TITLE: Midbrain dopaminergic differentiation of human pluripotent stem cells
AUTHORS: Tilo Kunath, Szuping Chiu, Sophie Glendinning, Yixi Chen, Nicola Drummond, Maurice Canham

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Differentiation of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) into midbrain dopaminergic (mDA) neurons is described in this protocol. This protocol has been adapted from published protocols from three laboratories, Lorenz Studer (New York), Malin Parmar/Agnete Kirkeby (Lund), and Su-Chun Zhang (Madison). The relevant papers from these labs are PMIDs: 20362538, 22056989, 22813745, 28094017, 28858290, 22696177. This protocol instructs hESCs and hiPSCs to adopt a midbrain floor plate radial glia-like identity before maturation into mDA neurons with mixed substania nigra pars compacta and ventral tegemental area identity. It has been applied to more than 10 hESC and hiPSC lines in our laboratory, with some cell lines producing exceptional results, and others less so. Our version the protocol is published here PMID: 30472757.</div></div>

[STEPS]
?. [Preparing reagents for mDA differentiation]
Prepare a 10 mM SB431542 solution.
?. [Preparing reagents for mDA differentiation]
To prepare 1190 µl of 10 mM SB431542:Add   (2 x ) of DMSO (Sigma Aldrich, cat no. D2650) directly to vial containing   of SB431542 (Merck Millipore, cat no. 616461-5MG) and mix by pipetting until powder is completely dissolved.Prepare -  aliquots of 10 mM SB431542 in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year. Once thawed, keep SB431542 aliquot at  in the dark for up to two weeks.
1190 µl
595 µl
5 mg
20 µl
50 µl
-80 °C
0 Room temperature
?. [Preparing reagents for mDA differentiation]
?. [Preparing reagents for mDA differentiation]
Prepare 10 mM (100,000x) and 0.1 mM (1000x) stocks of LDN193189. C25H22N6. MW: 406.48 g/mol.
?. [Preparing reagents for mDA differentiation]
To prepare 492 µl of 10 mM LDN193189:Add  of DMSO (Sigma Aldrich, cat no. D2650) directly to vial containing  of LDN193189 (Miltenyi Biotec, cat no. 130-103-925) to obtain a  solution. Warm briefly (3-5min) at  to dissolve powder.Alternatively, LDN193189 can be obtained in solution at 10 mM in DMSO (Miltenyi Biotec, cat no. 130-106-540).Dilute 10 mM LDN193189 at 1 in 100 with DMSO (Sigma Aldrich, cat no. D2650) to create  (1000x) stocks.Prepare -  aliquots of 0.1 mM LDN193189 in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year. Once thawed, keep LDN193189 aliquot at  in the dark for up to two weeks.
492 µl
2 mg
37 °C
20 µl
50 µl
-80 °C
0 Room temperature
?. [Preparing reagents for mDA differentiation]
Prepare 600 µg/ml (1000x) stock of Sonic Hedgehog (SHH-C24II).
?. [Preparing reagents for mDA differentiation]
To prepare 833 µl of 600 µg/ml SHH-C24II:Add volume of  of  DPBS without Mg2+ and Ca2+  (Thermo Fisher, Cat no 14190086)  directly to vial containing  of SHH-C24II (R+D Systems, cat no. 1845-SH-500). Add  of 7.5% BSA (Sigma Aldrich, cat no. A8412-100ML) directly to the vial.  Mix by pipetting up and down several times until the powder is dissolved.  Prepare -  aliquots of   SHH in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year. Once thawed, keep SHH aliquot at  for up to two weeks.
822 µl
500 µg
11 µl
20 µl
50 µl
-80 °C
4 °C
?. [Preparing reagents for mDA differentiation]
Prepare 10 mM stock of CHIR99021.
?. [Preparing reagents for mDA differentiation]
To prepare  2414 µl of 10mM CHIR99021:Add  of DMSO (Sigma Aldrich, cat no. D2650) directly to vial containing  of CHIR99021 (Miltenyi Biotech, Cat no 130-103-926) to obtain a  solution. Mix by pipetting up and down several times to dissolve the powder. If precipitate is observed, warm briefly (2-5min) at .Alternatively, CHIR99021 can be obtained in solution at 10 mM in DMSO (Miltenyi Biotech, Cat no. 130-106-539)Prepare aliquots of 10 mM CHIR99021 in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year. Once thawed, keep CHIR99021 aliquot at  in the dark for up to two weeks.
