Midbrain-like Organoids generation from hiPSCs

Hariam Raji; michela.deleidi

Mar 30, 2023

Midbrain-like Organoids generation from hiPSCs

DOI

https://dx.doi.org/10.17504/protocols.io.kqdg39d5qg25/v1

Hariam Raji¹,
michela.deleidi ¹

¹German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany

Federico Bertoli

ASAP

DOI: https://dx.doi.org/10.17504/protocols.io.kqdg39d5qg25/v1

Protocol Citation: Hariam Raji, michela.deleidi 2023. Midbrain-like Organoids generation from hiPSCs. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg39d5qg25/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: March 28, 2023

Last Modified: May 31, 2024

Protocol Integer ID: 79544

Keywords: Midbrain-like Organoids, human induced pluripotent stem cells (hiPSCs), Matrigel embedding, ASAPCRN, like organoids generation from hipsc, pluripotent stem cell, induced pluripotent stem cell, like organoids generation, human midbrain, like organoid, hipsc, midbrain, cell

Abstract

In this protocol we describe the differentiation of human induced pluripotent stem cells (hiPSCs) into human midbrain-like organoids (hMLOs). This protocol has been developed based from several published protocols.

Attachments

366-820.pdf

517KB

Materials

 Day 0-Medium composition:
AB
DMEM F12 (w/o HEPES) /Neurobasal (1:1)
N2 0.5%250 µL/50 mL
B27 1%500 µL/50 mL
NEAA 1%500 µL/50 mL
B-MercaptoEtOH  0.1%
Heparin1 µg/mL (100 mg/mL) 1:100.000
SB43154210 µM (10 mM) 1:1000
Noggin200 ng/mL (200 µg/mL) 1:1000 or (LDN 1:100.000)
CHIR990210.7 µM (3 mM) 1:4285 11.7 µL/50 mL
Rock Inhibitor (APOI)50 µM (10 mM) 1:200
 
Day 4-Medium composition:
AB
DMEM F12 (w/o HEPES)/Neurobasal (1:1)
N2 0.5%250 µL/50 mL
B27 1%500 µL/50 mL
NEAA 1%500 µL/50 mL
B-MercaptoEtOH  0.1%
Heparin1 µg/mL (100 mg/mL) 1:100.000
SB43154210 µM (10 mM) 1:1000
Noggin200 ng/mL (200 µg/mL) 1:1000 or (LDN 1:100.000)
CHIR990217.5 µM (3 mM) 1:400
SHH-C25II100 ng/mL (100 µg/mL) 1:1000
FGF8100 ng/mL (100 µg/mL) 1:1000
PMA1 µM (Stock: 1mM)
 
Day 7-Medium composition:  
AB
Neurobasal
N20.50%
B271%
Glutamax1%
NEAA1%
B-MercaptoEtOH0.1%
Insulin2.5 µg/mL (5 mg/mL) 1:2000
Laminin200 ng/mL (100 µg/mL) 1:500
SHH-C25II100 ng/mL (100 µg/mL) 1:1000
CHIR990217.5 µM (3 mM) 1:400
FGF8100 ng/mL (100 µg/mL) 1:10.000
PMA1 µM (Stock: 1 mM)
 
 Final Differentiation Medium composition:
AB
Neurobasal
N2(250 µL/50 mL)
B27(500 µL/50 mL)
Glutamax1% (500 µL/50 mL)
NEAA1% (500 µL/50 mL)
B-MercaptoEtOH0.1% (50 µL/50 mL)
BDNF10 ng/mL (20 µg/mL) 1:2000
GDNF10 ng/mL (20 µg/mL) 1:2000
Ascorbic Acid100 µM (200 mM) 1:2000
db-cAMP125 µM (500 mM) 1:4000
CHIR990217.5 µM (3 mM) 1:400
 
