May 07, 2020
Midbrain dopaminergic differentiation of human pluripotent stem cells
- Tilo Kunath,
- Szuping Chiu,
- Sophie Glendinning1,
- Yixi Chen1,
- Nicola Drummond1,
- Maurice Canham1
- 1University of Edinburgh
External link: https://onlinelibrary.wiley.com/doi/full/10.1111/ejn.14286
Protocol Citation: Tilo Kunath, Szuping Chiu, Sophie Glendinning, Yixi Chen, Nicola Drummond, Maurice Canham 2020. Midbrain dopaminergic differentiation of human pluripotent stem cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bddpi25n
Manuscript citation:
Chen Y, Dolt KS, Kriek M, Baker T, Downey P, Drummond NJ, Canham MA, Natalwala A, Rosser S, Kunath T. (2019) Engineering synucleinopathy-resistant human dopaminergic neurons by CRISPR-mediated deletion of the SNCA gene. Eur J Neurosci. 49:510-524. doi: 10.1111/ejn.14286.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our group and it is working.
Created: March 08, 2020
Last Modified: May 07, 2020
Protocol Integer ID: 33935
Keywords: hESC, iPSC, induced pluripotent stem cells, human embryonic stem cells, neuronal, midbrain, dopaminergic, mesencephalic
Abstract
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.
Materials
MATERIALS
Biolaminin 111 LNBiolaminaCatalog #LN111
B27 supplement without retinoic acid (50x)Gibco - Thermo Fisher ScientificCatalog #17504044
24-well cell culture plateCostarCatalog #3524
Neurobasal Medium (1x)Gibco - Thermo Fisher ScientificCatalog #21103049
StemMACS™ iPS-Brew XF human: Basal medium and supplementMiltenyi BiotecCatalog #130-104-368
StemMACS™ CHIR99021Miltenyi BiotecCatalog #130-103-926
Poly-L-Ornithine Merck MilliporeSigma (Sigma-Aldrich)Catalog #P4957
Dulbecco’s PBS (with calcium magnesium)Gibco - Thermo Fisher ScientificCatalog #14040083
DMEM/F12Gibco - Thermo Fisher ScientificCatalog #21331020
UltraPure 0.5 M EDTA pH8Invitrogen - Thermo FisherCatalog #15575020
Dulbecco’s PBS (without calcium magnesium)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8537
N2 supplement (100x supplement)Gibco - Thermo Fisher ScientificCatalog #17502048
L-Glutamine (200mM)Thermo Fisher ScientificCatalog #25030024
Recombinant human BDNF peprotechCatalog #450-02
Recombinant human GDNFpeprotechCatalog #450-10
Recombinant human FGF-8bpeprotechCatalog #100-25
Recombinant Human Sonic Hedgehog/Shh (C24II) N-TerminusR&D SystemsCatalog #1845-SH-500
L-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
DAPTTocrisCatalog #2634
Dibutyryl cAMPMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0627
StemMACS™ LDN-193189Miltenyi BiotecCatalog #130-103-925
Heparin sodium salt from porcine intestinal mucosaMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3149
Accutase® solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A6964
Trypan BlueMerck MilliporeSigma (Sigma-Aldrich)Catalog #T8154
STEM-CELLBANKER - GMP GradeamsbioCatalog #11890
SB431542Merck Millipore (EMD Millipore)Catalog #616461-5MG
7.5% Bovine Serum Albumin solution in DPBSMerck MilliporeSigma (Sigma-Aldrich)Catalog #A8412
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #D2650
Biolaminin 521 LNBiolaminaCatalog #LN521
StemMACS™ LDN-193189 in solutionMiltenyi BiotecCatalog #130-106-540
Y-27632 dihydrochlorideTocrisCatalog #1254
200 mM L-glutamine Invitrogen - Thermo FisherCatalog #25030-024
Before start
Before starting it is helpful to create a relatively large cryopreserved bank of hESCs or hiPSCs at one particular passage (20 or more vials). Upon thawing each vial perform the mDA differention protocol within one or two passages of recovering the cells. It is not advisable to keep the cells 'ticking' over, and initiating mDA differentiations at different passages of the cell line, as this reduces reproducibility This method of working is well-described by Mark Tomishima's CryoPause publication. 