2149 µl
10 mg
37 °C
10 µl
-80 °C
0 Room temperature
?. [Preparing reagents for mDA differentiation]
Prepare 100 µg/ml (1000x) stock of recombinant FGF8b
?. [Preparing reagents for mDA differentiation]
To prepare 250 µl of 100 µg/ml FGF8b:Add  of  DPBS without Mg2+ and Ca2+  (Thermo Fisher, Cat no 14190086) directly to vial containing  of FGF8b (R+D Systems, Cat no 423-F8-025) Add of 7.5% BSA (Sigma Aldrich, cat no. A8412-100ML) directly to the vial.  Mix by pipetting up and down several times to dissolve the powder. Prepare -  aliquots of 100 µg/ml FGF8b in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year. Once thawed, keep FGF8b aliquots at  in the dark for up to two weeks.
246 µl
25 µg
3.4 µl
20 µl
50 µl
-80 °C
4 °C
?. [Preparing reagents for mDA differentiation]
Prepare 1 mg/ml (1000x) stock of Heparin Sodium Salt from porcine intestinal mucosa
?. [Preparing reagents for mDA differentiation]
To prepare 55 ml of 1 mg/ml Heparin:Add  of  DPBS without Mg2+ and Ca2+  (Thermo Fisher, Cat no 14190086) directly to vial containing  of Heparin (Sigma Aldrich, Cat no H3149).Mix by pipetting up and down several times to dissolve the powder. Filter the reconstitued Heparin solution using an appropriatly sized syringe for the volume and 0.33µM filterPrepare  aliquots of   Heparin in sterile 1.5-ml Eppendorfs tubes and store at  for up to two years.
55 ml
55 mg
500 µl
4 °C
?. [Preparing reagents for mDA differentiation]
Prepare 20 µg/ml (1000x) stock of recombinant Bone-Derived Growth Factor (BDNF).
?. [Preparing reagents for mDA differentiation]
To prepare 5 ml of 20 µg/ml BDNF:Add  of  DPBS without Mg2+ and Ca2+  (Thermo Fisher, Cat no 14190086) directly to vial containing  of BDNF (PeproTech, Cat no 450-02).Add of 7.5% BSA (Sigma Aldrich, cat no. A8412-100ML) directly to the vial.  Mix by pipetting up and down several times to dissolve the powder. Prepare -  aliquots of   BDNF in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year. Once thawed, keep BDNF aliquots at  for up to two weeks.
4930 µl
100 µg
70 µl
20 µl
50 µl
-80 °C
4 °C
?. [Preparing reagents for mDA differentiation]
Prepare 20 µg/ml (2000x) stock of recombinant Glial Derived Growth Factor (GDNF).
?. [Preparing reagents for mDA differentiation]
To prepare 5 ml of 20 µg/ml GDNF:Add  of  DPBS without Mg2+ and Ca2+  (Thermo Fisher, Cat no 14190086) directly to vial containing  of GDNF (PeproTech, Cat no 450-10).Add of 7.5% BSA (Sigma Aldrich, cat no. A8412-100ML) directly to the vial.  Mix by pipetting up and down several times to dissolve the powder. Prepare -  aliquots of   GDNF in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year. Once thawed, keep GDNF aliquots at  for up to two weeks.
4930 µl
100 µg
70 µl
20 µl
50 µl
-80 °C
4 °C
?. [Preparing reagents for mDA differentiation]
Prepare 0.2 M (1000x) stock of Ascorbic Acid by Sigma Aldrich. C6H8O6, MW: 176.12 g/mol.
?. [Preparing reagents for mDA differentiation]
To prepare 2841 µl 0.2 M Ascorbic Acid:Add  of sterile water directly to vial containing  of Ascorbic Acid (Sigma Aldrich,  Cat no A4403).Mix by pipetting up and down several times to dissolve the powder. Filter the reconstitued Ascorbic Acid solution using an appropriatly sized syringe for the volume and a 0.33µM filter. Prepare -  aliquots of   Ascorbic Acid in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year. Once thawed, keep Ascorbic Acid aliquots at  in the dark for up to two weeks.
