Sep 14, 2022
Midbrain organoid generation from mfNPC
- Rachel Bates1
- 1UCL
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
Funders Acknowledgement:
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
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Protocol materials
Stemolecule LDN-193189Stemgent - Bio-connectCatalog #04-0074
Step 1.1
L-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403
In 5 steps
CHIR99021R&D SystemsCatalog #4423
In 3 steps
Neurobasal Plus MediumGibco, ThermoFisherCatalog #A3582901
In 5 steps
N-2 max supplementR&D SystemsCatalog #AR009
In 5 steps
Db-cAMP (dibutyryl-cyclic AMP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D0627
Step 15.1
Cultrex Reduced Growth Factor Basement Membrane ExtractR&D SystemsCatalog #343301001
In 2 steps
Ultra-low attachment 6-well plateCorningCatalog #CLS3471
Step 12
SB431542Cell Signaling TechnologyCatalog #14775
Step 1.1
MEM, NEAA, no glutamineThermo FisherCatalog #10370021
In 5 steps
recombinant human SHH proteinQkine
In 4 steps
N21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
In 5 steps
InsulinMerck MilliporeSigma (Sigma-Aldrich)Catalog #I2643-25MG
Step 10.1
FGF8Novus BiologicalsCatalog #423-F8-025
In 4 steps
SAG 1 mg
STEMCELL Technologies Inc.Catalog #73412
In 2 steps
Glutamax (100x)Gibco - Thermo FischerCatalog #35050-061
In 5 steps
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membraneMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020
Step 10.1
BDNFQkineCatalog #QK050
Step 15.1
GDNFQkineCatalog #QK051
Step 15.1
DMEM/F-12Thermo FisherCatalog #11320033
In 3 steps
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 organoids in maintenance medium.
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mfNPC maintenance medium
50 % volume DMEM/F-12Sigma AldrichCatalog #11320033
50 % volume Neurobasal Plus MediumSigma AldrichCatalog #A3582901
1:50 N21-MAX Media Supplement (50X)Sigma AldrichCatalog #AR008
1:100 N-2 max supplementSigma AldrichCatalog #AR009
1 % volume Glutamax (100x)Sigma AldrichCatalog #35050-061
1 % volume MEM, NEAA, no glutamineSigma AldrichCatalog #10370021
10 micromolar (µM) SB431542Sigma AldrichCatalog #14775
0.5 micromolar (µM) SAG 1 mg
Sigma AldrichCatalog #73412
250 millimolar (mM) Stemolecule LDN-193189Sigma AldrichCatalog #04-0074
200 micromolar (µM) L-Ascorbic acidSigma AldrichCatalog #A4403
3 micromolar (µM) CHIR99021Sigma AldrichCatalog #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
Day 2
Change the medium to mfNPC medium supplemented with 0.0001 mg/mL FGF8Sigma AldrichCatalog #423-F8-025 0.0001 mg/mL recombinant human SHH proteinSigma Aldrich being careful not to touch the organoid.
Day 4
Day 4
Change the medium to patterning I medium adding 300 µL per well.
patterning I medium
50 % volume DMEM/F-12Sigma AldrichCatalog #11320033
50 % volume Neurobasal Plus MediumSigma AldrichCatalog #A3582901
1:50 N21-MAX Media Supplement (50X)Sigma AldrichCatalog #AR008
1:100 N-2 max supplementSigma AldrichCatalog #AR009
1 % volume Glutamax (100x)Sigma AldrichCatalog #35050-061
1 % volume MEM, NEAA, no glutamineSigma AldrichCatalog #10370021
0.5 micromolar (µM) SAG 1 mg
Sigma AldrichCatalog #73412
200 micromolar (µM) L-Ascorbic acidSigma AldrichCatalog #A4403
3 micromolar (µM) CHIR99021Sigma AldrichCatalog #4423
0.0001 mg/mL FGF8Sigma AldrichCatalog #423-F8-025
0.0001 mg/mL recombinant human SHH proteinSigma Aldrich
Day 6
Day 6
Change medium to patterning II medium with reduced CHIRR adding 300 µL per well.
Patterning II medium
50 % volume DMEM/F-12Sigma AldrichCatalog #11320033
50 % volume Neurobasal Plus MediumSigma AldrichCatalog #A3582901
1:50 N21-MAX Media Supplement (50X)Sigma AldrichCatalog #AR008
1:100 N-2 max supplementSigma AldrichCatalog #AR009
1 % volume Glutamax (100x)Sigma AldrichCatalog #35050-061
1 % volume MEM, NEAA, no glutamineSigma AldrichCatalog #10370021
200 micromolar (µM) L-Ascorbic acidSigma AldrichCatalog #A4403
0.7 micromolar (µM) CHIR99021Sigma AldrichCatalog #4423
0.0001 mg/mL FGF8Sigma AldrichCatalog #423-F8-025
0.0001 mg/mL recombinant human SHH proteinSigma Aldrich
Thaw Cultrex Reduced Growth Factor Basement Membrane ExtractSigma AldrichCatalog #343301001 Overnight at 4 °C ready for next day.
Day 8
Day 8
30m
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 ExtractSigma AldrichCatalog #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 MediumSigma AldrichCatalog #A3582901
1:50 N21-MAX Media Supplement (50X)Sigma AldrichCatalog #AR008
1:100 N-2 max supplementSigma AldrichCatalog #AR009
1 % volume Glutamax (100x)Sigma AldrichCatalog #35050-061
1 % volume MEM, NEAA, no glutamineSigma AldrichCatalog #10370021
200 micromolar (µM) L-Ascorbic acidSigma AldrichCatalog #A4403
0.0001 mg/mL FGF8Sigma AldrichCatalog #423-F8-025
0.0001 mg/mL recombinant human SHH proteinSigma Aldrich
0.00025 mg/mL Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membraneSigma AldrichCatalog #L2020
0.025 mg/mL InsulinSigma AldrichCatalog #I2643-25MG
Day 9
Day 9
30m
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 plateSigma AldrichCatalog #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
Day 10
Change medium to Differentiation medium.
Differentiation medium
100 % volume Neurobasal Plus MediumSigma AldrichCatalog #A3582901
1:50 N21-MAX Media Supplement (50X)Sigma AldrichCatalog #AR008
1:100 N-2 max supplementSigma AldrichCatalog #AR009
1 % volume Glutamax (100x)Sigma AldrichCatalog #35050-061
1 % volume MEM, NEAA, no glutamineSigma AldrichCatalog #10370021
200 micromolar (µM) L-Ascorbic acidSigma AldrichCatalog #A4403
125 micromolar (µM) Db-cAMP (dibutyryl-cyclic AMP)Sigma AldrichCatalog #D0627
10ng/mLBDNFSigma AldrichCatalog #QK050
10ng/mLGDNFSigma AldrichCatalog #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.2016.07.005Mohamed 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/fcab223Step 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/s41598-017-05633-1Step 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