Nov 30, 2023

dopaminergic neurons from iPSC

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6xynzlqe/v1
  • Rachel Bates1
  • 1UCL
  • Organoid and Assembloid
rachel.bates Bates
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6xynzlqe/v1
Protocol Citation: Rachel Bates 2023. dopaminergic neurons from iPSC. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xynzlqe/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: November 30, 2023
Last Modified: May 31, 2024
Protocol  Integer ID: 91648
Keywords: ASAPCRN, dopaminergic neurons from ipsc, dopaminergic neuron, neural progenitor cell, neuron, human midbrain, human floor plate, ipsc, cell
Funders Acknowledgements:
Asap
Abstract
This protocol describes our method for the differentiation of human floor plate neural progenitor cells into human midbrain-dopaminergic neurons. This protocol has been developed using a combination of several published protocols.

Adapted from
Citation
Jo J, Xiao Y, Sun AX, Cukuroglu E, Tran HD, Göke J, Tan ZY, Saw TY, Tan CP, Lokman H, Lee Y, Kim D, Ko HS, Kim SO, Park JH, Cho NJ, Hyde TM, Kleinman JE, Shin JH, Weinberger DR, Tan EK, Je HS, Ng HH (2016). Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons. Cell stem cell.
https://doi.org/10.1016/j.stem.2016.07.005
LINK

Citation
Mohamed NV, Sirois J, Ramamurthy J, Mathur M, Lépine P, Deneault E, Maussion G, Nicouleau M, Chen CX, Abdian N, Soubannier V, Cai E, Nami H, Thomas RA, Wen D, Tabatabaei M, Beitel LK, Singh Dolt K, Karamchandani J, Stratton JA, Kunath T, Fon EA, Durcan TM (2021). Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy. Brain communications.
https://doi.org/10.1093/braincomms/fcab223
LINK

Protocol materials
DMEM/F-12Thermo FisherCatalog #11320033
Stemolecule LDN-193189Stemgent - Bio-connectCatalog #04-0074
L-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
N21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
SB431542Cell Signaling TechnologyCatalog #14775
CHIR99021R&D SystemsCatalog #4423
Neurobasal Plus MediumGibco - Thermo Fisher ScientificCatalog #A3582901
MEM, NEAA, no glutamineThermo FisherCatalog #10370021
SAG 1 mg STEMCELL Technologies Inc.Catalog #73412
N-2 max supplementR&D SystemsCatalog #AR009
Glutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
FGF8Novus BiologicalsCatalog #423-F8-025
recombinant human SHH proteinQkine
InsulinMerck MilliporeSigma (Sigma-Aldrich)Catalog #I2643-25MG
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membraneMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020
BDNFQkineCatalog #QK050
Db-cAMP (dibutyryl-cyclic AMP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D0627
GDNFQkineCatalog #QK051
Day 0
Human floor plate neuronal progenitor cells (mfNPC) were derived using Smits 2019 protocol. They were maintained for a minimum of 5 passages before being used to generate dopaminergic neurons.


Citation
Fedele S, Collo G, Behr K, Bischofberger J, Müller S, Kunath T, Christensen K, Gündner AL, Graf M, Jagasia R, Taylor V (Invalid date). Expansion of human midbrain floor plate progenitors from induced pluripotent stem cells increases dopaminergic neuron differentiation potential. Scientific reports.
https://doi.org/10.1038/s41598-017-05633-1
LINK

Citation
Smits LM, Reinhardt L, Reinhardt P, Glatza M, Monzel AS, Stanslowsky N, Rosato-Siri MD, Zanon A, Antony PM, Bellmann J, Nicklas SM, Hemmer K, Qing X, Berger E, Kalmbach N, Ehrlich M, Bolognin S, Hicks AA, Wegner F, Sterneckert JL, Schwamborn JC (Invalid date). Modeling Parkinson's disease in midbrain-like organoids. NPJ Parkinson's disease.
https://doi.org/10.1038/s41531-019-0078-4
LINK

mfNPC maintenance medium

50 % volume DMEM/F-12Thermo FisherCatalog #11320033
50 % volume Neurobasal Plus MediumGibco - Thermo Fisher ScientificCatalog #A3582901
1:50 N21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
1:100 N-2 max supplementR&D SystemsCatalog #AR009
1 % volume Glutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
1 % volume MEM, NEAA, no glutamineThermo FisherCatalog #10370021
10 micromolar (µM) SB431542Cell Signaling TechnologyCatalog #14775
0.5 micromolar (µM) SAG 1 mg STEMCELL Technologies Inc.Catalog #73412
250 millimolar (mM) Stemolecule LDN-193189Stemgent - Bio-connectCatalog #04-0074
200 micromolar (µM) L-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
3 micromolar (µM) CHIR99021R&D SystemsCatalog #4423



mfNPCs were detached using accutase at 37 °C for 00:03:00 .

