Development and Characterization of Human iPSC-Derived Midbrain Organoids for Neurological Research

Introduction

Studying the human brain, particularly in the context of neurodevelopment and disease, presents a significant challenge for researchers. Traditional methods like animal models and post-mortem brain tissue analysis have provided useful insights but have clear limitations. While animal models are valuable, they don’t fully replicate the human brain’s unique features, especially for conditions like schizophrenia and autism. Post-mortem analysis provides only a snapshot of the brain at the time of death, and obtaining human brain tissue for research is not as straightforward as accessing other types of biological samples. To address these challenges, researchers turned to in vitro models like 2D cell cultures to study human cells. While these models have been crucial for many breakthroughs, they don’t fully replicate the complex 3D structure and neural circuits that are vital to understanding brain function.

In recent years, the neuroscience field has made major advances with the development of brain organoids. These 3D cultures, derived from human pluripotent stem cells (hPSCs), offer a new way to model human brain development in vitro. Unlike earlier models, brain organoids replicate the complex structure and function of specific brain regions, providing a more accurate platform for studying neurological disorders and development. This technology addresses many of the limitations of previous models, allowing researchers to observe dynamic processes in a human-specific context.

Madeline Lancaster and Juergen Knoblich pioneered the development of self-organizing cerebral organoids, laying the groundwork for this breakthrough. In 2013, they introduced protocols for creating organoids that form spontaneously from hPSCs without external patterning cues. These cerebral organoids, or "mini-brains," mimic the early developmental stages of the human brain, especially the cerebral cortex, which is responsible for higher cognitive functions. Their work showed that stem cells could be guided to form 3D structures resembling human brain tissue, providing a valuable tool for studying brain development and disease.

Lancaster and Knoblich's pioneering work was a significant breakthrough in brain modeling. Their development of self-organizing organoids allowed for the formation of multiple brain regions within a single culture, providing a comprehensive model of early brain development. This work laid the foundation for subsequent advancements, as researchers have since refined techniques to create organoids that represent specific brain regions, further enhancing the ability to study particular functions and diseases in more detail.

The Development of Brain Region-Specific Organoids

Brain region-specific organoids have greatly improved how we model human brain development and disease. By focusing on individual brain regions, researchers can now explore the unique features and vulnerabilities of each area, gaining insights into diseases that affect specific parts of the brain. For example, dorsal forebrain organoids mimic the cerebral cortex—the brain's outer layer responsible for higher cognitive functions like reasoning, problem-solving, and sensory perception. These models are particularly useful for studying neurodevelopmental disorders like autism and schizophrenia, where cortical dysfunction plays a key role.

Midbrain organoids, on the other hand, help researchers study motor control and sensory processing. The midbrain is critical for functions like eye movement and auditory and visual processing. These organoids are especially relevant for neurodegenerative diseases like Parkinson's, where the loss of midbrain dopaminergic neurons leads to motor control problems.

Ventral forebrain organoids, which model the hypothalamus and basal ganglia, are used to study emotional regulation, motivation, and autonomic functions like hunger and body temperature. These models are valuable for understanding affective disorders and conditions linked to basal ganglia dysfunction, such as Huntington's disease.

Hindbrain organoids provide insights into regions like the cerebellum, pons, and medulla oblongata, which are involved in coordinating movement, balance, and essential autonomic functions such as heart rate and respiration. These models help study disorders that affect motor coordination and the autonomic nervous system, areas that are difficult to replicate in traditional models.

Thalamic organoids offer a window into the thalamus, which directs sensory information to the right areas of the cerebral cortex, while hippocampal organoids are key for studying memory formation and spatial navigation, making them essential for research into conditions like Alzheimer's disease. The ability to model each brain region separately allows researchers to delve deeper into the functions and diseases associated with these areas.

