Sep 14, 2022

Midbrain organoid generation from mfNPC

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvr4x1pgmk/v1
  • Rachel Bates1
  • 1UCL
  • Organoid and Assembloid
rachel.bates Bates
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvr4x1pgmk/v1
Protocol Citation: Rachel Bates 2022. Midbrain organoid generation from mfNPC. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4x1pgmk/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: September 07, 2022
Last Modified: May 31, 2024
Protocol  Integer ID: 69682
Keywords: ASAPCRN, midbrain organoid generation from mfnpc, midbrain organoid generation, neural progenitor cell, human midbrain, like organoid, human floor plate, mfnpc, cell
Funders Acknowledgements:
ASAP
Grant ID: ASAP2022
Abstract
This protocol describes our method for the differentiation of human floor plate neural progenitor cells into human midbrain-like organoids (hMLOs). 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
recombinant human SHH proteinQkine
FGF8Novus BiologicalsCatalog #423-F8-025
Cultrex Reduced Growth Factor Basement Membrane ExtractR&D SystemsCatalog #343301001
Ultra-low attachment 6-well plateCorningCatalog #CLS3471
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
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 organoids in maintenance medium.


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.
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 (2019). 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 9,000 cell/well in ultra-low attachment U-bottomed 96 well plates.
Note
BIOFLOAT plate from Facellitate work best for us compared to Corning or Nunc at producing uniform EBs.

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 being careful not to touch the organoid.



Day 4
Change the medium to patterning I medium adding 300 µL 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 300 µL 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
Thaw Cultrex Reduced Growth Factor Basement Membrane ExtractR&D SystemsCatalog #343301001 Overnight at 4 °C ready for next day.

Day 8
30m
Carefully remove as much medium as possible from each well being careful not to touch the organoid.
Add 15 µL ofCultrex Reduced Growth Factor Basement Membrane ExtractR&D SystemsCatalog #343301001 to each well and return to 37 °C incubator for 00:30:00 .

30m
Carefully add 300 µL of tissue induction medium to each well.
Note
Some organoids may float others with remain attached to plate, this does not affect the organoid.


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
Using sterilised scissors or a scalpel blade cut a pasteur pipette at the widest point.
Note
this will provide a sufficient bore to pick up the embedded organoid without damaging the cultrex.

Either add 2 mL of Tissue induction medium to Ultra-low attachment 6-well plateCorningCatalog #CLS3471 or add 500 µL to Ultra-low attachment 48 well plate.

using the cut pasteur pipette carefully pick up each organoid and move to new plate. For 6 well plate add up to 6 organoids per well or 1 per well in the 48-well plate.
Incubate plates at 37 °C on a orbital shaker set to 70 rpm .

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.

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
Step  1
Fedele S, Collo G, Behr K, Bischofberger J, Müller S, Kunath T, Christensen K, Gündner AL, Graf M, Jagasia R, Taylor V. Expansion of human midbrain floor plate progenitors from induced pluripotent stem cells increases dopaminergic neuron differentiation potential.
https://doi.org/10.1038/s41598-017-05633-1
Step  1
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. Modeling Parkinson's disease in midbrain-like organoids.
https://doi.org/10.1038/s41531-019-0078-4