2841 µl
100 mg
20 µl
50 µl
-80 °C
4 °C
?. [Preparing reagents for mDA differentiation]
Prepare 0.5 M (1000x) stock of dcAMP (N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt) by Sigma Aldrich. C18H23N5O8PNa, MW: 491.3 g/mol.
?. [Preparing reagents for mDA differentiation]
To prepare 4070 µl 0.5 M dcAMP:Add  of sterile water directly to vial containing  of dcAMP (Sigma Aldrich, Cat no D0627).Mix by pipetting up and down several times to dissolve the powder. Filter the reconstitued dcAMP solution using an appropriatly sized syringe for the volume and a 0.33µM filter.Prepare -  aliquots of   dcAMP in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year. Once thawed, keep dcAMP aliquots at  in the dark for up to two weeks.
4070 µl
1000 mg
20 µl
50 µl
-80 °C
4 °C
?. [Preparing reagents for mDA differentiation]
Prepare 10 mM (10,000x) stock of DAPT C23H26F2N2O4, MW: 432.46 g/mol.
?. [Preparing reagents for mDA differentiation]
To prepare 2312 µl 10 mM DAPT:Add  of  DMSO (Sigma Aldrich, cat no. D2650) directly to vial containing  of DAPT (Tocris, Cat no 2634).Mix by pipetting up and down several times to dissolve the powder. If precipitate is observed, warm the solution to  for 2-5 mins. Prepare  aliquots of   DAPT in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year. Once thawed, keep DAPT aliquots at  in the dark for up to 1 month.
2312 µl
10 mg
37 °C
20 µl
-80 °C
4 °C
?. [Preparing reagents for mDA differentiation]
Prepare 10 mM (1000x) stock of Y-27632 dihydrochlorideC14H21N3O.2HCl. MW: 320.26 g/mol.
?. [Preparing reagents for mDA differentiation]
To prepare 15.61 ml 10 mM Y-27632:Add  of DPBS without Mg2+ and Ca2+  (Thermo Fisher, Cat no 14190086) directly to vial containing  of Y-27632 dihydrochloride (Tocris, Cat no 1254).Mix by pipetting up and down several times to dissolve the powder.Prepare aliquots of   Y-27632 dihydrochloride in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year. Prepare smaller  aliquots for working use and larger aliquots which can be aliquoted out at a later date.  Once thawed, keep Y-27632 aliquots at  in the dark for up to 1 month.
15.61 ml
50 mg
-80 °C
50 µl
500 µl
4 °C
?. [Preparation for mDA D0]
On mDA day 0 cells hPSCs are lifted as clumps using EDTA and seeded onto plates that have been pre-coated with Laminin-111. Plates need to be coated with Laminin 111 at least 12 hours prior to the beginning the differentiation.
?. [Preparation for mDA D0]
Pre-Coating plates with Laminin 111:Laminin-111 stocks (0.1mg/ml) (Biolamina, Cat no LN111) should be thawed at RT and then diluted 1:20 in DPBS (with Ca2+ and Mg2+) (ThermoFischer, cat no. 14040083) to create a final working stock solution of   Laminin-111. The diluted laminin should be mixed thoroughly and added to the wells at a volume recommended for the well size (see below table).  ABC1  Plate type  Approx Area of Well (cm2)Volume of 5 µg/mll L521 (ul) required for optimal coating2  24 well plate    2    300  3  48 well plate    1    150  Move the plate gently around to ensure the well is evenly coveredWrap the plate in parafilm to reduce evaporationIncubate the plate at  overnight for at least 12 hours (overnight incubation is recommended).Coated plates can be stored at   for 3 weeks.
ABC1  Plate type  Approx Area of Well (cm2)Volume of 5 µg/mll L521 (ul) required for optimal coating2  24 well plate    2    300  3  48 well plate    1    150
4 °C
4 °C
?. [Preparation for mDA D0]
Preparation of Neural Induction Medium (NIM):50% DMEM/F12 + 50% Neurobasal + 1x N2 + 1x B27 + 2mM L-Glutamine. Between mDA D0 and mDA D4 cells are cultured within NIM.The components which form the base media NIM (N2 supplement, B27 supplement, DMEM/F12 and Neurobasal media) are in themselves only stable for 2 weeks due to the presence of light sensitive components. Thus, fresh base media should be made up at the start of each differentiation and kept in the dark at  for up to 2 weeks.