The Integration of Brain Regions with Assembloids

While brain region-specific organoids are excellent tools for studying individual brain areas, the brain's true complexity lies in the interactions between these regions. To address this, researchers like Sergiu Pa?ca have developed assembloids, which combine multiple region-specific organoids (2018). These assembloids allow scientists to study how different parts of the brain communicate, providing a more complete model of brain function.

For example, pairing a cortical organoid with a thalamic organoid helps researchers understand sensory information processing and how disruptions in this process might lead to conditions like schizophrenia. Similarly, combining a cortical organoid with a midbrain or spinal cord organoid allows the study of motor functions and their deterioration in diseases like Parkinson’s.

Assembloids represent an important advancement, offering a more connected and accurate model of the brain. This approach is key for understanding how different regions work together to support normal function, and how their miscommunication can result in neurological and psychiatric disorders. By merging the precision of region-specific organoids with the complexity of assembloids, researchers are uncovering new insights in neuroscience and opening doors to more targeted treatments for brain-related diseases.

Summary

Brain region-specific organoids and assembloids have revolutionized neuroscience research by providing detailed insights into the complexities of the human brain. These models allow researchers to examine specific brain regions individually, offering a clearer understanding of each area's unique roles and vulnerabilities. The shift from self-organizing cerebral organoids to more precisely directed, region-specific models has enabled scientists to study diseases in a way that more accurately reflects human physiology, leading to more relevant discoveries.

Assembloids take this research further by allowing scientists to study how different brain regions interact, offering a more comprehensive view of brain function and dysfunction. Together, these technologies are advancing our understanding of brain development and disease, while also paving the way for more effective, targeted therapies. As the field progresses, brain organoids and assembloids will continue to play a key role in driving breakthroughs in both basic research and clinical applications.

Human iPSC-Derived Midbrain Organoids from STEMCELL Technologies

STEMCELL Technologies has developed Human iPSC-Derived Midbrain Organoids (Catalog no. 200-0790, 200-0791, 200-0792, and 200-0793) for academic and commercial research purposes. These organoids were generated from the Healthy Control Human iPSC Line, Female, SCTi003-A (Catalog #200-0511), using the STEMdiff? Midbrain Organoid Differentiation Kit (Catalog #100-1096). The organoids recapitulate key features of midbrain development, including the presence of dopaminergic neurons, which are crucial for motor control and are prominently involved in diseases such as Parkinson’s. The organoids also display robust expression of midbrain markers FOXA2 and LMX1A, and dopaminergic markers TH and NURR1.

The Product Information Sheet (PIS) for Human iPSC-Derived Midbrain Organoids can be found here.

Advantages:

• Start your experiments faster with ready-to-use midbrain organoids.
• Obtain high-quality organoids derived from the highly characterized iPSC control line, SCTi003-A.
• Achieve physiologically relevant results with validated neuronal activity.
• Maintain organoids with STEMdiff? Neural Organoid Maintenance Kit.

Midbrain Organoid Generation and Maturation Process

The generation and maturation of Human iPSC-Derived Midbrain Organoids follow a structured and reproducible process designed to ensure high quality and consistency. SCTi003-A iPSCs (Catalog #200-0511), maintained in mTeSR? Plus (Catalog #100-0276), are dissociated and seeded in AggreWell?800 plates (Catalog #34860) at a density of 3 x 10^6 cells per well. This initial step is critical for the formation of uniform embryoid bodies, which serve as the foundation for organoid development.

Over the first six days, the cultures are fed daily with a formation medium, promoting the early stages of organoid growth. Following this, the organoids are transferred to a suspension culture environment, where they continue to grow and undergo patterning specific to midbrain development.

The organoids can be harvested at two key developmental stages, depending on the experimental requirements:

• Differentiated Organoids (Day 42-49): At this stage, the organoids have undergone significant development and are suitable for studies requiring midbrain-specific neuronal populations.
• Mature Organoids (Day 90-97): These organoids have reached a more advanced stage of development, exhibiting complex cellular structures and functions. They are ideal for research focused on mature midbrain characteristics and long-term studies.

