Apr 30, 2024
Differentiation of hPSCs into Dopamine neurons
- 11Stephen and Denise Adams Center for Parkinson’s Disease Research of Yale School of Medicine, New Haven, CT 06511, USA;
- 2Aligning Science Across Parkinson’s Collaborative Research Network, Chevy Chase, MD 20815, USA
Protocol Citation: Beatrice Weykopf 2024. Differentiation of hPSCs into Dopamine neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71849gwz/v1
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 and it's working
Created: April 22, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 98607
Keywords: ASAPCRN
Abstract
This protocol describes the differentiation of hPSCs into dopaminergic neurons modified from Kriks et al., 2011 and Ryan et al., 2013
Materials
SRM Medium Dopa Mat Basal medium
79% Knockout DMEM 100% Neurobasal
20% KOSR 1x B27 w/o vitamin A
1% L-GlutaMAX 1% L-GlutaMAX
1% Pen/Strep 1% Pen/Strep
Dopa Mat Medium
100% Dopa mat basal medium
20 ng/ml BDNF
20 ng/ml GDNF
10 µM DAPT
221 µM LAAP
0.5 mM dbcAMP
1 ng/ml TGF-ß-III
| Reagent | Concentration | Vendor | |
| Y-27632 | 10µM | Stem cell Technologies #72304 | |
| TGFß-III | 2 µg/ml | Peprotech #100-36E | |
| Shh (C25II) | 100 ng/ml | R&D Systems #464-SH-200 | |
| SB431542 | 10 µM | Stemcell Technologies #72232 | |
| Purmorphamine | 2 µM | Emdmillipore #540220-5MG | |
| Poly-D-Lysine hydrobromide | 100 µg/ml | Sigma-Aldrich #P1024-50MG | |
| Matrigel | 1:20 - 1:30 | Corning #354234 | |
| LDN-1931189 | 200 nM | Stemgent #04-0046 | |
| Laminin | 10µg/ml | R&D Systems #3400-010-02 | |
| LAAP | 221 µM | Sigma-Aldrich #A8960-5G | |
| GDNF | 10 µg/ml | Peprotech #450-10 | |
| FGF-8b | 100 µg/ml | Peprotech #100-18B-50UG | |
| dbcAMP | 0.5 mM | Enzo BML-CN125-0100 | |
| DAPT | 10 µM | Tocris #2634 | |
| CHIR99021 | 3 µM | Milteniy Biotec #130-103-926 | |
| AraC Cytosine ß-D-arabinofuranoside | 3µM | Sigma-Aldrich #C1768 | |
| Accutase | 1x | Gibco #A1110501 | |
| mTeSR plus | 1x | Stemcell Technologies #100-027 | |
| Knockout Serum Replacement | n/a | Gibco #10828028 | |
| KnockOut DMEM | n/a | Gibco #10829018 | |
| Neurobasal | n/a | Gibco #21103049 | |
| B27 w/o vitamin A | n/a | Gibco #12587010 | |
| L-GlutaMAX | n/a | Gibco #35050 | |
| Penicillin-Streptomycin | n/a | Gibco #15140122 |
Day -1: seed 200K cell/cm2 in mTeSR+ medium supplemented with 10µM Rock inhibitor (RI) onto Martigel coated TC plates
Day 0: Differentiation initiation. Aspirate mTeSR+ medium and add SRM media supplemented with 200nM LDN193189 + 10µM SB431542
Day 1: SRM + LDN + SB supplemented with 100 ng/ml FGF8b, 100 ng/ml ShhC25II and 2µM Purmorphamine
Day 2: SRM + LDN + SB+ FGF8b + ShhC25II + Pur
Day 3: SRM + LDN + SB+ FGF8b + ShhC25II + Pur +3 µM CHIR99021
Day 4: no feed if medium is not consumed
Day 5: 75% SRM + 25% Neurobasal + LDN + SB+ FGF8b + ShhC25II + Pur +CHIR
Day 6: no feed if medium is not consumed
Day 7: 50% SRM + 50% Neurobasal + LDN + CHIR
Day 8: no feed if medium is not consumed
Day 9: 25% SRM + 75% Neurobasal + LDN + CHIR
Day 10: no feed if medium is not consumed
Day 11: 100% Dopa mat basal medium + CHIR
Day 12: no feed
Day 13: Passage cells in a ratio of 1:1 onto matrigel-coated dishes with 30-60min Accutase treatment. Spin down the cells in Dopa mat basal and resuspend in Dopa mat medium supplemented with 10µM Y27632 and CHIR.
Day 20-24: Cells are plated on Poly-D-lysine and laminin coated plates in a density of 2-2.5M cells / 6 well. Cells are dissociated using 60-90min Accutase supplemented with 10µM Y27632.
Day 25-27: 3µM cytosine arabinoside is added to Dopa mat media. Day 27 cells are washed twice with Dopa mat basal medium to remove any cytosine arabinoside residues.
Around Day 30: Cells are dissociated using Accutase supplemented with 10µM Y27632 and plated in onto Poly-D-lysine and laminin coated dishes. The final density depends on the assay.
Protocol references
Kriks S, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z, Carrillo-Reid L, Auyeung G, Antonacci C, Buch A, Yang L, Beal MF, Surmeier DJ, Kordower JH, Tabar V, Studer L. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature. 2011 Nov 6;480(7378):547-51. doi: 10.1038/nature10648. PMID: 22056989; PMCID: PMC3245796.
Ryan, S. D., Dolatabadi, N., Chan, S. F., Zhang, X., Akhtar, M. W., Parker, J., … Lipton, S. A.(2013). Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1alpha transcription. Cell, 155, 1351–1364.
Weykopf B, Haupt S, Jungverdorben J, Flitsch LJ, Hebisch M, Liu GH, Suzuki K, Belmonte JCI, Peitz M, Blaess S, Till A, Brüstle O. Induced pluripotent stem cell-based modeling of mutant LRRK2-associated Parkinson's disease. Eur J Neurosci. 2019 Feb;49(4):561-589. doi: 10.1111/ejn.14345. PMID: 30656775; PMCID: PMC7114274.
Fonseca-Ornelas L, Stricker JMS, Soriano-Cruz S, Weykopf B, Dettmer U, Muratore CR, Scherzer CR, Selkoe DJ. Parkinson-causing mutations in LRRK2 impair the physiological tetramerization of endogenous α-synuclein in human neurons. NPJ Parkinsons Dis. 2022 Sep 16;8(1):118. doi: 10.1038/s41531-022-00380-1. PMID: 36114228; PMCID: PMC9481630.