CryoPause_Tomishima.pdf
CryoPause_Tomishima.pdf Preparing reagents for mDA differentiation
Preparing reagents for mDA differentiation
Prepare 10 mM (1000x) stock of SB431542.
C22H16N4O3•2H2O. MW: 420.42 g/mol.
To prepare 1190 µl of 10 mM SB431542:
Add 1190 µL (2 x 595 µL ) of DMSO (Sigma Aldrich, cat no. D2650) directly to vial containing 5 mg of SB431542 (Merck Millipore, cat no. 616461-5MG) and mix by pipetting until powder is completely dissolved.
Prepare 20 µL -50 µL aliquots of 10 mM SB431542 in sterile 1.5-ml Eppendorfs tubes and store at -80 °C for up to one year. Once thawed, keep SB431542 aliquot at Room temperature in the dark for up to two weeks.
Prepare 10 mM (100,000x) and 0.1 mM (1000x) stocks of LDN193189.
C25H22N6. MW: 406.48 g/mol.
To prepare 492 µl of 10 mM LDN193189:
Add 492 µL of DMSO (Sigma Aldrich, cat no. D2650) directly to vial containing 2 mg of LDN193189 (Miltenyi Biotec, cat no. 130-103-925) to obtain a 10 millimolar (mM) solution. Warm briefly (3-5min) at 37 °C 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 0.1 millimolar (mM) (1000x) stocks.
Prepare 20 µL -50 µL aliquots of 0.1 mM LDN193189 in sterile 1.5-ml Eppendorfs tubes and store at -80 °C for up to one year. Once thawed, keep LDN193189 aliquot at Room temperature in the dark for up to two weeks.
Prepare 600 µg/ml (1000x) stock of Sonic Hedgehog (SHH-C24II).
To prepare 833 µl of 600 µg/ml SHH-C24II:
Add volume of 822 µL of DPBS without Mg2+ and Ca2+ (Thermo Fisher, Cat no 14190086) directly to vial containing 500 µg of SHH-C24II (R+D Systems, cat no. 1845-SH-500).
Mix by pipetting up and down several times until the powder is dissolved.
Prepare 20 µL -50 µL aliquots of 600 μg/ml SHH in sterile 1.5-ml Eppendorfs tubes and store at -80 °C for up to one year. Once thawed, keep SHH aliquot at 4 °C for up to two weeks.
Prepare 10 mM stock of CHIR99021.
To prepare 2414 µl of 10mM CHIR99021:
Add 2149 µL of DMSO (Sigma Aldrich, cat no. D2650) directly to vial containing 10 mg of CHIR99021 (Miltenyi Biotech, Cat no 130-103-926) to obtain a 10 millimolar (mM) solution. Mix by pipetting up and down several times to dissolve the powder. If precipitate is observed, warm briefly (2-5min) at 37 °C .
Alternatively, CHIR99021 can be obtained in solution at 10 mM in DMSO (Miltenyi Biotech, Cat no. 130-106-539)
Prepare 10 µL aliquots of 10 mM CHIR99021 in sterile 1.5-ml Eppendorfs tubes and store at -80 °C for up to one year. Once thawed, keep CHIR99021 aliquot at Room temperature in the dark for up to two weeks.
Prepare 100 µg/ml (1000x) stock of recombinant FGF8b
To prepare 250 µl of 100 µg/ml FGF8b:
Add 246 µL of DPBS without Mg2+ and Ca2+ (Thermo Fisher, Cat no 14190086) directly to vial containing 25 µg of FGF8b (R+D Systems, Cat no 423-F8-025)
Mix by pipetting up and down several times to dissolve the powder.
Prepare 20 µL -50 µL aliquots of 100 µg/ml FGF8b in sterile 1.5-ml Eppendorfs tubes and store at -80 °C for up to one year. Once thawed, keep FGF8b aliquots at 4 °C in the dark for up to two weeks.
