≥2 times before beginning a differentiation experiment, or until the cell line displays normal morphology and growth rate characteristics.
CRITICAL: Routine mycoplasma testing is essential to maintaining high-quality hPSC stocks that will consistently differentiate. The quality of stem cells will dictate differentiation efficiency and should be closely monitored by testing, morphology and immunostaining (e.g. ≥ 95% OCT4+). In addition, we do not recommend using high passage number hPSC.
?. [BEFORE YOU BEGIN: Preparing Differentiation Media ]
Prepare media as outlined in the Materials and Equipment section below (Tables 3 and 4).
Note: Always prepare media as close to use as possible and use within 2 weeks. It is best to make Induction Media the day of the experiment and Maturation Media at day 11. Where possible, add each factor (SHH, CHIR, etc.) to the base media on the day to minimize degradation.
?. [Plating hPSC for Differentiation]
Timing: 1 hThis section describes the passaging of hPSC cultures into monolayers appropriate for differentiation.
?. [Plating hPSC for Differentiation]
Coat a 48-well plate with  and leave in the incubator for ≥ (see Tables 1 and 2 for laminin-521 dilution).
[Laminin-521]
Note: Differentiation can proceed in any size multi-well plate or flask. Adjust the Laminin-521 dilution, seeding density, and media volume accordingly.
?. [Plating hPSC for Differentiation]
Passage hPSC at 70%–80% confluency by washing once in PBS-/-, adding , and placing in the incubator for –. Monitor the morphology of the cells closely during this period. Once colonies show a characteristic bright halo and are close to detaching, gently pipette using a P1000 until ≥90% of the flask has detached.
[Accutase]
?. [Plating hPSC for Differentiation]
Rapidly dilute Accutase solution in ≥ and centrifuge at .
[ mTeSR1]
Centrifuge: 300 34, 3 min, 21 10
?. [Plating hPSC for Differentiation]
Aspirate supernatant, resuspend the cell pellet in fresh  mTeSR1 supplemented with  and count live cells using a hemocytometer.
[warmed]
[Y27632]
Note: Resuspend hPSC at 1–2 x 106 cells/mL to enable rapid, accurate counting. Typically, we resuspend hPSC in 1 mL/5–10 cm2 . Each cell line grows at a different density and therefore the correct volume to resuspend cells in should be determined individually.
?. [Plating hPSC for Differentiation]
Seed 0.3 x 106 cells/cm2 (48 well surface area = ∼1 cm2) in ≥. Rock the plate in a ‘‘figure eight’’ movement to evenly distribute cells and leave to settle for – before placing in the incubator.
[mTeSR1 + Y27632]
Note: It is crucial to start the differentiation with an even monolayer of cells. Carefully dissociating to a single cell solution is important as it allows for an accurate count and an even distribution upon seeding. It is equally important not to prolong exposure of the hPSCs to enzymatic dissociation as this will impair survival. The duration of enzymatic treatment and the balance between optimal dissociation and survival may vary between cell lines.
Note: Initial seeding density should be optimized for each cell line to ensure confluency 24 h after seeding. Typically, we seed 0.2–0.4 x 106 cells/cm2 . Trypan blue exclusion dye can be utilized during cell counting to ensure that only viable cells are included in the final cell seeding calculation.
CRITICAL: Seeding too many cells can lead to early detachment during differentiation, while seeding an insufficient number of cells can lead to poor survival and off-target differentiation.
?. [vmDA Progenitor Differentiation]
Timing: 13 daysThis step describes the steps needed to differentiate hPSCs into vmDA progenitors.See Figure 1A for a schematic of the differentiation.
?. [vmDA Progenitor Differentiation]
Day 0: 24 h after seeding check under the microscope that hPSC have survived and appear as a single, confluent (≥95%) monolayer with no obvious differentiation or uneven distribution.
Note: No (or very few small) gaps should be present the day after seeding the cells. If it is hard to see, wash twice with PBS-/- and check under the microscope. In addition, there should be very little death. If there is a large amount of death or cells are not confluent, it is best to discard the cells and start again.
?. [vmDA Progenitor Differentiation]
Prepare Induction Media and supplement with  and , SMAD inhibitors of the Transforming Growth Factor-b and Bone Morphogenetic Protein pathways, respectively.
[SB431542]
[LDN193189]
?. [vmDA Progenitor Differentiation]
Gently wash each well twice with PBS-/- to remove residual mTeSR and replace media with .