-4 °C
?. [Preparation for mDA D0]
To prepare 50ml of NIM: Add of DMEM/F12 (Thermo Fisher, Cat no 21331020) +  of NeurobasalTM Medium (Thermo Fisher, Cat no 21103049) +  of 100x N2 (Thermo Fisher, Cat no 17502048) +  of 50x B27 (Thermo Fisher, Cat no 17504044) +  of 200mM L-Glutamine (Thermo Fisher, Cat no 25030032).
24 ml
24 ml
500 µl
1 ml
500 µl
?. [mDA Day 0 ]
mDA Day 0 plating On mDA Day 0 cells are lifted as clumps using 0.5mM EDTA and then seeded onto Laminin-111 coated plates.Before beginning; self-renewing cultures of hPSCs must be free of spontaneous differentiation and confluency should be between 70% and 90% before use.Prior to mDA D0 seeding, human ESC/iPSC cultures are grown on Laminin-521 (5μg/ml) matrix coated 6-well plates with daily feeding using 2 ml StemMACS iPS-BrewXF (Miltenyi Biotec). Each well of a 6-well plate should be pre-coated with 1200 μl of Laminin-521 overnight at 4oC before use to ensure even hPSC adherence and efficient growth. One well of a 6-well plate should provide enough cells to seed between 6 and 10 wells of a 24-well platefor mDA differentiation.
?. [mDA Day 0 ]
Preparation of wash media for lifted hPSCs:Per each well of a 6 well plate of hPSCs prepare 4ml of wash media.Wash media: 50% iPS-Brew XF medium (self renewal media for hPSCs) + 50% DMEM/F12 + 10 μM Y27632.For example when lifting one well of hPSCs from a 6 well plate prepare: iPS-Brew XF medium (Miltenyibiotec, Cat no 130-104-368) +  DMEM/F12 (Thermo Fisher, Cat no 21331020) +  10mM Y27632.
2 ml
2 ml
2 µl
?. [mDA Day 0 ]
Prepare an adequate volume of plating medium: Neural Induction Media (NIM) +  10 μM Y27632 + 10 μM SB431542, 100nM LDN, 600 ng/ml Shh and between 0.7 and 1.2 μM CHIR99021*. To start differentiation in 24-well plates, you will need 400 μl per well.*The concentration of CHIR99021 recommended can vary between cell lines. A change in 0.1 μM can have a suprisingly large effect on the puritiy of cells obtained. Therefore it is recommended when first starting to try out and compare a range of CHIR99021 concentrations.
?. [mDA Day 0 ]
Lifting hPSCs with EDTA Aspirate the iPS-Brew XF medium from the hPSCs (in 6-well plate).Wash cells once with  of  EDTA (prepared step 16.1). Add  of  EDTA to the cells and incubate at  for approximately 5-7 minutes or when most of the cells appear ‘refractile’ by light microscopy. *The incubation time should depend on the confluency of the cells.After the incubation period; remove the EDTA and use of the Wash medium (prepared in step 18.2) to lift the cells gently from the well. Aim to lift cells in a few pipettes as possible to ensure optimal surivial. Transfer the lifted cells to the tube with remaining Wash medium. Gently invert the tube a few times to ensure the cells are thoroughly mixed for cell counting.
1 ml
1 ml
37 °C
1 ml
?. [mDA Day 0 ]
Counting the cells:By using EDTA the cells are lifted as clumps. For more accurate counting of the cells take an aliquot to be broken into single cells.To count the cells, take 2 x 10 μl aliquots of cells into new 0.5ml Eppendorfs. Mix each with of Trypan Blue (Sigma Aldrich, Cat no T8154). Count the cells using a haemacytometer*. Calculate the amount of cells within the wash media.
