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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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-based therapeutic strategies.

Traditionally, two-dimensional (2D) neuronal cultures derived from iPSCs have been the cornerstone of in vitro studies aimed at mimicking human neuronal physiology and pathology. These monolayer cultures have been instrumental in enhancing our understanding of neurological diseases and in the discovery of neuroprotective agents2,3,4,5,6,7. Despite their utility, these 2D systems fall short in emulating the true cellular diversity and three-dimensional (3D) structure of the human brain.

The advent of brain organoid technology represents a significant leap in in vitro modeling, providing a tool that closely mimics the human brain's intricate biology within a physiologically relevant framework8. Early techniques in brain organoid development capitalized on the inherent regulatory properties of iPSCs to spontaneously form ectoderm derivatives, thereby mimicking the early stages of brain development5,9,10,11,12. Given the complex networks formed by neurons with other neuronal and non-neuronal cells, 3D modeling becomes essential for accurately studying neurodegenerative diseases. 3D cultures provide a more representative in vivo environment, facilitating accelerated neuronal differentiation and network formation12,13,14, and promote a broader expression of neuronal genes compared to 2D cultures15. Neurons developed in 3D contexts attain morphologies and physiological attributes more reminiscent of their in vivo counterparts16.

Recent advancements in 3D brain models have been pivotal in capturing the spatial and functional complexity of the human brain more effectively. Over the last decade, the advancement of diverse protocols for generating both whole-brain organoids and region-specific brain models11,17,18,19,20,21,22, proven invaluable in modeling neurological disorders such as microcephaly11, Alzheimer's disease14,23, and Parkinson's disease24. The goal of this protocol is to develop and refine a method for creating 3D human MOs derived from human iPSCs. This protocol is specifically aimed at generating organoids that are enriched with dopaminergic neurons and spatially organized in a manner that closely mimics the natural structure and functionality of the human midbrain. The primary purpose of these organoids is to serve as advanced in vitro models for studying Parkinson's disease, providing a more accurate and physiologically relevant system for exploring the neurodevelopmental processes and pathologies associated with this condition. By leveraging patient-derived iPSCs to generate these midbrain-specific organoids, the protocol seeks to enhance the understanding of Parkinson's disease mechanisms, facilitate the discovery of potential therapeutic targets, and improve the development of cell-based therapies. Through this innovative approach, the protocol aims to overcome the limitations of traditional 2D neuronal cultures and contribute significantly to the field of neurodegenerative disease research by offering a potent tool for in vitro disease modeling and the exploration of novel treatment strategies.

Over the past 10 years, various research teams have developed midbrain organoids (MOs)8,21,25,26,27,28, utilizing methodologies that exhibit several key similarities. In our pursuit to deepen the understanding of Parkinson's disease, we have developed a protocol for creating 3D human MOs derived from human iPSCs. These organoids, enriched with spatially arranged dopaminergic neurons, present an ideal model for studying Parkinson's disease. Our development and refinement of midbrain-specific organoid protocols have yielded advanced in vitro models that have significantly contributed to our understanding of neurodevelopmental processes and neurodegenerative diseases. These models, particularly when applied to patient-derived MOs, have demonstrated their efficacy as potent tools for in vitro disease modeling, offering new perspectives and methodologies in the field of advanced 3D cell culture.

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 plate and essential 8 (E8) medium preparation
    1. Thaw the basement membrane matrix vial on ice overnight. Dilute 1:100 in cold Advanced Dulbecco's Modified Eagle's Medium (DMEM)/Ham's F12 DMEM/F12 (1% final concentration) (see Table of Materials and Table 1). Store aliquots at -20 °C for long-term use.
    2. Thaw the diluted basement membrane at 4 °C; coat a 6-well plate with 1 mL of solution per well. Incubate at 37 °C for 1 h.
    3. Remove the plate from the incubator and allow it to equilibrate to room temperature (RT).
      NOTE: Plates can be refrigerated for a maximum of 2 weeks (ensure warming to RT before use). For prolonged storage, add 1 mL of E8 Medium (see Table of Materials and Table 1) to the coated plate to avoid the gel drying out.
  2. E8 medium preparation
    1. In a sterile environment, combine E8 basal medium with E8 supplement (see Table of Materials and Table 1).
      NOTE: This can be stored at 4 °C for 2 weeks. Thaw the frozen E8 supplement overnight at 4 °C prior to preparing the full medium. Do not thaw at 37 °C, as this may compromise its stability. Before use, warm only the required volume of E8 Medium to room temperature (RT) until it is no longer cool to the touch.