[supplemented Induction Media]
?. [vmDA Progenitor Differentiation]
Day 1: Change media to  supplemented with , , as well as ventralizing factors  and .
[Induction Media ]
[SB431542]
[LDN193189]
[C25II SHH]
[purmorphamin]
?. [vmDA Progenitor Differentiation]
Days 2, 3 and 4: Change media to   supplemented with , , ,  and  to caudalize developing progenitors.
500 µl
[SB431542]
[LDN193189]
[C25II SHH]
[purmorphamine]
[Wnt-agonist CHIR99021]
Note: Media is changed every 24 h and 500 mL/48-well is added at days 0–4. At day 5 and beyond media is changed every other day and the volume is increased to 1 mL/48-well.
?. [vmDA Progenitor Differentiation]
Day 5: Change media to  supplemented with , ,  and .
[Induction Media]
[LDN193189]
[ C25II SHH]
[purmorphamine]
[CHIR99021]
?. [vmDA Progenitor Differentiation]
Days 7 and 9: Change media to  supplemented with  and .
[Induction Media]
[LDN193189]
[CHIR99021]
?. [vmDA Progenitor Differentiation]
Day 11: Wash cultures twice in PBS-/- and switch base media to Maturation Media with , , , ,  , and , supplemented with .
[rhBDNF]
[rhGDNF]
[dibutyryl cAMP]
[Ascorbic Acid]
[rhTGFb3]
[DAPT (Maturation Media + BGCATD)]
[CHIR99021]
Optional: Analyze cells at the end of progenitor patterning, day 13, for markers of vmDA progenitors by fixation and immunofluorescence or collection of cells for RT-qPCR. See below for the immunostaining protocol. By day 13 the majority (≥80%) of cells should co-express OTX2, FOXA2 and Nestin (Figure 2A; Table 5). Together, these markers indicate correct specification of ventral midbrain progenitors.
Note: If vmDA progenitors are too dense at day 13 for accurate quantification, it is possible to passage the cells with Accutase. If so, replate cells in Maturation Media + BGCATD, supplemented with    at 0.3–0.45 x 106/cm2 . Fix 24 h later, after cells have attached, for analysis or continued culture.
CRITICAL: Developing neural progenitors are extremely sensitive to CHIR99021 concentration. Take care when diluting new vials of CHIR99021 and precisely pipette the correct volume during the experiment. It is best to always add CHIR99021 on the day if possible.
?. [Terminal Differentiation and Long-Term Culture of vmDA Neurons]
Timing: 12+ daysThis step describes the long-term culture of vmDA progenitors and the generation of vmDA neurons.
?. [Terminal Differentiation and Long-Term Culture of vmDA Neurons]
Days 13–30. Change media every other day to Maturation Media + BGCATD.
Optional: Transplantation of vmDA progenitors is optimal between days 13–22. Wash cells twice in PBS-/-, add 400 mL/48-well of Accutase and incubate for  –  at  . Monitor the wells closely under a microscope and test detachment every few minutes by gently pipetting the Accutase directly onto the cells. Once cells begin to detach, gently pipette using a P1000 to collect ≥90% of the well and further dissociate vmDA progenitors. Accutase can be left on for up to 20 min for slowly detaching, more dense cultures. Arrest enzymatic digestion when cells have dissociated into small (5–20 cell) aggregates by adding Maturation Media + BGCATD, supplemented with   and centrifuge at  . Aspirate the supernatant, resuspend the cell pellet in (1 mL/cm2) Maturation Media + BGCATD +   and count viable progenitors using a hemocytometer and Trypan blue exclusion. Centrifuge at  , aspirate supernatant, and resuspend at 0.1–0.2 x 106/μL in Maturation Media + BGCATD +    and store   prior to transplantation.
Note: From day 18–20, vmDA neurons will become increasingly abundant and can be indirectly observed by the formation of neuronal bunches and axonal projections. By day 25, these neuronal bundles become dense and highly arborized (Figure 1B), making dissociation challenging and resulting in poor survival after plating and following transplantation. Viability when counting cells for further culture or transplantation should be ≥90%. Cells should not be stored on ice for >5 h.
