Nov 30, 2023

Public workspacedopaminergic neurons from iPSC

DOI
dx.doi.org/10.17504/protocols.io.bp2l6xynzlqe/v1
  • Rachel Bates1
  • 1UCL
  • Organoid and Assembloid
rachel.bates Bates
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DOI: 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
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.
LINK
https://doi.org/10.1016/j.stem.2016.07.005

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.
LINK
https://doi.org/10.1093/braincomms/fcab223

Protocol materials
ReagentDMEM/F-12Thermo FisherCatalog #11320033
ReagentStemolecule LDN-193189Stemgent - Bio-connectCatalog #04-0074
ReagentL-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
ReagentN21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
ReagentSB431542Cell Signaling TechnologyCatalog #14775
ReagentCHIR99021R&D SystemsCatalog #4423
ReagentNeurobasal Plus MediumGibco - Thermo Fisher ScientificCatalog #A3582901
ReagentMEM, NEAA, no glutamineThermo FisherCatalog #10370021
ReagentSAG 1 mg STEMCELL Technologies Inc.Catalog #73412
ReagentN-2 max supplementR&D SystemsCatalog #AR009
ReagentGlutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
ReagentFGF8Novus BiologicalsCatalog #423-F8-025
ReagentN21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
ReagentSAG 1 mg STEMCELL Technologies Inc.Catalog #73412
ReagentCHIR99021R&D SystemsCatalog #4423
Reagentrecombinant human SHH proteinQkine
ReagentMEM, NEAA, no glutamineThermo FisherCatalog #10370021
ReagentL-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
ReagentN-2 max supplementR&D SystemsCatalog #AR009
ReagentGlutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
ReagentDMEM/F-12Thermo FisherCatalog #11320033
ReagentNeurobasal Plus MediumGibco - Thermo Fisher ScientificCatalog #A3582901
ReagentFGF8Novus BiologicalsCatalog #423-F8-025
ReagentN-2 max supplementR&D SystemsCatalog #AR009
ReagentMEM, NEAA, no glutamineThermo FisherCatalog #10370021
ReagentNeurobasal Plus MediumGibco - Thermo Fisher ScientificCatalog #A3582901
ReagentN21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
ReagentGlutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
Reagentrecombinant human SHH proteinQkine
ReagentDMEM/F-12Thermo FisherCatalog #11320033
ReagentL-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
ReagentCHIR99021R&D SystemsCatalog #4423
ReagentInsulinMerck MilliporeSigma (Sigma-Aldrich)Catalog #I2643-25MG
ReagentGlutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
ReagentMEM, NEAA, no glutamineThermo FisherCatalog #10370021
ReagentN-2 max supplementR&D SystemsCatalog #AR009
ReagentL-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
ReagentNeurobasal Plus MediumGibco - Thermo Fisher ScientificCatalog #A3582901
Reagentrecombinant human SHH proteinQkine
ReagentLaminin from Engelbreth-Holm-Swarm murine sarcoma basement membraneMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020
ReagentN21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
ReagentFGF8Novus BiologicalsCatalog #423-F8-025
ReagentNeurobasal Plus MediumGibco - Thermo Fisher ScientificCatalog #A3582901
ReagentN-2 max supplementR&D SystemsCatalog #AR009
ReagentMEM, NEAA, no glutamineThermo FisherCatalog #10370021
ReagentBDNFQkineCatalog #QK050
ReagentN21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
ReagentGlutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
ReagentDb-cAMP (dibutyryl-cyclic AMP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D0627
ReagentGDNFQkineCatalog #QK051
ReagentL-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
ReagentFGF8Novus BiologicalsCatalog #423-F8-025
Reagentrecombinant human SHH proteinQkine
Day 0
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 (2017). Expansion of human midbrain floor plate progenitors from induced pluripotent stem cells increases dopaminergic neuron differentiation potential.. Scientific reports.
LINK
https://doi.org/10.1038/s41598-017-05633-1

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 (2019). Modeling Parkinson's disease in midbrain-like organoids.. NPJ Parkinson's disease.
LINK
https://doi.org/10.1038/s41531-019-0078-4

mfNPC maintenance medium

Concentration50 % volume ReagentDMEM/F-12Sigma AldrichCatalog #11320033
Concentration50 % volume ReagentNeurobasal Plus MediumSigma AldrichCatalog #A3582901
1:50 ReagentN21-MAX Media Supplement (50X)Sigma AldrichCatalog #AR008
1:100 ReagentN-2 max supplementSigma AldrichCatalog #AR009
Concentration1 % volume ReagentGlutamax (100x)Sigma AldrichCatalog #35050-061
Concentration1 % volume ReagentMEM, NEAA, no glutamineSigma AldrichCatalog #10370021
Concentration10 micromolar (µM) ReagentSB431542Sigma AldrichCatalog #14775
Concentration0.5 micromolar (µM) ReagentSAG 1 mg Sigma AldrichCatalog #73412
Concentration250 millimolar (mM) ReagentStemolecule LDN-193189Sigma AldrichCatalog #04-0074
Concentration200 micromolar (µM) ReagentL-Ascorbic acidSigma AldrichCatalog #A4403
Concentration3 micromolar (µM) ReagentCHIR99021Sigma AldrichCatalog #4423



mfNPCs were detached using accutase at Temperature37 °C for Duration00:03:00 .

