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

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.

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.

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 agents^2,3,4,5,6,7. 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 development^5,9,10,11,12. 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 formation^12,13,14, 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 models^{11,17,18,19,20,21,22}, proven invaluable in modeling neurological disorders such as microcephaly¹¹, Alzheimer's disease^14,23, 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.

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.

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, employing SB2431542 as a key compound²⁹. The ensuing stage involves the cultivation of 3D neurospheres in a suspension culture. This is facilitated by an orbital shaker, enhancing nutrient and oxygen access³⁰, crucial for organoid development.

We meticulously monitored the morphological progression of these organoids using brightfield microscopy, as detailed in Figure 2. Our observations capture a differentiation from the initial iPSC colonies (Figure 2A) to the emergence of early organoid structures (Figure 2B), then progressing to mid-stage organoids with distinct boundaries (Figure 2C) and culminating in the formation of densely structured, mature organoids (Figure 2D,E).

A cornerstone of our study is the extensive use of immunostaining analyses to ascertain the regional identity of the organoids. We observed no expression of FOXG1, typically a marker specific to the forebrain³¹, indicating an absence of forebrain characteristics in the organoids' patterning or differentiation. Further immunostaining studies revealed the expression of FOXA2 (Figure 3C) as the marker indicative of ventral midbrain development³² and mature neuron markers MAP2 (Figure 3F), respectively. The presence of OTX2 (Figure 3B) also emerged, marking the midbrain-hindbrain boundary³³. At Day 90, the organoids demonstrated co-expression of DA neural marker TH and colocalization with nature neural marker MAP2 (Figure 4), signifying the successful generation of neuronal cell types pertinent to Parkinson's disease research³⁴.

Employing advanced single-cell RNA sequencing technology, we further dissected the cellular composition of 3-month-old MOs. A Uniform Manifold Approximation and Projection (UMAP) plot (Figure 5A) effectively categorized cells into distinct clusters based on gene expression, unveiling a rich diversity of cell types including dopaminergic neurons, oligodendrocytes, and radial glial cells. This highlights the organoids' remarkable cellular heterogeneity and differentiation capabilities. UMAP showed positive expression of the midbrain marker genes EN1 and LMX1A (Figure 5B,C), further suggesting a midbrain lineage in our differentiation protocol.

After dissociating the midbrain organoids and culturing them as a monolayer for one month, the neurons displayed co-expression of TH and Dopamine Transporter (DAT) (Figure 6). This co-expression indicates the successful differentiation of dopaminergic neuronal cell types from iPSC-derived MOs.

Figure 1: Schematic representation of the differentiation of the MOs from iPSCs. Phase I-Neural Induction (Day 0 to Day 5): iPSCs are cultured in a CDM with the addition of SB431542, NAC, and AMP-activated protein kinase inhibitor Compound C, promoting the formation of neural spheres. Phase II-MO Patterning (Day 5 to Day 19): Neural spheres are embedded in Matrigel droplets and further cultured in CDM supplemented with FGF-8b and patterning medium PM within an incubator, resulting in MO differentiation. Phase III-MO Termination (Day 19 to ≥3 months): The differentiated MOs are then terminated and further matured in CDM containing BDNF and GDNF on culture dishes, leading to final MO differentiation. Abbreviations: MO = midbrain organoid; iPSCs = induced pluripotent stem cells; CDM = chemically defined medium; NAC = N-acetyl cysteine; FGF-8b = Fibroblast Growth Factor 8b; PM = patterning medium; BDNF = Brain-derived Neurotrophic Factor, GDNF = Glial cell line-Derived Neurotrophic Factor. This figure was created using BioRender. Please click here to view a larger version of this figure.

Figure 2: Flow chart of the iPSC differentiation and representative images of the cells during the differentiation and characterization of the iPSC-derived MOs. (A) iPSC colonies: At the outset, iPSCs form tight, compact colonies, indicative of an undifferentiated state. (B) Early MO Formation: An early MO shows the beginnings of spherical organization, a critical step towards complex organoid development. (C) Mid-stage MO development: The organoid structure becomes more defined, with a smoother and more homogeneous appearance. (D) Advanced MO growth: This stage features a significantly larger and more opaque organoid, suggesting advanced cell differentiation and organoid maturation. (E) Mature MOs: The final stage displays a fully formed midbrain organoid with a dense and uniform structure. Scale bar = 100 µm (A-D), 50 µm (E). Abbreviations: MO = midbrain organoid; iPSCs = induced pluripotent stem cells. Please click here to view a larger version of this figure.