2. Seeding iPSCs

  1. Prewarm the matrix-coated plates and E8 Medium at RT or incubator at 37 °C.
  2. Aspirate culture medium; rinse iPSCs, which are at 60%-70% confluency, with Dulbecco's Phosphate-Buffered Saline without calcium and magnesium (DPBS Ca2+/Mg2+-free,4 mL per well in a 6-well plate).
  3. Add ethylenediaminetetraacetic acid (EDTA, 0.5 mM, 1 mL per well, see Table of Materials and Table 1); incubate at 37 °C until the colony edges detach (3-5 min).
  4. Aspirate the dissociation solution; to detach colonies, carefully pipette the prewarmed E8 Medium (4 mL per well) directly onto the colonies with a stronger-than-usual force from the pipette, which generates a localized fluid motion over the cell surface.
  5. Split the cells at a 1:2 ratio by transferring them into two new wells of a matrix-coated 6-well plate, adding 2 mL of medium per well. Then, incubate at 37 °C.
  6. Exchange the media daily until the colonies reach 80% confluence.
  7. Using a microscope at 10x magnification, observe the colonies to ensure they are of an appropriate size, typically characterized by a distinct, round shape with a diameter ranging from 0.5 to 1 mm. Confirm that colonies are dense, compact structures featuring delineated, smooth borders.

3. Neural induction

  1. Medium preparation and Day 0 setup
    1. Prepare 500 mL each of Chemically Defined Medium (CDM), Neural Induction Medium (NIM), and Neural Stem Cell Serum-free (NSC SF) medium, referring to the Table of Materials and Table 1.
    2. Rinse the cells in a 6-well plate with 4 mL of DPBS (1x) (Ca2+/Mg2+- free) per well, followed by the addition of 3 mL of NIM per well for Day 0 setup.
    3. Replace NIM (3 mL per well) on Days 1, 3, and 5; observe daily under a microscope.
    4. On Day 6, detach the neural rosettes into a suspension culture using the following steps.
      1. Gently wash once with DPBS (1x) (Ca2+/Mg2+-free) (4 mL per well in a 6-well plate). Add collagenase IV (1 mL per well in a 6-well plate) and incubate for 1 min. Remove the collagenase IV, and delicately rinse once with DPBS (1x) (Ca2+/Mg2+-free) (4 mL per well in a 6-well plate).
      2. Add 2 mL of NSC SF Medium to each well of a 6-well plate. Detach the cells by scraping the well bottoms, creating grids with a 200 µL pipette tip.
      3. Transfer the cell suspension from the 6-well plate into a 10 cm non-adherent dish. Adjust the volume to 12 mL with NSC SF Medium.
      4. Place the non-adherent dish on shaker (ssm1 compact orbital shaker) at 85 rpm at 0° angle in an incubator set to 37 °C with 5% CO2 to prevent aggregation.

4. Patterning of midbrain

  1. On Day 7, add 12 mL of CDM with 100 ng/mL FGF-8b; place on orbital shaker incubator.
  2. On Days 8-13, change the medium every 2 days; observe under a microscope daily.
  3. On Day 14, add 12 mL of CDM with 100 ng/mL FGF-8b and 1 µM purmorphamine (PM); place on orbital shaker.
  4. On Days 15-20, change the medium every 2 days; observe under a microscope daily.

5. Matrigel embedding and MO termination and maturation

  1. Thaw Matrigel on ice at a temperature between 2 °C and 8 °C for a duration of 1-2 h.
    NOTE: Thaw an adequate amount of Matrigel to supply 15 µL for each embryoid body (EB). Keep Matrigel chilled on ice to avoid early polymerization. Cool all plasticware that will be in contact with Matrigel at -20 °C for a minimum of 30 min before utilization.
  2. Place a sterile organoid embedding sheet into an empty dish.
  3. Using a wide-bore 200 µL pipette tip, gently take out the organoid from the dish and transfer it to the embedding surface with one organoid per well. Repeat this process until 12-16 EBs are collected on the embedding surface.
  4. Carefully remove the excess medium from each EB and then add 15 µL of cold Matrigel dropwise onto each EB. With the help of a pipette, reposition the EB to the center of the Matrigel droplet.
  5. Incubate the plate in an incubator set at 37 °C for 15-20 min to allow the Matrigel to polymerize.
  6. Using sterile forceps, lift the embedding sheet with the Matrigel droplets. Hold the sheet directly above one well in a 6-well ultra-low adherent plate. Pipette up 3 mL of CDM with 10 ng/mL Brain-Derived Neurotrophic Factor (BDNF) and 10 ng/mL Glial cell line-Derived Neurotrophic Factor (GDNF) and gently rinse the Matrigel droplets from the sheet into each well. Repeat this process until all 6-8 Matrigel droplets are transferred into one well.
  7. Place the plates on an orbital shaker in a 37 °C incubator at 85 rpm and change the CDM with 10 ng/mL BDNF and 10 ng/mL GDNF every 3 days.
    NOTE: Maintain differentiating cultures for up to 3 months; neural morphology appears after 2 weeks post termination. BDNF and GDNF are not necessary for culturing beyond 3 months.