Prepare 1 mg/ml (1000x) stock of Heparin Sodium Salt from porcine intestinal mucosa
To prepare 55 ml of 1 mg/ml Heparin:
Add 55 mL of DPBS without Mg2+ and Ca2+ (Thermo Fisher, Cat no 14190086) directly to vial containing 55 mg 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 filter
Prepare 500 µL aliquots of 1 mg/ml Heparin in sterile 1.5-ml Eppendorfs tubes and store at 4 °C for up to two years.
Prepare 20 µg/ml (1000x) stock of recombinant Bone-Derived Growth Factor (BDNF).
To prepare 5 ml of 20 µg/ml BDNF:
Add 4930 µL of DPBS without Mg2+ and Ca2+ (Thermo Fisher, Cat no 14190086) directly to vial containing 100 µg of BDNF (PeproTech, Cat no 450-02).
Mix by pipetting up and down several times to dissolve the powder.
Prepare 20 µL - 50 µL aliquots of 20 μg/ml BDNF in sterile 1.5-ml Eppendorfs tubes and store at -80 °C for up to one year. Once thawed, keep BDNF aliquots at 4 °C for up to two weeks.
Prepare 20 µg/ml (2000x) stock of recombinant Glial Derived Growth Factor (GDNF).
To prepare 5 ml of 20 µg/ml GDNF:
Add 4930 µL of DPBS without Mg2+ and Ca2+ (Thermo Fisher, Cat no 14190086) directly to vial containing 100 µg of GDNF (PeproTech, Cat no 450-10).
Mix by pipetting up and down several times to dissolve the powder.
Prepare 20 µL - 50 µL aliquots of 20 μg/ml GDNF in sterile 1.5-ml Eppendorfs tubes and store at -80 °C for up to one year. Once thawed, keep GDNF aliquots at 4 °C for up to two weeks.
Prepare 0.2 M (1000x) stock of Ascorbic Acid by Sigma Aldrich. C6H8O6, MW: 176.12 g/mol.
To prepare 2841 µl 0.2 M Ascorbic Acid:
Add 2841 µL of sterile water directly to vial containing 100 mg 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 20 µL - 50 µL aliquots of 0.2 Molarity (M) Ascorbic Acid in sterile 1.5-ml Eppendorfs tubes and store at -80 °C for up to one year. Once thawed, keep Ascorbic Acid aliquots at 4 °C in the dark for up to two weeks.
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.
To prepare 4070 µl 0.5 M dcAMP:
Add 4070 µL of sterile water directly to vial containing 1000 mg 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 20 µL - 50 µL aliquots of 0.5 Molarity (M) dcAMP in sterile 1.5-ml Eppendorfs tubes and store at -80 °C for up to one year. Once thawed, keep dcAMP aliquots at 4 °C in the dark for up to two weeks.
Prepare 10 mM (10,000x) stock of DAPT
C23H26F2N2O4, MW: 432.46 g/mol.
To prepare 2312 µl 10 mM DAPT:
Add 2312 µL of DMSO (Sigma Aldrich, cat no. D2650) directly to vial containing 10 mg 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 37 °C for 2-5 mins.
Prepare 20 µL aliquots of 10 millimolar (mM) DAPT in sterile 1.5-ml Eppendorfs tubes and store at -80 °C for up to one year. Once thawed, keep DAPT aliquots at 4 °C in the dark for up to 1 month.
Prepare 10 mM (1000x) stock of Y-27632 dihydrochloride
C14H21N3O.2HCl. MW: 320.26 g/mol.
To prepare 15.61 ml 10 mM Y-27632:
Add 15.61 mL of DPBS without Mg2+ and Ca2+ (Thermo Fisher, Cat no 14190086) directly to vial containing 50 mg of Y-27632 dihydrochloride (Tocris, Cat no 1254).
Mix by pipetting up and down several times to dissolve the powder.