Figure 3: Immunofluorescence characterization of midbrain markers in 3-month-old midbrain organoids (MOs) derived from iPSCs. (A-C) Immunofluorescence staining of key neural markers, showcasing the diversity of cell types and the maturity of the neural structures within the organoids. The absence of FOXG1 staining indicates a lack of forebrain development. OTX2 staining reveals intense red fluorescence, indicating the presence of midbrain-hindbrain boundary-specific progenitor cells. FOXA2 staining is seen in red, highlighting the ventral midbrain regions and the development of dopaminergic neuron lineages within the organoid. MAP2 is displayed in cyan fluorescence, revealing the mature neuronal cytoarchitecture and indicating the presence of post-mitotic neurons. DAPI is used to stain the nuclei, shown in blue. Scale bars = 50 µm (10 µm for FOXA2). (D) Immunostaining specificity in samples, showing isotype controls alongside positive controls for TH and DAPI. DAPI confirms cell presence, while the isotype control (scale bar = 50 µm and 200 µm) demonstrates antibody specificity with no non-specific binding. Figure 3B,C were taken from Chen et al.³⁵. Abbreviations: MOs = midbrain organoids; iPSCs = induced pluripotent stem cells; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

Figure 4: Immunocytochemical analysis of midbrain neural markers in MOs derived from iPSCs and hESCs. (A) A series of immunofluorescence images illustrating the expression of the midbrain marker TH and the mature neuronal marker MAP2 in 3-month-old iPSC-derived MOs. Green fluorescence indicates PAX6, marking regions within the organoid that contain progenitor cells. Scale bar = 100 µm. (B) TH and MAP2 expression in 3-month-old hESC-derived MOs. Scale bar = 50 µm. Abbreviations: MO = midbrain organoid; iPSCs = induced pluripotent stem cells; TH = tyrosine hydroxylase; DAPI = 4',6-diamidino-2-phenylindole; hESCs = human pluripotent stem cells. Please click here to view a larger version of this figure.

Figure 5: Single-cell RNA sequencing analysis of a 3-month-old iPSC-derived MOs. This figure illustrates the cellular heterogeneity within a 3-month-old MO as revealed by single-cell RNA sequencing analysis. B. The UMAP visualization demonstrates the distinct clusters representing various cell types identified based on their gene expression profiles. C. The UMAP visualization demonstrates the expression profile of the EN1 and Lmx1A genes. C. The gene expression of EN1 and Lmx1 with mean and standard deviation (SD). Abbreviations: MOs = midbrain organoids; iPSCs = induced pluripotent stem cells; UMAP = the Uniform Manifold Approximation and Projection; DA = Dopaminergic neurons. Please click here to view a larger version of this figure.

Figure 6: Immunocytochemical characterization of midbrain neural markers in DA neurons. This figure displays a series of immunofluorescence images that highlight the expression of midbrain neural marker TH and DAT in 3-month-old MOs derived DA neurons from iPSCs. DAPI is used to stain the nuclei, shown in blue. Scale bar = 10 µm. Abbreviations: TH = tyrosine hydroxylase; iPSCs = induced pluripotent stem cells; DA neurons = dopaminergic neurons; DAT = Dopamine transporter, DAPI = 4',6-diamidino-2-pheynlindole. Please click here to view a larger version of this figure.

Table 1: Detailed recipes for various culture mediums and stock solutions essential for iPSC culture and differentiation. Please click here to download this Table.

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 cultures to three-dimensional neurospheres constitutes a substantial advancement in organoid technology, providing a more physiologically relevant context for investigating cellular dynamics and tissue architecture. The maintenance of these neurospheres in suspension with the aid of an orbital shaker was pivotal, ensuring homogenous growth and differentiation through improved nutrient and oxygen distribution.

