Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Summary

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Abstract

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

Introduction

Mitochondrial diseases represent the largest class of inborn errors of metabolism¹. They are caused by genetic mutations disrupting different mitochondrial processes, including oxidative phosphorylation (OXPHOS)², respiratory chain assembly, mitochondrial dynamics, and mitochondrial DNA transcription or replication³. Tissues with energy requirements are particularly affected by mitochondrial dysfunction⁴. Accordingly, patients with mitochondrial diseases typically develop early-onset neurological manifestations.

There are currently no treatments ....

Protocol

NOTE: The use of human iPSCs may require an ethical approval. iPSCs used in this study were derived from healthy control individuals following local ethical approval (#2019-681). All cell culture procedures must be performed under a sterile cell culture hood, carefully disinfecting all reagents and consumables before transferring under the hood. Human iPSCs used for differentiation should have a passage number below 50 to avoid potential genomic aberrations that may occur upon extensive culture. The pluripotent state of the cells should be validated before organoid generation, for example, by monitoring the expression of pluripotency-associated markers such as NANOG o....

Results

The protocol described here facilitates the robust generation of round organoids (Figure 1A). The generated organoids contain mature neurons that can be visualized using protein markers specific for axons (SMI312) and dendrites (microtubule-associated protein 2 (MAP2)) (Figure 1B). Mature organoids contain not only neuronal cells (MAP2-positive) but also glial cells (e.g., positive for the astrocyte marker S100 .......