10 µl
?. [mDA Day 0 ]
Plating cells:On mDA Day 0 we recommend using seeding into a 24 well plate.  From the cell count, estimate the total volume of cells you will need to initiate differentiation at a density of 40,000 cells/cm2*.  For example, for 4 wells of a 24-well plate, you will need 4 (number of wells) x 2 (area of each well) x 40,000 (required density) = 320,000 cells.*Note the optimal plating density can vary depending on the cell line used. For example RC17 a plating density of 40,000 cells/cm2 is recommended but for AST18 a plating density of 60,000cells/cm2 is recommended. Transfer 110% of the required volume (an extra 10% is transferred to account for any cell death) from the remaining  cell suspension within the wash media (that has not been triturated) to a new tube for centrifugation 300 g for 3 minutes.Aspirate the wash media and resuspend the colonies gently in the plating medium (prepared in step 18.2). It is recommended to plate cells into the wells of a 24 well plate in a volume of 400 μl per well. E.g if 4 wells of a 24-well plate are being seeding for example then cells should be resuspended in 4 x 400 μl.Aspirate the Laminin-111 from the coated plates and seed the cell suspension into the well. Shake the plate with  to ensure an even distribution of cells.
?. [mDA Day 2]
mDA Day 2 media change. Fresh medium should be prepared from base media NIM and growth factors.
?. [mDA Day 2]
Prepare an adequate volume of fresh day 2 medium: Neural Induction Media (NIM) + 10 μM SB431542, 100nM LDN, 600 ng/ml SHH and between 0.6 and 1.0 μM CHIR99021. Prepare medium according to the number of the wells to be fed, remember to account for extra dead volume.For each well of a 24-well plate of fresh media is required.
400 µl
?. [mDA Day 4]
mDA Day 4 media changeBetween mDA Day 4 and mDA Day 11 cells are no longer cultured within NIM as a base media but instead are cultured within neural proliferation media NPM. *You should observe the cells becoming more confluent day by day. We expect cells to be close to 100% confluent between mDA D3 and mDA D7. How quickly cells become confluent varies between cell lines. The components which form the base media NPM (N2 supplement, B27 supplement, DMEM/F12 and Neurobasal media) are in themselves only stable for 2 weeks due to the presence of light sensitive components. Thus, fresh base media should be made up at the start of each differentiation and kept in the dark at  for up to 2 weeks.
4 °C
?. [mDA Day 4]
Preparation of NPM50% NIM + 25% Neurobasal + 25% DMEM/F12 + 2mM L-Glutamine. To prepare NPM from NIM:For example, to make NPM = NIM + DMEM/F12 (Thermo Fisher, Cat no 21331020) + NeurobasalTM Medium  (Thermo Fisher, Cat no 21103049) + 200mM L-Glutamine (Thermo Fisher, Cat no 25030032).Altnatively to make NPM de novo:50% Neurobasal, 50% DMEM/F12 + 0.5x N2 + 0.5x B27 + 2mM L-Glutamine. For example, to make  NPM=  of DMEM/F12 (Thermo Fisher, Cat no 21331020) + of NeurobasalTM Medium (Thermo Fisher, Cat no 21103049)  +  of 100x N2 (Thermo Fisher, Cat no 17502048) +  of 50x B27 (Thermo Fisher, Cat no 17504044) +  of 200mM L-Glutamine (Thermo Fisher, Cat no 25030032).
50 ml
25 ml
12.25 ml
12.25 ml
250 µl
50 ml
24.4 ml
24.4 ml
250 µl
500 µl
500 µl
?. [mDA Day 4]
Prepare an adequate volume of fresh day 4 medium: NPM + 10 μM SB431542, 100nM LDN, 600 ng/ml Shh and between 0.6 and 1.0 μM CHIR99021. Prepare medium according to the number of the wells to be fed, remember to account for extra dead volume.For each well of a 24-well plate  of fresh media is required.
600 µl
?. [mDA Day 7]
mDA Day 7 media change. Cells should be confluent by mDA Day 7 at the latest.
?. [mDA Day 7]
Prepare an adequate volume of fresh day 7 medium: NPM + 10 μM SB431542, 100 nM LDN, 600 ng/ml Shh and between 0.6 and 1.0 μM CHIR99021. Prepare medium according to the number of the wells to be fed, remember to account for extra dead volume.For each well of a 24-well plate of fresh media is required.
600 µl
?. [mDA Day 9]
mDA Day 9 media change
?. [mDA Day 9]
Prepare an adequate volume of fresh medium: NPM + 100 ng/ml FGF8b + 1 μM Heparin solution. Prepare medium according to the number of the wells to be fed, remember to account for extra dead volume.For each well of a 24-well plate,  of fresh media is required.