Prepare aliquots of 10 millimolar (mM) Y-27632 dihydrochloride in sterile 1.5-ml Eppendorfs tubes and store at -80 °C for up to one year. Prepare smaller 50 µL aliquots for working use and larger 500 µL aliquots which can be aliquoted out at a later date.
Once thawed, keep Y-27632 aliquots at 4 °C in the dark for up to 1 month.
Prepare aliquots of N2
N2 stocks should be stored at -20 °C . To avoid freeze thawing it is helpful to create working aliquots.
Prepare 0.5 mL aliquots of 100x N2 (Thermo Fisher, Cat 17502048) in sterile 1.5-ml Eppendorfs tubes and store at -20 °C for up to one year.
Prepare aliquots of B27 (without RA)
B27 stocks should be stored at -20 °C . To avoid freeze thawing it is helpful to create working aliquots.
Prepare 1 mL aliquots of 50x B27 (without RA) (Thermo Fisher, Cat no 17504044) in sterile 1.5-ml Eppendorfs tubes and store at -20 °C for up to one year.
Preparation for mDA D0
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.
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 5 μg/ml 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).
| Plate type | Approx Area of Well (cm2) | Volume of 5 µg/mll L521 (ul) required for optimal coating | |
| 24 well plate | 2 | 300 | |
| 48 well plate | 1 | 150 |
Move the plate gently around to ensure the well is evenly covered
Wrap the plate in parafilm to reduce evaporation
Incubate the plate at 4 °C overnight for at least 12 hours (overnight incubation is recommended).
Coated plates can be stored at 4 °C for 3 weeks.
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).
To prepare 50ml of 0.5mM EDTA:
Take an 50 µL aliquot of 50 millimolar (mM) Ultra Pure EDTA (ThermoFisher, Cat no 15575020), and add to a 50ml falcon tube.
Add 50 mL of DPBS (without Ca2+ and Mg2+) (ThermoFischer, cat no. 14040083) directly to the 50ml falcon tube and mix to create a 0.5 millimolar (mM) EDTA stock
Filter the diluted EDTA through a 0.33μM filter into a fresh 50ml falcon tube.
Label and store 0.5 mM EDTA stock atRoom temperature .
0.5mM EDTA can be kept at Room temperature for 6 months.
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 -4 °C for up to 2 weeks.
To prepare 50ml of NIM:
Add 24 mL of DMEM/F12 (Thermo Fisher, Cat no 21331020) + 24 mL of NeurobasalTM Medium (Thermo Fisher, Cat no 21103049) + 500 µL of 100x N2 (Thermo Fisher, Cat no 17502048) + 1 mL of 50x B27 (Thermo Fisher, Cat no 17504044) + 500 µL of 200mM L-Glutamine (Thermo Fisher, Cat no 25030032).
mDA Day 0
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 plate
for mDA differentiation.
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:
2 mL iPS-Brew XF medium (Miltenyibiotec, Cat no 130-104-368) + 2 mL DMEM/F12 (Thermo Fisher, Cat no 21331020) + 2 µL 10mM Y27632.
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.
Lifting hPSCs with EDTA
Aspirate the iPS-Brew XF medium from the hPSCs (in 6-well plate).
Wash cells once with 1 mL of 0.5 millimolar (mM) EDTA (prepared step 16.1).
Add 1 mL of 0.5 millimolar (mM) EDTA to the cells and incubate at 37 °C 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 1 mL 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.
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 with10 µL of Trypan Blue (Sigma Aldrich, Cat no T8154).
Count the cells using a haemacytometer*. Calculate the amount of cells within the wash media.
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
mDA Day 2 media change.
Fresh medium should be prepared from base media NIM and growth factors.
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 400 µL of fresh media is required.
mDA Day 4
mDA Day 4
mDA Day 4 media change
Between 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 4 °C for up to 2 weeks.
Preparation of NPM
50% NIM + 25% Neurobasal + 25% DMEM/F12 + 2mM L-Glutamine.