Discussion

This paper describes the reproducible generation of human iPSC-derived brain organoids and their use for mitochondrial disease modeling. The protocol described here is modified based on a previously published work²⁰. One major advantage of the present protocol is that it does not require the manual embedding of each organoid into a scaffolding matrix. In fact, the matrix solution is simply dissolved into the cell culture medium. Moreover, there is no need to employ expensive bioreactors, as organo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-mercaptoethanol	Gibco	31350-010
Affinity Designer	Serif (Europe) Ltd		Layout software; Vector graphics editor
Alexa Fluor 488 donkey anti-guinea pig	Sigma Aldrich	SAB4600033-250UL	1:300
Alexa Fluor 488 donkey anti-mouse	Thermo Fisher Scientific	A-31571	1:300
Antimycin A	Sigma Aldrich	1397-94-0
Anti-β-Tubulin III (TUJ-1)	Sigma Aldrich	T8578	1:2000
Argon Laser	Melles Griot		Any other Laser, e.g., diode lasers emitting 488 is fine, too
Ascorbic acid	Sigma	A92902
B-27 with Vitamin A	Gibco	17504044
Bacto Agar	Becton Dickinson		3% in PBS, store solution at -20 °C
BDNF	Miltenyi Biotec	130-093-0811
cAMP	Sigma	D0627
Cell Star cell culture 6 well plate	Greiner-Bio-One	657160
Chemically Defined Lipid Concentrate	Gibco	11905031
Confocal laser scanning microscope C1	Nikon Microscope Solutions		Modular confocal microscope system
Corning Matrigel Growth Factor Reduced (GFR) Basement membrane matrix, Phenol Red-free, LDEV-free	Corning	356231	Matrix component
CyQUANT Cell Proliferation Assay Kit	Thermo Fisher	C7026
DMEM/F12	ThermoFisher	31330038
DMSO	Sigma	D2660-100ML
Donkey anti-goat Cy3	Merck Millipore	AP180C	1:300
Donkey anti-mouse Cy3	Merck Millipore	AP192C	1:300
Donkey anti-rabbit Cy3	Merck Millipore	AP182C	1:300
DPBS	Gibco	14190250
DS-Q1Mc camera	Nikon Microscope Solutions
Eclipse 90i upright widefield microscope	Nikon Microscope Solutions
Eclipse E 600FN upright microscope	Nikon Microscope Solutions
Eclipse Ts2 Inverted Microscope	Nikon Microsope Solutions
EZ-C1 Silver Version 3.91	Nikon Microscope Solutions		Imaging software for confocal microscope
FCCP	Sigma Aldrich	370-86-5
Fetal Bovine Serum	Gibco	10270-106
GDNF	Miltenyi Biotec	130-096-291
Glasgow MEM	Gibco	11710-035
Glass Pasteur pipette	Brand	747715	Inverted
Glutamax	Gibco	35050-061
Helium-Neon Laser	Melles Griot		Every other Laser, e.g., diode lasers emitting 594 is fine, too
Heparin	Merck	H3149-25KU
HERACell 240i CO2 Incubator	Thermo Scientific	51026331
Hoechst 33342	Invitrogen	H3570	1:2500
Image J 1.53c	Wayne Rasband National Institute of Health		Image processing Software
Injekt Solo 10 mL/ Luer	Braun	4606108V
Knockout Serum Replacement	Gibco	10828010
Laser (407 nm)	Coherent		Any other Laser, e.g., diode lasers emitting 407 is fine, too
Map2	Synaptic Systems	No. 188004	1:1000
Maxisafe 2030i
MEM NEAA	Gibco	11140-050
mTeSR Plus	Stemcell Technology	85850	iPSC medium
Multifuge X3R Centrifuge	Thermo Scientific	10325804
MycoAlert Mycoplasma Detection Kit	Lonza	# LT07-218
N2 Supplement	Gibco	17502-048
Needle for single usage (23G x 1” TW)	Neoject	10016
NIS-Elements Aadvanced Research 3.2	Nikon		Imaging software
Oligomycin A	Sigma Aldrich	75351
Orbital Shaker Heidolph Unimax 1010	Heidolph	543-12310-00
PAP Pen	Sigma	Z377821-1EA	To draw hydrophobic barrier on slides.
Papain Dissociation System kit	Worthington	LK003150
Paraformaldehyde	Merck	818715	4% in PBS, store solution at -20 °C
Pasteur pipette 7mL	VWR	612-1681	Graduated up to 3 mL
Penicillin-Streptomycin	Gibco	15140-122
Plan Apo VC 20x / 0.75 air DIC N2  ∞/0.17 WD 1.0	Nikon Microscope Solutions		Dry Microscope Objective
Plan Apo VC 60x / 1.40 oil DIC N2 ∞/0.17 WD 0.13	Nikon Microscope Solutions		Oil Immersion Microscope Objective
Polystyrene Petri dish (100 mm)	Greiner Bio-One	664161
Polystyrene round-bottom tube with cell-strainer cap (5 mL)	Falcon	352235
Potassium chloride	Roth	6781.1
ProLong Glass Antifade Moutant	Invitrogen	P36980
Qualitative filter paper	VWR	516-0813
Rock Inhibitior	Merck	SCM075
Rotenone	Sigma	83-794
S100β	Abcam	Ab11178	1:600
SB-431542	Cayman Chemical Company	13031
Scalpel blades	Heinz Herenz Hamburq	1110918
SMI312	Biolegend	837904	1:500
Sodium bicarbonate	Merck/Sigma	31437-1kg-M
Sodium chloride	Roth	3957
Sodium dihydrogen phosphate	Applichem	131965
Sodium Pyruvate	Gibco	11360070
SOX2	Santa Cruz Biotechnology	Sc-17320	1:100
StemPro Accutase Cell Dissociation Reagent	Gibco/StemPro	A1110501	Reagent A
Super Glue Gel	UHU	63261	adhesive gel
SuperFrost Plus	VWR	631-0108
Syringe for single usage (1 mL)	BD Plastipak	300015
TB2 Thermoblock	Biometra
TC Plate 24 Well	Sarstedt	83.3922
TC Plate 6 Well	Sarstedt	83.392
TGFbeta3	Miltenyi Biotec	130-094-007
Tissue Culture Hood	ThermoFisher	51032711
TOM20	Santa Cruz Biotechnology	SC-11415	1:200
Triton-X	Merck	X100-5ML
UltraPure 0.5M EDTA	Invitrogen	15575020
Vibratome Microm HM 650 V	Thermo Scientific		Production terminated, any other adjustable microtome is fine, too.
Vibratome Wilkinson Classic Razor Blade	Wilkinson Sword	70517470
Whatman Benchkote	Merck/Sigma	28418852
Wnt Antagonist I	EMD Millipore Corp	3378738
XF 96 extracellular flux analyser	Seahorse Bioscience	100737-101
XF Assay DMEM Medium	Seahorse Bioscience	103680-100
XF Calibrant Solution	Seahorse Bioscience	100840-000
XFe96 FluxPak (96-well microplate)	Seahorse Bioscience	102416-100