600 µl
?. [mDA Day 11: preparation]
mDA Day 11On mDA day 11 cells can either be replated or frozen for use later on.  From mDA D11 onwards cells are cultured within new base media; Neural differentiation medium (NDM). mDA cells should be replated onto new plates freshly coated Laminin-111.
?. [mDA Day 11: preparation]
Pre-coating plates with Laminin-111Plates should be coated with L111 following the same protocol as step ?
?. [mDA D11 replating]
mDA D11 replating:On day 11 cells can be (i) lifted and replated for continued differentiation, or (ii) cryopreservedTo replate; mDA D11 cells are lifted as single cells using Acctuase and replated into a new L111-coated plate at a density of 800,000cells/cm2.
?. [mDA D11 replating]
Preparation of wash medium:NPM + 10 μM Y27632For each well of a 24 well plate prepare wash media. *This volume is 5x the volume of Acctuase added per well.
1.2 ml
?. [mDA D11 replating]
Preparation of plating medium:NDM + 10 μM Y-27632 + 20 ng/ml BDNF + 10 ng/ml GDNF 100 ng/ml FGF8b + 0.2 mM Ascorbic Acid + 1 μg/ml heparin solution.Prepare plating medium according to the number of the wells to be re-plated, remember to account for extra dead volume. It is recommended to re-plate cells into either a 24 or 48 well formatt.Volume for day 11 plating:  ABC1µl/wellarea (cm2)Volume (µl)224 well plate2600348 well plate1300
ABC1µl/wellarea (cm2)Volume (µl)224 well plate2600348 well plate1300
?. [mDA D11 replating]
Lifting cells as single cells with Accutase Wash the cells once briefly Accutase (300 µl/well for 24-well plate) (Sigma Aldrich, Cat no A6964)Add 300µl/well of Accutase (Sigma Aldrich, Cat no A6964) and incubate  for 10min. Following the incubation period, to dissociate into single cells pipette up and down. **Place a P1000 tip in a P200 tip, set the P1000 to 600. Tilt the plate towards you, place the pipette tip in the bottom corner of the well . Pipetting the cells up and down against this corner will aid in breaking up the cells. Pipetting ~15 times is usually sufficient to break into single cells. Try to avoid the production of bubbles where possible. Transfer the lifted cells to the wash media.Repeat the process for each well.
37 °C
?. [mDA D11 replating]
Counting cellsTo count the cells, take 2 x 10 μl aliquots of cells into new 0.5ml Eppendorfs. Mix each with of Trypan Blue (Sigma Aldrich, Cat no T8154). Count the cells using a haemacytometer*. Calculate the amount of cells within the wash media. *Alternatively, can use automatic cell counter to count the cells. E.g TC20TM Automated Cell Counter Biorad (BioRad, Cat no 1450102).
10 µl
?. [mDA D11 replating]
Plating cells:From the cell count, estimate the total volume of cells needed for re-plating. For mDA day 11 replate at a density of 800,000 cells/cm2.For example: cells needed = (number of wells) x 2cm2 (area of a well in a 24-well plate) x 800,000 cells/cm2 (required density). The volume to spin down = cells needed / cell count.Transfer the required volume from the remaining  cell suspension within the wash media (that has not been triturated) to a new tube for centrifugation 300 g for 3 minutes.Aspirate the wash media and resuspend the colonies gently in the mDA Day 11 plating medium (prepared in step 24.2). E.g if 4 wells of a 24-well plate are being seeding for example then cells should be resuspended in 4 x 600 μl.Aspirate the Laminin-111 from the coated plates and seed the cell suspension into the wells. Shake the plate to ensure even distribution of cells.
?. [mDA D11 replating]
Alternative to replating: freezing mDA Day 11 cellsInstead of replating cells can be cyropreserved at this stage. See cryopreservation protocol described in: https://www.biorxiv.org/content/10.1101/2020.02.11.944272v1.
?. [mDA Day 14]
mDA Day 14 media change.