3m
Re-suspend cells in d0 induction medium and plate 0.1 x 105 cells per well 12 well plate coated with Ultimatrix for 1 hour beforehand.


Day 2
Change the medium to mfNPC medium supplemented with 0.0001 mg/mL FGF8Novus BiologicalsCatalog #423-F8-025 0.0001 mg/mL recombinant human SHH proteinQkine .



Day 4
Change the medium to patterning I medium adding 1 mL per well.

patterning I medium

50 % volume DMEM/F-12Thermo FisherCatalog #11320033
50 % volume Neurobasal Plus MediumGibco - Thermo Fisher ScientificCatalog #A3582901
1:50 N21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
1:100 N-2 max supplementR&D SystemsCatalog #AR009
1 % volume Glutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
1 % volume MEM, NEAA, no glutamineThermo FisherCatalog #10370021
0.5 micromolar (µM) SAG 1 mg STEMCELL Technologies Inc.Catalog #73412
200 micromolar (µM) L-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
3 micromolar (µM) CHIR99021R&D SystemsCatalog #4423
0.0001 mg/mL FGF8Novus BiologicalsCatalog #423-F8-025
0.0001 mg/mL recombinant human SHH proteinQkine
Day 6
Change medium to patterning II medium with reduced CHIRR adding 1 mL per well.

Patterning II medium

50 % volume DMEM/F-12Thermo FisherCatalog #11320033
50 % volume Neurobasal Plus MediumGibco - Thermo Fisher ScientificCatalog #A3582901
1:50 N21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
1:100 N-2 max supplementR&D SystemsCatalog #AR009
1 % volume Glutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
1 % volume MEM, NEAA, no glutamineThermo FisherCatalog #10370021
200 micromolar (µM) L-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
0.7 micromolar (µM) CHIR99021R&D SystemsCatalog #4423
0.0001 mg/mL FGF8Novus BiologicalsCatalog #423-F8-025
0.0001 mg/mL recombinant human SHH proteinQkine
Day 8
30m
Carefully add 1 mL of tissue induction medium to each well.



Tissue induction medium


100 % volume Neurobasal Plus MediumGibco - Thermo Fisher ScientificCatalog #A3582901
1:50 N21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
1:100 N-2 max supplementR&D SystemsCatalog #AR009
1 % volume Glutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
1 % volume MEM, NEAA, no glutamineThermo FisherCatalog #10370021
200 micromolar (µM) L-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
0.0001 mg/mL FGF8Novus BiologicalsCatalog #423-F8-025
0.0001 mg/mL recombinant human SHH proteinQkine
0.00025 mg/mL Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membraneMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020
0.025 mg/mL InsulinMerck MilliporeSigma (Sigma-Aldrich)Catalog #I2643-25MG

Day 9
30m
Add 2 mL of Tissue induction medium to 12 well plate.

Incubate plates at 37 °C .

Day 10
Change medium to Differentiation medium.
Differentiation medium

100 % volume Neurobasal Plus MediumGibco - Thermo Fisher ScientificCatalog #A3582901
1:50 N21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
1:100 N-2 max supplementR&D SystemsCatalog #AR009
1 % volume Glutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
1 % volume MEM, NEAA, no glutamineThermo FisherCatalog #10370021
200 micromolar (µM) L-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
125 micromolar (µM) Db-cAMP (dibutyryl-cyclic AMP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D0627
10ng/mLBDNFQkineCatalog #QK050
10ng/mLGDNFQkineCatalog #QK051

After day 10 perform a 75% medium change every 2-3 days.
Use neurons after a further 45 days differentiation.
Citations
Jo J, Xiao Y, Sun AX, Cukuroglu E, Tran HD, Göke J, Tan ZY, Saw TY, Tan CP, Lokman H, Lee Y, Kim D, Ko HS, Kim SO, Park JH, Cho NJ, Hyde TM, Kleinman JE, Shin JH, Weinberger DR, Tan EK, Je HS, Ng HH. Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons.
https://doi.org/10.1016/j.stem.2016.07.005
Mohamed NV, Sirois J, Ramamurthy J, Mathur M, Lépine P, Deneault E, Maussion G, Nicouleau M, Chen CX, Abdian N, Soubannier V, Cai E, Nami H, Thomas RA, Wen D, Tabatabaei M, Beitel LK, Singh Dolt K, Karamchandani J, Stratton JA, Kunath T, Fon EA, Durcan TM. Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy.
https://doi.org/10.1093/braincomms/fcab223