Long-term maintenance and further maturation of the midbrain organoids can be achieved using the STEMdiff? Neural Organoid Maintenance Kit (Catalog #100-0120). This detailed and controlled process ensures the production of highly reproducible midbrain organoids that are ready for immediate use in a variety of research applications. Figure 1 provides a schematic overview of the stages involved in midbrain organoid generation and maturation, highlighting the shipping windows for both differentiated and mature organoids.

Figure 1. Schematic for Human iPSC-Derived Midbrain Organoid Development

SCTi003-A iPSCs maintained in mTeSR? Plus are dissociated and seeded at a density of 3 x 10^6 cells/well in seeding medium in AggreWell?800 plates. Thereafter, cultures are fed daily with formation medium. After 6 days, the organoids are transferred and cultured in suspension, allowing growth and subsequent patterning to the midbrain. Long-term maintenance and further maturation of midbrain organoids can be achieved using the STEMdiff? Neural Organoid Maintenance Kit.

Midbrain Organoid Morphology

Midbrain organoids derived from SCTi003-A iPSCs (Catalog #200-0511) exhibit consistent and high-quality morphological characteristics throughout their development. These organoids are generated using the STEMdiff? Midbrain Organoid Differentiation Kit (Catalog #100-1096) and display a uniform size and spherical morphology across different stages of maturation. At Day 25, the organoids are relatively small, reflecting early stages of differentiation. By Day 43, the organoids increase in size, maintaining a well-defined spherical shape, indicative of robust growth and homogeneity. By Day 120, the organoids reach their mature form, showcasing a more compact and dense structure, crucial for studies involving midbrain development and related pathologies. This consistent morphology underlines the reproducibility and quality of the midbrain organoids, making them an ideal model for investigating midbrain-specific functions and diseases. Figure 2 demonstrates the high-quality and homogeneous morphology of iPSC-derived midbrain organoids at different stages of in vitro development, emphasizing their suitability for various experimental applications.

Figure 2. Human iPSC-Derived Midbrain Organoids Exhibit High-Quality Homogeneous Morphology

Representative phase contrast images of midbrain organoids derived from SCTi003-A iPSCs using STEMdiff? Midbrain Organoid Differentiation Kit show uniform size and spherical morphology at (A) Day 25, (B) Day 43, and (C) Day 120 in vitro.

Brain Region-Specific Patterning in Midbrain Organoids

Human iPSC-Derived Midbrain Organoids exhibit precise brain region-specific patterning, as demonstrated by the expression of key midbrain markers at various stages of development. This patterning is essential for modeling the unique cellular environment of the midbrain and for studying diseases specific to this region.

Figure 3 shows the fold change in expression for several key markers:

• FOXA2: A marker for midbrain floorplate precursors, showing elevated expression at both Day 60 and Day 100, indicating the organoids' commitment to a midbrain identity.
• TH: A marker for mature dopaminergic neurons, essential for dopamine synthesis, which is significantly expressed at both time points, highlighting the differentiation towards a dopaminergic lineage.
• PITX3: Another important dopaminergic marker, crucial for the development and maintenance of dopaminergic neurons, also shows increased expression, reinforcing the midbrain-specific differentiation.
• MAP2: A general neuronal marker that is elevated, indicating the presence of mature neurons within the organoids.
• GFAP: A marker for glial cells, specifically astrocytes, which shows increased expression at Day 100, reflecting the emergence of supporting glial populations as the organoids mature.

These gene expression patterns confirm the successful differentiation of the organoids into midbrain-specific neural populations, making them highly relevant for modeling neurodegenerative diseases such as Parkinson’s disease, which primarily affects dopaminergic neurons in the midbrain.

In Figure 4, the spatial organization and expression of these markers are visually depicted in the organoids at Day 60 and Day 120:

• FOXA2: Continues to mark the midbrain region's progenitor cells.
• TH: Indicates the increasing presence and maturation of dopaminergic neurons, particularly evident at Day 120.
• MAP2: Shows the distribution of mature neurons throughout the organoid.