6. Immunofluorescence staining

  1. Use a 1,000 µL pipette to gently retrieve the organoid from the medium and place it gently on a microscope slide with minimal medium.
  2. Allow it to air-dry completely at RT. Add 4% paraformaldehyde (PFA) (300 µL per sample) for 30 min.
    NOTE: PFA is hazardous and may have carcinogenic properties. Handle it with precaution, refrain from direct contact with skin and eyes, and ensure usage within a chemical fume hood.
  3. Add 30% sucrose solution (300 µL per sample) to the slides and let them incubate overnight at 4 °C.
  4. Block and permeate the organoids by adding blocking buffer (BB) (300 µL per sample), which includes PBS (with Ca2+/Mg2+), 0.3% Triton X-100, and 10% normal goat serum, for 2 h at RT (or overnight at 4 °C). Use a hydrophobic barrier pen to delineate the organoids and confine the buffer on the slide.
  5. Cover the samples with an appropriate volume of primary antibody in blocking buffer (300 µL per sample), anti-forkhead box protein G1 (FOXG1, 1:200), anti-orthodenticle homeobox 2 (OTX2, 1:100), anti-Forkhead Box A2 (FOXA2, 1:100), anti-Tyrosine Hydroxylase (TH, 1:100), anti-SRY (sex determining region Y)-box 2 (SOX2, 1:100), anti-paired box protein (PAX6, 1:100), and anti-microtubule associated protein 2 (MAP2 1:500), and incubate overnight at 4 °C in the dark (see Table of Materials).
  6. Wash the samples in 1x PBS for 3 h with two to three buffer changes on a gently rocking platform.
  7. Incubate with the secondary antibody (300 µL per sample), anti-Alexa Fluor 488 (1:800), anti-Alexa Fluor 594 (1:800), and anti-Alexa Fluor 647 (1:800) overnight at 4 °C in a humidified darkroom. Add Hoechst 33342 (1:5,000) nuclear stain simultaneously (see Table of Materials).
  8. Remove the antibody, rinse quickly with PBS, and subsequently add PBS containing 0.01% sodium azide for 1-2 days at 4 °C to inhibit contamination.
  9. Aspirate the PBS, remove any excess solution, and mount the organoids using a mounting medium (refer to the Table of Materials). Place a coverslip, ensuring to prevent air bubble formation, and store in a dark room at RT for at least 12 h to allow the mounting medium to fully polymerize.
    NOTE: The specimen is now ready for observation using a fluorescence microscope.

Results

In this study, we introduce a pioneering protocol for the derivation of MOs from iPSCs. Central to our methodology is the innovative use of dual-SMAD inhibition, synergistically combined with FGF-8b and sonic hedgehog (SHH) pathway agonist purmorphamine (PM). This approach is depicted in Figure 1. The differentiation process begins by steering iPSCs towards neuroectoderm lineage, forming neuroepithelial or neural rosette colonies. This is achieved through dual SMAD signaling inhibition, empl...

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...

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

Figure 1 is created using BioRender.com. We thank University of Bergen Meltzers Høyskolefonds (project number: 103517133), Gerda Meyer Nyquist Guldbrandson, and Gerdt Meyer Nyquists legat (project number: 103816102) for funding.