Optional: We routinely characterize our cultures at days 13, 16, and 25 to confirm the degree of vmDA specification and neuronal identity within the cultures. If desired, fix cultures or recover cells from wells at these stages for immunofluorescence and/or RT-qPCR. vmDA progenitors co-expressing LMX1A, EN1, NURR1 should be apparent by D16, and vmDA neurons co-expressing FOXA2 along with the postmitotic lineage markers PITX3 and TH should be readily apparent by D25 (Figure 2B; Table 5).NestinOTX2FOXA2LMX1AEN1NURR1PITX2BARHL1(PITX3)eGFPTH1:10001:10001:5001:2001:501:2001:5001:5001:10001:1000Day 13++++++++++++/--/++/-+/---Day 16++++++++++++++++/-+/-+/-+/-Day 25+++++++++++/-++/-+/-++++Day 60+/-++++++/-+/-+/-+/-++++++
?. [Terminal Differentiation and Long-Term Culture of vmDA Neurons]
From day 30 onwards, perform half-media changes, such that ∼ is removed from each well and ∼ of fresh, equilibrated media is added to maintain ∼/48-well. At these late stages of the differentiation, media changes can be done 2–3 times per week.
400
500
1 mL
Note: For in vitro studies of DA development, homeostasis, disease modelling, or for drug screening approaches, vmDA neurons can be cultured until at least day 60, at which point electrophysiologically active vmDA neurons will be present.
?. [Cryopreservation of vmDA Progenitors]
Timing: 1 hThis step describes the protocol for generating frozen stocks of vmDA progenitors for downstream applications.
Note: Cryopreserving vmDA progenitors between days 13 and 19 is ideal to balance lineage acquisition and survival.
?. [Cryopreservation of vmDA Progenitors]
Label cryovials and cool freezing container to .
4 °C
?. [Cryopreservation of vmDA Progenitors]
Prepare freeze media containing  and  and  + BGCATD + .
[DMSO]
[KnockOut serum replacement]
[fresh Maturation Media]
[Y27632]
Note:   and   are needed per 1 mL cryovial. Typically, we cryopreserve vmDA progenitors at 3 x 106/mL.
?. [Cryopreservation of vmDA Progenitors]
Wash wells twice in PBS-/- and add /48-well of Accutase. Place in incubator for –. Monitor cell morphology closely under the microscope.
400
?. [Cryopreservation of vmDA Progenitors]
Dissociate cells by gentle pipetting using a P1000 and dilute Accutase in Maturation Media + BGCATD + Y27632.
?. [Cryopreservation of vmDA Progenitors]
Centrifuge cells at , aspirate supernatant and resuspend in Maturation Media + BGCATD +  (1 mL/cm2) at approximately 1 mL x 106/mL for accurate counting using a hemocytometer and Trypan blue exclusion.
Centrifuge: 300 34, 3 min
[Y27632]
?. [Cryopreservation of vmDA Progenitors]
Centrifuge at , aspirate supernatant and resuspend in fresh Maturation Media + BGCATD +  at 6 x 106/mL.
Centrifuge: 300 34, 3 min
[Y27632]
?. [Cryopreservation of vmDA Progenitors]
Transfer  to each cryovial and add 500 mL of freeze media, pipetting twice to gently mix.
[of the cell solution]
?. [Cryopreservation of vmDA Progenitors]
Rapidly move vials to the freezing container and place at .
-80 °C
?. [Cryopreservation of vmDA Progenitors]
After ≥, transfer cryovials to LN2 for long-term storage.
Pause Point: vmDA progenitors can be stored for periods ≥1 year.
CRITICAL: Ensure cells are exposed to Accutase for the minimum required time that still allows for accurate quantification. Maximizing the presence of small aggregates (2–10 cells) will improve cell survival. In contrast, over-dissociation in Accutase, leading to a high number of single cells, will significantly impair survival during thawing.
?. [Recovery of Cryopreserved vmDA Progenitors]
Timing: 30 minSee Figure 3 for an overview of vmDA progenitor maturation following thawing.This step describes how to thaw cryopreserved vmDA progenitors for use in downstream applications.
?. [Recovery of Cryopreserved vmDA Progenitors]
Coat 48-well plates with  and place in incubator for ≥.
[Laminin-521]
?. [Recovery of Cryopreserved vmDA Progenitors]
Equilibrate  + BGCATD +  at  for ≥.
[Maturation Media]
[Y27632]
37 °C
?. [Recovery of Cryopreserved vmDA Progenitors]
Rapidly thaw a cryovial of vmDA progenitors in a  water bath and transfer the contents to using a P1000 pipette.
37 °C
[prewarmed media]
?. [Recovery of Cryopreserved vmDA Progenitors]
Centrifuge at , aspirate supernatant and resuspend in Maturation Media +BGCATD + .