These images demonstrate the structural organization and regional specificity of the organoids, with more defined and mature dopaminergic neurons evident by Day 120. This level of organization within the organoids underscores their utility as a robust model for studying midbrain-related functions and disorders.

This comprehensive analysis of marker expression and organization within the midbrain organoids reinforces their potential as a powerful tool for advancing our understanding of midbrain development and associated pathologies. The ability to observe and manipulate specific brain regions in vitro provides researchers with unprecedented opportunities to investigate the complexities of human brain development and disease.

Figure 3. Human iPSC-Derived Midbrain Organoids Express Key Markers of Brain-Region-Specific Patterning

Single organoids differentiated from SCTi003-A iPSCs were harvested for RNA at Day 60 and Day 100. Fold change is reported relative to parent hPSC and the TBP housekeeping gene control. (A) Midbrain floorplate precursor marker FOXA2 was elevated in midbrain organoids at Day 60 and Day 100. More mature marginal zone dopaminergic markers, (B) TH, (C) PITX3, and general neuronal marker (D) MAP2 were elevated at Day 60 and Day 100. (E) Glial marker GFAP expression is elevated in the organoids at Day 100. Colours represent separate differentiations.

Figure 4. Human iPSC-Derived Midbrain Organoids Exhibit Brain-Region-Specific Marker Expression and Organization

Representative immunofluorescence images of Human iPSC-Derived Midbrain Organoids at (A) Day 60 and (B) Day 120 show expression of key markers involved in midbrain development. DAPI staining highlights nuclei, while FOXA2 marks floorplate progenitors, and TH indicates dopaminergic neurons. MAP2, a general neuronal marker, demonstrates the differentiation of neurons over time. Organoids at Day 120 show more organized and pronounced expression of these markers, particularly an increase in TH and MAP2, reflecting maturation and dopaminergic neuron development.

Neuronal Activity in Midbrain Organoids Demonstrated Using MEA Assay

To evaluate the functional activity of pre-made iPSC-derived midbrain organoids, an MEA (multielectrode array) assay was performed in collaboration with Axion BioSystems. Human iPSC-Derived Midbrain Organoids (Day 125) were cultured on an MEA plate (CytoView MEA? 6, Axion Biosystems) to record neuronal activity in response to different treatments. The organoids were shipped live from Canventa Life Sciences (Emeryville, CA, U.S.) to Axion Biosystems (Atlanta, GA, U.S.), via next-day shipping, ensuring the data was directly obtained from a live-shipped batch of organoids, demonstrating their viability post-shipment. The organoids were maintained in STEMdiff? Neural Organoid Maintenance Medium (Catalog #100-0120), and their neuronal activity was measured after one hour of treatment with 4-Aminopyridine (4-AP), Rotenone, or DMSO as a control using the Maestro Pro? MEA and impedance system (Axion Biosystems).

4-AP was used to induce increased neuronal excitability by blocking potassium channels, thereby enhancing neurotransmitter release and overall activity. This compound is often used to model hyperexcitability conditions such as epilepsy. Rotenone, a mitochondrial complex I inhibitor, was selected for its known neurotoxic effects, commonly used to model neurodegenerative diseases such as Parkinson’s. DMSO was used as a control to assess baseline neuronal activity without inducing any specific excitatory or toxic effects.

Figure 5 highlights the results of this assay. Panel (A) shows a brightfield image of a Day 125 midbrain organoid on the MEA plate. Panels (B-D) display raster plots of spike activity, where detected spikes (black lines) and network bursts (orange boxes) indicate neuronal firing patterns under different conditions. Organoids treated with 4-AP (100 μM) demonstrated increased network bursting, signifying enhanced excitability, while Rotenone (100 nM) treatment reduced network bursting, indicating neurotoxicity.