Cytokines and factors: 
ABCD
CompanyCat.Nr.NameSize
ThermoScientific12-587-010B2710 ml
ThermoScientific17502-048N25 ml
R&D System3400-010-03Laminin I1 mg
Corning354230Matrigel10 ml
SelleckchemS1049ApoI (Y-27632 2HCl)
AppliChemA0455,1000dbCAMP (C)1 mg
Sigma-Aldrich540220-5MGPurmorphamine (PMA)
R&D System1614SB 43154210 mg
PeproTech450-02-500BDNF500 µg
PeproTech450-10-500GDNF500 µg
PeproTech100-25-500FGF8500 µg
PeproTech100-45-100Sonic hedgehog100 µg
Sigma-AldrichA6964-100 mlAccutase100 ml
Gibco11320074DMEM/F12 w/o HEPES500 ml
Gibco21103049Neurobasal Medium500 ml
Gibco11140035MEM NEAA100 ml
Gibco35050038Glutamaxx100 ml
SigmaSML1046-5MGCHIR 990213 mM
Sigma25556-4L-Ascorbic Acid200 mM
ThermoScientific12585014Insulin, human recomb.4 mg/ml
Axon1509LDN 193189HCl100 µM
 ReagentB-27™ Supplement (50X) minus vitamin AThermo Fisher ScientificCatalog #B-27™ Supplement (50X), minus vi  
ReagentN-2 Supplement (100X)Thermo FisherCatalog #17502048 
ReagentCultrex Stem Cell Qualified Laminin I PathclearR&D SystemsCatalog #3400-010-03 
ReagentGrowth Factor Reduced (GFR) Matrigel®CorningCatalog #354230 
ReagentY-27632SelleckchemCatalog #S1049 
ReagentN6-2-O-Dibutyryl-Adenosine 35-Cyclophosphate Sodium Salt 1-hydrate BioChemicaPanreac AppliChemCatalog # A0455,1000 
ReagentPurmorphamineMerck Millipore (EMD Millipore)Catalog #540220 
ReagentSB 431542R&D SystemsCatalog #1614 
ReagentBDNFpeprotechCatalog #450-02-500 
ReagentGDNFpeprotechCatalog #450-10-500 
ReagentFGF8peprotechCatalog #100-25-500 
ReagentSonic hedgehogpeprotechCatalog #100-45-100 
ReagentAccutase® solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A6964 
ReagentDMEM/F-12Thermo FisherCatalog #11320074 
ReagentNeurobasal medium Gibco - Thermo Fisher ScientificCatalog #21103049 
ReagentMEM Non-Essential Amino Acids Solution (100X)Thermo Fisher ScientificCatalog #11140035 
ReagentGlutaMAXGibco - Thermo Fisher ScientificCatalog #35050038 
ReagentCHIR99021Merck MilliporeSigma (Sigma-Aldrich)Catalog #SML1046-5MG 
ReagentL-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #255564 
ReagentInsulin, human recombinant, zinc solutionThermo FisherCatalog #12585014 
ReagentLDN 193189 hydrochlorideAxon MedchemCatalog #1509 

Troubleshooting

Day 0

Dissociate iPSC colonies to single cells with Accutase for Duration00:07:00   at Temperature37 °C  .

Re-suspend cells in day0 medium and plate 8.000 cells/well in 96-Wells Plate U-round-Bottom Low Attachment.

Day 4

Carefully exchange the medium, without touching the EBs.

Day 7: Matrigel embedding

1d 0h 30m

Dilute Matrigel in a 3:2 ratio with day 4 medium (used as an embedding mixture).

Wash EBs in day 4 medium. Transfer and mix 5-8 EBs into the embedding mixture and plate onto a 6-well ultra-low-attachment plate.

Incubate for Duration00:30:00   at Temperature37 °C   and add day 7 medium.

30m

Incubate at least Duration24:00:00   between d7 and d8.

Day 8

Add fresh differentiation medium without disrupting the embedded EBs.

After Day 8 change medium twice a week. 

Day 10-13

At day 10 reduce CHIR to Concentration3.0 micromolar (µM)   (1:1000).

Day 13-15

Reduce CHIR to Concentration0.7 micromolar (µM)  . Remove CHIR from medium at day 15 and onwards.

Day 20-25

Manually dissociate organoids from Matrigel, using two surgical needles.

Place on orbital shaker after dissociation (Shaker80 rpm ).