Markers including OTX2, FOXA2, and PAX6 substantiate the authenticity of our midbrain patterning, confirming the establishment of an appropriate progenitor state. Subsequent detection of MAP2 in later developmental stages of the organoids underscores the successful neuronal maturation, a critical attribute for a genuine MO model. After a differentiation period of 90 days, the identification of cells co-expressing TH and MAP2 is especially significant, indicating the successful generation of DA neurons characterized by TH⁺/DAT⁺ expression within the organoids. These neurons are crucial for neural function and are particularly pertinent to the exploration of neurodegenerative diseases such as Parkinson's disease²⁴.

At the 3-month milestone, our single-cell RNA sequencing analysis confirms the differentiation of iPSCs into a spectrum of cell types characteristic of the midbrain. This cellular heterogeneity is crucial as it enables the organoids to function as versatile model systems for dissecting disease pathogenesis, assessing therapeutic interventions, and exploring cell-based treatments. When compared with seminal studies such as those by Jo et al.²¹, Kwak et al.²², and Mohamed et al.²⁰, our protocol demonstrates enhanced specificity and efficiency in the generation of midbrain cell types. Jo et al.²¹ initially demonstrated the generation of functional dopaminergic neurons within midbrain-like organoids. Subsequently, Kwak et al.²² refined this approach by engineering homogeneously structured MOs specifically tailored for modeling Parkinson's disease, including susceptibility to neurotoxins. Mohamed et al.²⁰ extended this work by incorporating genetic modifications to effectively model synucleinopathies. Our protocol offers a refined methodology that facilitates the differentiation of iPSCs into mature, functionally diverse midbrain organoids, positioning it as a significant advancement in the field.

Despite these advancements, there remains an imperative to enhance the homogeneity and maturation of organoids, as well as to develop methodologies for their scalable production. Future investigations will focus on incorporating assessments of neuronal functionality, specifically activity and connectivity, to deepen our understanding of midbrain dynamics and pathologies.

The protocol we employ for generating MOs from iPSCs marks a significant advance in modeling human neurological development and diseases. However, it is essential to acknowledge certain limitations. One notable limitation of our approach, similar to those reported in studies^20,21,22, is the potential variability in organoid size and structure, which may affect the consistency of experimental outcomes. While this variability challenge persists across the field, our study does not explicitly tackle the production of neuromelanin or the modeling of synucleinopathies, presenting potential areas for future research, particularly relevant to Parkinson's disease.

Variability between different iPSC lines in their differentiation propensities can impact the reproducibility of organoid generation, necessitating line-specific optimization of protocols. The extent of neuronal maturation within the organoids may not fully reflect the mature state of human midbrain tissues, limiting the model's applicability for studying late-onset neurodegenerative disorders.

Furthermore, inherent heterogeneity in organoid cultures can lead to variability in cell type composition and structural organization, posing challenges for standardized analysis. The absence of vascularization in organoids restricts nutrient and oxygen diffusion, potentially resulting in necrotic cores within larger organoids. The use of Matrigel droplets for structural support does not mimic the complex extracellular matrix environment of the human brain and may influence cellular behaviors. Current protocols do not systematically incorporate functional assessments of neural activity, such as electrophysiological measurements, which restricts the utility of organoids in modeling functional neural circuits and neurological disease phenotypes.

Scalability of MO production for high-throughput applications remains limited. The extensive culture period required for organoid maturation is resource-intensive, and sustaining long-term cultures presents significant challenges. Ethical considerations, particularly concerning the potential for sentience and the use of human genetic material, are paramount in research involving iPSCs and advanced organoid models.

While MOs serve as a powerful investigative tool for disease studies, they may not fully capture the complexity of diseases involving multiple cell types and systems beyond the midbrain. Addressing these limitations is critical for future studies aiming to utilize MOs as reliable models for disease mechanisms, drug discovery, and regenerative medicine. Continued advancements in organoid vascularization, maturation, and functional integration, along with the development of more standardized protocols for organoid generation and analysis, are expected to enhance the fidelity and utility of MOs as models of human neurological function and disease.

In summary, our research utilizes midbrain-specific organoids derived from human iPSCs. These organoids provide a valuable platform for exploring disease mechanisms and develop effective treatments for neurodegenerative disorders.

The authors have no conflicts of interest to disclose.

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.