To prepare NPM from NIM:
For example, to make 50 mL NPM = 25 mL NIM + 12.25 mL DMEM/F12 (Thermo Fisher, Cat no 21331020) + 12.25 mL NeurobasalTM Medium (Thermo Fisher, Cat no 21103049) +250 µL 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 50 mL NPM= 24.4 mL of DMEM/F12 (Thermo Fisher, Cat no 21331020) + 24.4 mL of NeurobasalTM Medium (Thermo Fisher, Cat no 21103049) + 250 µL of 100x N2 (Thermo Fisher, Cat no 17502048) + 500 µL of 50x B27 (Thermo Fisher, Cat no 17504044) + 500 µL of 200mM L-Glutamine (Thermo Fisher, Cat no 25030032).
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 600 µL of fresh media is required.
mDA Day 7
mDA Day 7
mDA Day 7 media change.
Cells should be confluent by mDA Day 7 at the latest.
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 600 µL of fresh media is required.
mDA Day 9
mDA Day 9
mDA Day 9 media change
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, 600 µL of fresh media is required.
mDA Day 11: preparation
mDA Day 11: preparation
mDA Day 11
On 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.
Pre-coating plates with Laminin-111
Plates should be coated with L111 following the same protocol as step ?
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 4 °C for up to 2 weeks.
Preparation of NDM:
Neurobasal + 1x B27 + 2mM L-Glutamine.
For example, to make 50 mL NDM = 48.5 mL of NeurobasalTM Medium (Thermo Fisher, Cat no 21103049) + 1 mL of 50x B27 (Thermo Fisher, Cat no 17504044) + 500 µL of 200mM L-glutamine (Thermo Fisher, Cat no 25030032).
mDA D11 replating
mDA D11 replating
mDA D11 replating:
On day 11 cells can be (i) lifted and replated for continued differentiation, or (ii) cryopreserved
To 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.
Preparation of wash medium:
NPM + 10 μM Y27632
For each well of a 24 well plate prepare 1.2 mL wash media. *This volume is 5x the volume of Acctuase added per well.
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:
| µl/well | area (cm2) | Volume (µl) | |
| 24 well plate | 2 | 600 | |
| 48 well plate | 1 | 300 |
Lifting cells as single cells with Accutase
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.
Counting cells
To count the cells, take 2 x 10 μl aliquots of cells into new 0.5ml Eppendorfs. Mix each with10 µL 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).
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.
Alternative to replating: freezing mDA Day 11 cells
Instead 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
mDA Day 14 media change.
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:
| Plate Type | area (cm2) | Volume (µl/ well) | |
| 24 well plate | 2 | 600 | |
| 48 well plate | 1 | 300 |
Preparation for mDA Day 16
Preparation for mDA Day 16
mDA Day 16 replating preparation
On 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.
Pre-coating plates with Laminin-111
Plates should be coated with L111 following the same protocol as step 15.
mDA Day 16
mDA Day 16
mDA Day 16 replating
On 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.
Preparation of wash media:
Wash media is prepared from NDM + 10 μM Y-27632.
For each well of a 24-well plate prepare 1.2 mL wash media.
For each well of a 48-well plate prepare 0.6 mL wash media.
*This volume is 5x the volume of Acctuase added per well.
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:
| Plate Type | area (cm2) | Volume (µl/ well) | |
| 24 well plate | 2 | 600 | |
| 48 well plate | 1 | 300 |
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 37 °C 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.
Counting cells
To count the cells, take 2 x 10 μl aliquots of cells into new 0.5ml Eppendorfs. Mix each with10 µL 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).
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.
Alternative to replating: freezing mDA Day 16 cells
Instead of replating, cells can be frozen at this stage.
mDA Day 18
mDA Day 18
mDA Day 18 media change
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:
| Plate Type | area (cm2) | Volume (µl/ well) | |
| 24 well plate | 2 | 600 | |
| 48 well plate | 1 | 300 |
mDA Day 18 onwards
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.
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:
| Plate Type | area (cm2) | Volume (µl/ well) | |
| 24 well plate | 2 | 600 | |
| 48 well plate | 1 | 300 |
mDA 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.