?. [mDA Day 14]
Prepare an adequate volume of fresh medium: NDM +  20 ng/ml BDNF + 10 ng/ml GDNF + 100 ng/ml FGF8b + 0.2 mM Ascorbic Acid + 1 μg/ml heparin solution.Prepare medium according to the number of the wells to be fed, remember to account for extra dead volume.Volume needed per well:  ABC1Plate Typearea (cm2)Volume (µl/ well)224 well plate2600348 well plate1300
ABC1Plate Typearea (cm2)Volume (µl/ well)224 well plate2600348 well plate1300
?. [Preparation for mDA Day 16]
mDA Day 16 replating preparationOn mDA day 16 cells can either be replated or cyropreserved for use later on. mDA cells should be replated onto new plates freshly coated Laminin-111.
?. [Preparation for mDA Day 16]
Pre-coating plates with Laminin-111Plates should be coated with L111 following the same protocol as step 15.
?. [mDA Day 16 ]
mDA Day 16 replatingOn day 16 cells can be (i) lifted and replated for continued differentiation, (ii) cryopreserved, or (iii) transplanted into an animal model.As with mDA Day 11 replating, mDA Day 16 cells are lifted as single cells using Acctuase and replated into a new L111-coated plate at a density of 800,000 cells/cm2.
?. [mDA Day 16 ]
Preparation of wash media:Wash media is prepared from NDM + 10 μM Y-27632.For each well of a 24-well plate prepare  wash media. For each well of a 48-well plate prepare  wash media. *This volume is 5x the volume of Acctuase added per well.
1.2 ml
0.6 ml
?. [mDA Day 16 ]
Lifting cells as single cells with Accutase Aspirate off the NDM from each well. Wash the cells once briefly Accutase (300 µl/well for 24-well plate, 150 µl/well for 48-well plate) (Sigma Aldrich, Cat no A6964) to remove excess medium. Add Accutase (300 µl/well for 24-well plate, 150 µl/well for 48-well plate) (Sigma Aldrich, Cat no A6964) and incubate for 10 mins.Following the incubation period, to dissociate into single cells pipette up and down. ******Place a P1000 tip in a P200 tip, set the P1000 to 600. Tilt the plate towards you, place the pipette tip in the bottom corner of the well pipetting the cells up and down against this corner will aid in breaking up the cells. Pipetting ~15 times is usually sufficient. Try to avoid the production of bubbles.Transfer the lifted cells to the wash media.Repeat the process for each well.
37 °C
?. [mDA Day 16 ]
Counting cellsTo count the cells, take 2 x 10 μl aliquots of cells into new 0.5ml Eppendorfs. Mix each with of Trypan Blue (Sigma Aldrich, Cat no T8154).Count the cells using a haemacytometer*. Calculate the amount of cells within the wash media. *Alternatively, can use automatic cell counter to count the cells. E.g TC20TM Automated Cell Counter Biorad (BioRad, Cat no 1450102).
10 µl
?. [mDA Day 16 ]
Plating cells:From the cell count, estimate the total volume of cells needed for re-plating. For mDA day 16 replate at a density of 800,000 cells/cm2.For example: cells needed = (number of wells) x 2 cm2 (area of a well in a 24-well plate) x 800,000 cells/cm2 (required density). The volume to spin down = cells needed / cell count.Transfer the required volume from the remaining  cell suspension within the wash media (that has not been triturated) to a new tube for centrifugation 300 g for 3 minutes.Aspirate the wash media and resuspend the colonies gently in the mDA Day 16 plating medium (prepared in step 27.2. E.g if 4 wells of a 24-well plate are being seeding for example then cells should be resuspended in 4 x 600 μl.Aspirate the Laminin-111 from the coated plates and seed the cell suspension into the wells with generous shaking to ensure even distribution.
?. [mDA Day 18]
mDA Day 18 media change
?. [mDA Day 18]
Prepare an adequate volume of fresh day 18 medium: NDM +  20 ng/ml BDNF + 10 ng/ml GDNF + 0.2 mM Ascorbic Acid + 0.5 mM dcAMP + 1 µM DAPT. Prepare medium according to the number of the wells to be fed, remember to account for extra dead volume. Volume for day 18 media change:   ABC1Plate Typearea (cm2)Volume (µl/ well)224 well plate2600348 well plate1300
ABC1Plate Typearea (cm2)Volume (µl/ well)224 well plate2600348 well plate1300
?. [mDA Day 18 onwards]
Media changes mDA day 18-day 45:From mDA Day 18 onwards cells should be fed every 2-3 days until day 45. At day 45 the mDA cells are expressing high levels of mature mDA markers e.g TH.