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
Day 2
Change the medium to mfNPC medium supplemented with Concentration0.0001 mg/mL ReagentFGF8Sigma AldrichCatalog #423-F8-025 Concentration0.0001 mg/mL Reagentrecombinant human SHH proteinSigma Aldrich .



Day 4
Day 4
Change the medium to patterning I medium adding Amount1 mL per well.

patterning I medium

Concentration50 % volume ReagentDMEM/F-12Sigma AldrichCatalog #11320033
Concentration50 % volume ReagentNeurobasal Plus MediumSigma AldrichCatalog #A3582901
1:50 ReagentN21-MAX Media Supplement (50X)Sigma AldrichCatalog #AR008
1:100 ReagentN-2 max supplementSigma AldrichCatalog #AR009
Concentration1 % volume ReagentGlutamax (100x)Sigma AldrichCatalog #35050-061
Concentration1 % volume ReagentMEM, NEAA, no glutamineSigma AldrichCatalog #10370021
Concentration0.5 micromolar (µM) ReagentSAG 1 mg Sigma AldrichCatalog #73412
Concentration200 micromolar (µM) ReagentL-Ascorbic acidSigma AldrichCatalog #A4403
Concentration3 micromolar (µM) ReagentCHIR99021Sigma AldrichCatalog #4423
Concentration0.0001 mg/mL ReagentFGF8Sigma AldrichCatalog #423-F8-025
Concentration0.0001 mg/mL Reagentrecombinant human SHH proteinSigma Aldrich
Day 6
Day 6
Change medium to patterning II medium with reduced CHIRR adding Amount1 mL per well.

Patterning II medium

Concentration50 % volume ReagentDMEM/F-12Sigma AldrichCatalog #11320033
Concentration50 % volume ReagentNeurobasal Plus MediumSigma AldrichCatalog #A3582901
1:50 ReagentN21-MAX Media Supplement (50X)Sigma AldrichCatalog #AR008
1:100 ReagentN-2 max supplementSigma AldrichCatalog #AR009
Concentration1 % volume ReagentGlutamax (100x)Sigma AldrichCatalog #35050-061
Concentration1 % volume ReagentMEM, NEAA, no glutamineSigma AldrichCatalog #10370021
Concentration200 micromolar (µM) ReagentL-Ascorbic acidSigma AldrichCatalog #A4403
Concentration0.7 micromolar (µM) ReagentCHIR99021Sigma AldrichCatalog #4423
Concentration0.0001 mg/mL ReagentFGF8Sigma AldrichCatalog #423-F8-025
Concentration0.0001 mg/mL Reagentrecombinant human SHH proteinSigma Aldrich
Day 8
Day 8
30m
30m
Carefully add Amount1 mL of tissue induction medium to each well.



Tissue induction medium


Concentration100 % volume ReagentNeurobasal Plus MediumSigma AldrichCatalog #A3582901
1:50 ReagentN21-MAX Media Supplement (50X)Sigma AldrichCatalog #AR008
1:100 ReagentN-2 max supplementSigma AldrichCatalog #AR009
Concentration1 % volume ReagentGlutamax (100x)Sigma AldrichCatalog #35050-061
Concentration1 % volume ReagentMEM, NEAA, no glutamineSigma AldrichCatalog #10370021
Concentration200 micromolar (µM) ReagentL-Ascorbic acidSigma AldrichCatalog #A4403
Concentration0.0001 mg/mL ReagentFGF8Sigma AldrichCatalog #423-F8-025
Concentration0.0001 mg/mL Reagentrecombinant human SHH proteinSigma Aldrich
Concentration0.00025 mg/mL ReagentLaminin from Engelbreth-Holm-Swarm murine sarcoma basement membraneSigma AldrichCatalog #L2020
Concentration0.025 mg/mL ReagentInsulinSigma AldrichCatalog #I2643-25MG

Day 9
Day 9
30m
30m
Add Amount2 mL of Tissue induction medium to 12 well plate.

Incubate plates at Temperature37 °C .

Day 10
Day 10
Change medium to Differentiation medium.
Differentiation medium

Concentration100 % volume ReagentNeurobasal Plus MediumSigma AldrichCatalog #A3582901
1:50 ReagentN21-MAX Media Supplement (50X)Sigma AldrichCatalog #AR008
1:100 ReagentN-2 max supplementSigma AldrichCatalog #AR009
Concentration1 % volume ReagentGlutamax (100x)Sigma AldrichCatalog #35050-061
Concentration1 % volume ReagentMEM, NEAA, no glutamineSigma AldrichCatalog #10370021
Concentration200 micromolar (µM) ReagentL-Ascorbic acidSigma AldrichCatalog #A4403
Concentration125 micromolar (µM) ReagentDb-cAMP (dibutyryl-cyclic AMP)Sigma AldrichCatalog #D0627
10ng/mLReagentBDNFSigma AldrichCatalog #QK050
10ng/mLReagentGDNFSigma AldrichCatalog #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