Centrifuge: 300 34, 3 min
[Y2732]
?. [Recovery of Cryopreserved vmDA Progenitors]
Plate progenitors at 0.3–0.45 x 106/cm2.
Optional: Count vmDA progenitors using a hemocytometer to ascertain an accurate count of surviving cells. Live/dead analysis using FACS can also be used with a subfraction of the cells, in addition to Trypan blue exclusion. Viability immediately post-thaw is typically 60%–70%, although a further proportion may undergo apoptosis after re-plating.
Optional: Transplant vmDA progenitors directly after thawing. Wash once in PBS-/- aftercounting, centrifuge, aspirate supernatant and resuspend in Maturation Media + BGCATD +  at 0.1–0.2 x 106/μL for transplantation.
Optional: For long-term culture of vmDA neurons for studying development, drug screening, and/or disease modeling, wash cells once in PBS-/- the day after thawing and replace media with   + BGCATD. Proceed with steps 28–29 to continue thedifferentiation.
Note: The concentration of rhGDNF and Y27632 can be doubled to   and  , respectively, if survival upon thawing is poor.
?. [Immunostaining of vmDA Progenitors and Neurons]
Timing: 2 daysThis step outlines how to undertake immunocytochemical profiling of generated vmDA progenitors.
?. [Immunostaining of vmDA Progenitors and Neurons]
Wash wells once with PBS-/- to remove debris and add . Leave at for .
[4% paraformaldehyde]
21 Room temperature
?. [Immunostaining of vmDA Progenitors and Neurons]
Aspirate paraformaldehyde and wash 3 times with PBS-/-, leaving wells in PBS-/- for at least between washes.
Pause Point: Fixed plates can be stored for several months at  . Addition of   to PBS-/- can reduce possible bacterial or fungal contamination if storing long-term.
?. [Immunostaining of vmDA Progenitors and Neurons]
Prepare blocking buffer by adding  (same as secondary antibodies) and  to .
[species-specific serum]
[10% Triton X-100]
[PBS-Azide 0.05%]
?. [Immunostaining of vmDA Progenitors and Neurons]
Add /48-well of blocking buffer to each well for –.
200
?. [Immunostaining of vmDA Progenitors and Neurons]
Dilute the primary antibodies in blocking buffer. Aspirate blocking buffer and add /48- well of primary antibody solution. Incubate at  for –.
200
4 °C
?. [Immunostaining of vmDA Progenitors and Neurons]
Aspirate paraformaldehyde.
?. [Immunostaining of vmDA Progenitors and Neurons]
Leave wells in PBS-/- for at least .
?. [Immunostaining of vmDA Progenitors and Neurons]
Wash with PBS-/-.
?. [Immunostaining of vmDA Progenitors and Neurons]
Leave wells in PBS-/- for at least .
?. [Immunostaining of vmDA Progenitors and Neurons]
Wash with PBS-/-.
?. [Immunostaining of vmDA Progenitors and Neurons]
Wash with PBS-/-.
?. [Immunostaining of vmDA Progenitors and Neurons]
Wash wells 3 times in PBS-/- to remove primary antibodies and block cells again for – by adding .
[blocking buffer]
?. [Immunostaining of vmDA Progenitors and Neurons]
Dilute appropriate secondary antibodies in blocking buffer and add  to each well. Incubate cultures in the dark at  for –.
[secondary antibody solution]
21 Room temperature
?. [Immunostaining of vmDA Progenitors and Neurons]
Wash wells in PBS-/- to remove primary antibodies.
?. [Immunostaining of vmDA Progenitors and Neurons]
Wash wells in PBS-/- to remove primary antibodies.
?. [Immunostaining of vmDA Progenitors and Neurons]
Wash wells in PBS-/- to remove primary antibodies.
?. [Immunostaining of vmDA Progenitors and Neurons]
Block cells again for – by adding .
[blocking buffer]
?. [Immunostaining of vmDA Progenitors and Neurons]
Wash wells in PBS-/- and add  for .
[DAPI (diluted in PBS-/-)]
?. [Immunostaining of vmDA Progenitors and Neurons]
Wash wells twice in PBS-/- and image.
Pause Point: Stained samples can be stored for a week at   before imaging. It is best to store them in PBS-/- +  .
Note: It is optimal to perform immunofluorescence immediately after fixation of the cells. If long-term storage is necessary, ensure sufficient PBS-Azide is added to each well ( ), to account for evaporation during refrigeration and minimize exposure to light to prevent fluorochrome fading.