?. [mDA Day 18 onwards]
Prepare an adequate volume of fresh medium: NDM +  20 ng/ml BDNF + 10 ng/ml GDNF + 0.2 mM ascorbic acid + 0.5 mM dcAMP + 1 µM DAPT. Prepare medium according to the number of the wells to be fed, remember to account for extra dead volume. Volume for media change:  ABC1Plate Typearea (cm2)Volume (µl/ well)224 well plate2600348 well plate1300mDA neurons can be kept in culture for over 1 year feeding once or twice a week. DAPT should be removed from the feeding medium after day 45.
ABC1Plate Typearea (cm2)Volume (µl/ well)224 well plate2600348 well plate1300
?. [mDA Day 16 ]
Alternative to replating: freezing mDA Day 16 cellsInstead of replating, cells can be frozen at this stage. See freezing protocol described in: https://www.biorxiv.org/content/10.1101/2020.02.11.944272v1.
?. [mDA Day 16 ]
Preparation of plating media:NDM + 10 μM Y27632 + 20 ng/ml BDNF + 10 ng/ml GDNF + 0.2 mM Ascorbic Acid + 0.5 mM dcAMP + 1 µM DAPT. Prepare plating medium according to the number of the wells to be re-plated, remember to account for extra dead volume. It is recommended to re-plate cells into either a 24 or 48-well formatt.Volume for day 16 plating:   ABC1Plate Typearea (cm2)Volume (µl/ well)224 well plate2600348 well plate1300
ABC1Plate Typearea (cm2)Volume (µl/ well)224 well plate2600348 well plate1300
?. [Preparing reagents for mDA differentiation]
Prepare aliquots of N2N2 stocks should be stored at . To avoid freeze thawing it is helpful to create working aliquots.
-20 °C
?. [Preparing reagents for mDA differentiation]
Prepare  aliquots of 100x N2  (Thermo Fisher, Cat 17502048) in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year.
0.5 ml
-20 °C
?. [Preparing reagents for mDA differentiation]
Prepare aliquots of B27 (without RA)B27 stocks should be stored at . To avoid freeze thawing it is helpful to create working aliquots.
-20 °C
?. [Preparing reagents for mDA differentiation]
Prepare  aliquots of 50x B27 (without RA) (Thermo Fisher, Cat no 17504044) in sterile 1.5-ml Eppendorfs tubes and store at  for up to one year.
1 ml
-20 °C
?. [Preparation for mDA D0]
Preparation of 0.5mM EDTA from ThermoFisher. To lift hPSCs as clumps for seeding, a stock of 0.5mM EDTA is prepared from 50mM Ultra Pure EDTA  (ThermoFisher, Cat no 15575020).
?. [Preparation for mDA D0]
To prepare 50ml of 0.5mM EDTA:Take an aliquot of  Ultra Pure EDTA (ThermoFisher, Cat no 15575020), and add to a 50ml falcon tube. Add of DPBS (without Ca2+ and Mg2+) (ThermoFischer, cat no. 14040083) directly to the 50ml falcon tube and mix to create a  EDTA stockFilter the diluted EDTA through a 0.33μM filter into a fresh 50ml falcon tube.Label and store 0.5 mM EDTA stock at. 0.5mM EDTA can be kept at  for 6 months.
50 µl
50 ml
0 Room temperature
0 Room temperature
?. [mDA Day 11: preparation]
Preparation of Neural Differentiation Medium (NDM):The components which form the base media NDM  (B27 supplement, and Neurobasal media) are in themselves only stable for 2 weeks due to the presence of light sensitive components. Thus, fresh base media should be made up at the start of each differentiation and kept in the dark at  for up to 2 weeks.
4 °C
?. [mDA Day 11: preparation]
Preparation of NDM:Neurobasal  + 1x B27 + 2mM L-Glutamine. For example, to make  NDM =  of NeurobasalTM Medium (Thermo Fisher, Cat no 21103049) +  of 50x B27  (Thermo Fisher, Cat no 17504044) +  of 200mM L-glutamine (Thermo Fisher, Cat no 25030032).
50 ml
48.5 ml
1 ml
500 µl
?. [Preparing reagents for mDA differentiation]
Prepare 10 mM (1000x) stock of SB431542. C22H16N4O3•2H2O. MW: 420.42 g/mol.