Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

Summary

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

Abstract

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.

The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain's inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.

The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson's disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

Introduction

The human brain, with its intricate architecture and complex cellular and molecular composition, presents formidable challenges in neuroscience research, particularly in the context of disease modeling and cellular therapy development¹. These challenges are further complicated by the limited availability of sophisticated in vitro models that truly represent the complexity of the human brain. However, recent advancements in human induced pluripotent stem cell (iPSC) technology, enabling controlled differentiation into specific neuronal subtypes, have opened promising avenues for exploring human brain development, disease pathogenesis, and cell-b....

Protocol

The iPSCs generated from human normal fibroblasts Detroit 551 and human embryonic stem cells (ESCs) hESC line 360 as previously described⁴. The iPSCs were obtained with the approval of the Western Norway Committee for Ethical Health Research REK nr. 2012/919. All cells were regularly monitored for Mycoplasma contamination using MycoAlert Mycoplasma Detection Kit. The Table of Materials contains information about all materials, reagents, and equipment used in this protocol. Table 1 describes the media and other stock used.

1. Thawing of iPSCs

1. Matrix-coated....

Discussion

In this investigation, we have developed a methodology for the differentiation of MO from iPSCs. Our protocol employs a dual-SMAD inhibition strategy enhanced with morphogenic factors, including FGF-8b and the SHH agonist PM. This approach closely simulates the developmental cues crucial for midbrain ontogeny. The differentiation pathway we have instituted prompts the formation of neuroepithelial structures reminiscent of neural rosettes observed during natural brain development. This transformation from bidimensional cu.......

Materials

Name	Company	Catalog Number	Comments
CCD Microscope Camera Leica DFC3000 G	Leica Microsystems, Germany
Chemically Defined Lipid Concentrate	Thermo Fisher Scientific	11905031	CDM ingredient
Collagenase  IV	Thermo Fisher Scientific	17104019	Reagent for gentle dissociation of human iPSCs
Corning non-treated culture dishes	Sigma-Aldrich	CLS430589	Suspension culture
DMEM/F-12, GlutaMAX supplement	Thermo Fisher Scientific	10565018	Astrocyte differentiation basal Medium
DPBS	Thermo Fisher Scientific	14190250	Used for a variety of cell culture wash
EDTA	Thermo Fisher Scientific	15575020	Reagent for gentle dissociation of human iPSCs
Essential 8 Basal Medium	Thermo Fisher Scientific	A1516901	Basal medium for iPSC culture
Essential 8 Supplement (50x)	Thermo Fisher Scientific	A1517101	Supplement for iPSC culture
FCCP	Abcam	ab120081	Eliminates mitochondrial membrane potential and TMRE staining
FGF-basic	PeproTech	100-18B	Astrocyte differentiation medium ingredient
Fluid aspiration system BVC control	Vacuubrand, Germany
Formaldehyde (PFA) 16%	Thermo Fisher Scientific	28908	Cell fixation
GDNF	Peprotech	450-10	DA neurons medium ingredient
Geltrex (Basement membrane matrix)	Thermo Fisher Scientific	A1413302	Used for attachment and maintenance of human iPSCs
GlutaMAX Supplement	Thermo Fisher Scientific	35050061	Supplement for NSC culture
Heracell 150i CO2 Incubators	Fisher Scientific, USA
IMDM	Thermo Fisher Scientific	21980032	Basal medium for CDM
InSolution AMPK Inhibitor	Sigma-Aldrich	171261	Neural induction medium ingredient
Insulin	Roche	1376497	CDM ingredient
iPSCs derived from Detroit 551 fibroblats	ATCC	CCL-110
Leica TCS SP8 STED confocal microscope	Leica Microsystems, Germany
Matrigel	Life Science	354230	Matrigel embedding
Monothioglycerol	Sigma-Aldrich	M6145	CDM ingredient
Normal goat serum	Thermo Fisher Scientific	PCN5000	Used for blocking buffer
Orbital shakers - SSM1	Stuart Equipment, UK
Organoid Embedding Sheet	STEMCELL Technologies	8579	Matrigel embedding
Organoid Embedding Sheet	STEMCELL Technologies	8579
PBS 1x	Thermo Fisher Scientific	18912014	Used for a variety of washes
Poly-D-lysine hydrobromide	Sigma-Aldrich	P7405	Promotes attachment and growth of neural cells in vitro
Poly-L-ornithine solution	Sigma-Aldrich	P4957	Promotes attachment and growth of neural cells in vitro
ProLong Gold Antifade Mountant	Thermo Fisher Scientific	P36930	Mounting the coverslip for confocal image
Purmorphamine	STEMCELL Technologies	72204	Promotes DA neuron differentiation
Recombinant Human/Mouse FGF-8b Protein	R&D Systems	423-F8-025/CF	Promotes DA neuron differentiation
SB 431542	Tocris Bioscience	TB1614-GMP	Neural Induction Medium ingredient
TRITON X-100	VWR International	9002-93-1	Used for cells permeabilization in immunostaining assays
SSM1 compact orbital shaker	Norrscope	51901-10 SSM1 Shaker, orbital, mini 230V	Rotator for organoid culturing.
Water Bath Jb Academy Basic Jba5 JBA5 Grant Instruments	Grant Instruments, USA
Antibodies used for immunostaining
Primary antibody
anti-DAT	Abcam	ab128848, RRID:AB_2665470	Rabbit; 1:100
anti-FOXA2	ProteinTech	22474-1-AP, RRID:AB_2879110	Rabbit; 1:100
anti-FOXG1	Abcam	ab196868, RRID:AB_2892604	Rabbit; 1:200
anti-LMX1	Abcam	ab139726, RRID:AB_2827684	Rabbit; 1:100
anti-MAP2	Abcam	ab5392 ,RRID:AB_2138153	Chicken; 1:500
anti-OTX2	ProteinTech	13497-1-AP, RRID:AB_2157176	Rabbit; 1:100
anti-TH	Abcam	ab75875, RRID:AB_1310786	Rabbit; 1:100
Secondary antibody			Dilution (μL)
Alexa Fluor  594 goat anti-rabbit IgG	Thermo Fisher Scientific	A-11012	1:400
Alexa Fluor  488 goat anti-rabbit IgG	Thermo Fisher Scientific	A- 11008	1:400
Alexa Fluor  488 goat anti-rabbit IgG	Thermo Fisher Scientific	A- 11008	1:400
Alexa Fluor  488 goat anti-rabbit IgG	Thermo Fisher Scientific	A- 11008	1:400
Alexa Fluor  594 goat anti-rabbit IgG	Thermo Fisher Scientific	A-11012	1:400
Alexa Fluor  594 goat anti-rabbit IgG	Thermo Fisher Scientific	A-11012	1:400
Alexa Fluor  647 goat anti-chicken IgG	Thermo Fisher Scientific	A-21469	1:400