Materials

NameCompanyCatalog NumberComments
CCD Microscope Camera Leica DFC3000 GLeica Microsystems, Germany
Chemically Defined Lipid ConcentrateThermo Fisher Scientific11905031CDM ingredient
Collagenase  IVThermo Fisher Scientific17104019Reagent for gentle dissociation of human iPSCs
Corning non-treated culture dishesSigma-AldrichCLS430589Suspension culture 
DMEM/F-12, GlutaMAX supplementThermo Fisher Scientific10565018Astrocyte differentiation basal Medium
DPBSThermo Fisher Scientific14190250Used for a variety of cell culture wash
EDTAThermo Fisher Scientific15575020Reagent for gentle dissociation of human iPSCs
Essential 8 Basal MediumThermo Fisher ScientificA1516901Basal medium for iPSC culture
Essential 8 Supplement (50x)Thermo Fisher ScientificA1517101Supplement for iPSC culture
FCCPAbcamab120081Eliminates mitochondrial membrane potential and TMRE staining
FGF-basicPeproTech100-18BAstrocyte differentiation medium ingredient
Fluid aspiration system BVC controlVacuubrand, Germany
Formaldehyde (PFA) 16%Thermo Fisher Scientific28908Cell fixation 
GDNFPeprotech450-10DA neurons medium ingredient
Geltrex (Basement membrane matrix)Thermo Fisher ScientificA1413302Used for attachment and maintenance of human iPSCs
GlutaMAX SupplementThermo Fisher Scientific35050061Supplement for NSC culture
Heracell 150i CO2 IncubatorsFisher Scientific, USA
IMDMThermo Fisher Scientific21980032Basal medium for CDM
InSolution AMPK InhibitorSigma-Aldrich171261Neural induction medium ingredient
InsulinRoche1376497CDM ingredient
iPSCs derived from Detroit 551 fibroblats ATCCCCL-110
Leica TCS SP8 STED confocal microscopeLeica Microsystems, Germany
MatrigelLife Science354230Matrigel embedding
MonothioglycerolSigma-AldrichM6145CDM ingredient
Normal goat serumThermo Fisher ScientificPCN5000Used for blocking buffer
Orbital shakers - SSM1Stuart Equipment, UK
Organoid Embedding SheetSTEMCELL Technologies8579Matrigel embedding
Organoid Embedding SheetSTEMCELL Technologies 8579
PBS 1xThermo Fisher Scientific18912014Used for a variety of washes
Poly-D-lysine hydrobromideSigma-AldrichP7405Promotes attachment and growth of neural cells in vitro
Poly-L-ornithine solutionSigma-AldrichP4957Promotes attachment and growth of neural cells in vitro
ProLong Gold Antifade MountantThermo Fisher ScientificP36930Mounting the coverslip for confocal image
PurmorphamineSTEMCELL Technologies72204Promotes DA neuron differentiation
Recombinant Human/Mouse FGF-8b Protein R&D Systems423-F8-025/CFPromotes DA neuron differentiation 
SB 431542Tocris BioscienceTB1614-GMPNeural Induction Medium ingredient
TRITON X-100VWR International9002-93-1Used for cells permeabilization in immunostaining assays
SSM1 compact orbital shaker Norrscope51901-10 SSM1 Shaker, orbital, mini 230VRotator for organoid culturing.
Water Bath Jb Academy Basic Jba5 JBA5 Grant InstrumentsGrant Instruments, USA
Antibodies used for immunostaining
Primary antibody
anti-DATAbcamab128848, RRID:AB_2665470Rabbit; 1:100 
anti-FOXA2ProteinTech22474-1-AP, RRID:AB_2879110Rabbit; 1:100 
anti-FOXG1Abcamab196868, RRID:AB_2892604Rabbit; 1:200
anti-LMX1Abcamab139726, RRID:AB_2827684Rabbit; 1:100 
anti-MAP2Abcamab5392 ,RRID:AB_2138153Chicken; 1:500
anti-OTX2ProteinTech13497-1-AP, RRID:AB_2157176Rabbit; 1:100 
anti-THAbcamab75875, RRID:AB_1310786Rabbit; 1:100 
Secondary antibodyDilution (μL)
 Alexa Fluor  594 goat anti-rabbit IgGThermo Fisher ScientificA-11012 1:400
Alexa Fluor  488 goat anti-rabbit IgGThermo Fisher ScientificA- 11008 1:400
Alexa Fluor  488 goat anti-rabbit IgGThermo Fisher ScientificA- 11008 1:400
Alexa Fluor  488 goat anti-rabbit IgGThermo Fisher ScientificA- 11008 1:400
Alexa Fluor  594 goat anti-rabbit IgG Thermo Fisher ScientificA-11012 1:400
Alexa Fluor  594 goat anti-rabbit IgG Thermo Fisher ScientificA-11012 1:400
Alexa Fluor  647 goat anti-chicken IgG Thermo Fisher ScientificA-21469 1:400

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