	A	B
	DMEM F12 (w/o HEPES) /Neurobasal (1:1)
	N2 0.5%	250 µL/50 mL
	B27 1%	500 µL/50 mL
	NEAA 1%	500 µL/50 mL
	B-MercaptoEtOH 0.1%
	Heparin	1 µg/mL (100 mg/mL) 1:100.000
	SB431542	10 µM (10 mM) 1:1000
	Noggin	200 ng/mL (200 µg/mL) 1:1000 or (LDN 1:100.000)
	CHIR99021	0.7 µM (3 mM) 1:4285 11.7 µL/50 mL
	Rock Inhibitor (APOI)	50 µM (10 mM) 1:200

	A	B
	DMEM F12 (w/o HEPES)/Neurobasal (1:1)
	N2 0.5%	250 µL/50 mL
	B27 1%	500 µL/50 mL
	NEAA 1%	500 µL/50 mL
	B-MercaptoEtOH 0.1%
	Heparin	1 µg/mL (100 mg/mL) 1:100.000
	SB431542	10 µM (10 mM) 1:1000
	Noggin	200 ng/mL (200 µg/mL) 1:1000 or (LDN 1:100.000)
	CHIR99021	7.5 µM (3 mM) 1:400
	SHH-C25II	100 ng/mL (100 µg/mL) 1:1000
	FGF8	100 ng/mL (100 µg/mL) 1:1000
	PMA	1 µM (Stock: 1mM)

	A	B
	Neurobasal
	N2	0.50%
	B27	1%
	Glutamax	1%
	NEAA	1%
	B-MercaptoEtOH	0.1%
	Insulin	2.5 µg/mL (5 mg/mL) 1:2000
	Laminin	200 ng/mL (100 µg/mL) 1:500
	SHH-C25II	100 ng/mL (100 µg/mL) 1:1000
	CHIR99021	7.5 µM (3 mM) 1:400
	FGF8	100 ng/mL (100 µg/mL) 1:10.000
	PMA	1 µM (Stock: 1 mM)

	A	B
	Neurobasal
	N2	(250 µL/50 mL)
	B27	(500 µL/50 mL)
	Glutamax	1% (500 µL/50 mL)
	NEAA	1% (500 µL/50 mL)
	B-MercaptoEtOH	0.1% (50 µL/50 mL)
	BDNF	10 ng/mL (20 µg/mL) 1:2000
	GDNF	10 ng/mL (20 µg/mL) 1:2000
	Ascorbic Acid	100 µM (200 mM) 1:2000
	db-cAMP	125 µM (500 mM) 1:4000
	CHIR99021	7.5 µM (3 mM) 1:400

A	B	C	D
Company	Cat.Nr.	Name	Size
ThermoScientific	12-587-010	B27	10 ml
ThermoScientific	17502-048	N2	5 ml
R&D System	3400-010-03	Laminin I	1 mg
Corning	354230	Matrigel	10 ml
Selleckchem	S1049	ApoI (Y-27632 2HCl)
AppliChem	A0455,1000	dbCAMP (C)	1 mg
Sigma-Aldrich	540220-5MG	Purmorphamine (PMA)
R&D System	1614	SB 431542	10 mg
PeproTech	450-02-500	BDNF	500 µg
PeproTech	450-10-500	GDNF	500 µg
PeproTech	100-25-500	FGF8	500 µg
PeproTech	100-45-100	Sonic hedgehog	100 µg
Sigma-Aldrich	A6964-100 ml	Accutase	100 ml
Gibco	11320074	DMEM/F12 w/o HEPES	500 ml
Gibco	21103049	Neurobasal Medium	500 ml
Gibco	11140035	MEM NEAA	100 ml
Gibco	35050038	Glutamaxx	100 ml
Sigma	SML1046-5MG	CHIR 99021	3 mM
Sigma	25556-4	L-Ascorbic Acid	200 mM
ThermoScientific	12585014	Insulin, human recomb.	4 mg/ml
Axon	1509	LDN 193189HCl	100 µM

Public workspaceMidbrain-like Organoids generation from hiPSCs

Midbrain-like Organoids generation from hiPSCs