The accompanying graphs (E-G) compare the mean firing rate, network burst frequency, and overall viability across treatments. Results show that 4-AP increases neuronal excitability, while Rotenone reduces both network activity and viability, correlating with its neurotoxic effects. These findings validate the functional activity of midbrain organoids and demonstrate their utility for modeling neurotoxicity and excitability in vitro.

This data further emphasizes the robustness of the midbrain organoids during live-shipment and their potential application in high-throughput screening platforms for neuroactive compounds and toxicology studies, making them a valuable tool for drug discovery and neurodevelopmental research.

Figure 5. Human iPSC-Derived Midbrain Organoids (Day 125) Demonstrate Treatment-Dependent Neuronal Activity Measured with the MEA Assay

Human iPSC-Derived Midbrain Organoids were plated on an MEA plate (CytoView MEA? 6, Axion BioSystems) and maintained with STEMdiff? Neural Organoid Maintenance Medium. Activities from 64 electrodes were recorded from organoids at Day 125, after one hour of treatment with 4-AP, Rotenone, or DMSO as a control, using The Maestro Pro? MEA and impedance system (Axion BioSystems). (A) Representative brightfield image of a Human iPSC-Derived Midbrain Organoid on the MEA plate. (B-D) Detected spikes (black lines), single channel bursts (blue lines; a collection of at least 5 spikes, each separated by an ISI of no more than 100 ms), and network bursts (orange boxes; a collection of at least 50 spikes from a minimum of 35% of participating electrodes, each separated by an ISI of no more than 100 ms) were recorded for each treatment. Raster plots of spike activity show the Day 125 organoids exhibit increased network bursting upon 4-AP treatment (100 μM) and a decrease in network bursting with rotenone treatment (100 nM). Graphs comparing (E) mean firing rate, (F) network burst frequency, and (G) viability are shown. ISI = inter-spike interval.

Live Shipment and Immediate Handling of Midbrain Organoids

Human iPSC-Derived Midbrain Organoids are shipped live at ambient temperature to ensure they retain their structural integrity and cellular composition during transit. Each shipment arrives in a CoolGuard? Advance single-use temperature-controlled shipper (Peli BioThermal), which combines phase change materials with vacuum insulation panels to reduce temperature excursions and increase compliance. All shippers are validated to the ISTA 7D test standard to ensure high performance. The shipper’s six insulation panels maintain a stable environment throughout the journey, ensuring optimal conditions for the organoids, which are securely packaged within a 96-well plate. The plate is double-bagged, with the lid secured by rubber bands and sealed with a breathable membrane to prevent contamination. CoolGuard? Advance shippers provide up to 120 hours of protection to enable safe transport over extended durations.

Upon receipt, it is crucial that the shipment is handled with care, avoiding unnecessary tilting or shaking of the package to protect the integrity of the organoids. Once unpacked, the plate should be centrifuged at 100 x g for 3 minutes to ensure that any organoids stuck to the sealing membrane during transport return to their respective wells. Centrifugation helps prevent damage and ensures that all organoids are properly settled before further handling.

Human iPSC-Derived Midbrain Organoids are available in both differentiated and mature stages, offering flexibility for various research applications. Upon receipt, the organoids should be immediately transferred to a sterile maintenance culture prepared with STEMdiff? Neural Organoid Maintenance Medium (Catalog #100-0120). The organoids should be transferred using wide-bore pipette tips to prevent mechanical damage. For differentiated organoids, standard 1000 μL pipette tips should be cut approximately 1.5 cm from the end to create a 3 mm bore. For mature organoids, the tip should be cut 2 cm from the end, creating a bore of 4-5 mm, allowing for gentle transfer.

The organoids are suitable for downstream applications after a two-week recovery period in maintenance culture, during which full-medium changes should be performed every 2-3 days. Long-term culture can be maintained using STEMdiff? Neural Organoid Maintenance Medium.

During shipment, the organoids are kept at ambient temperature and are not suitable for freezing upon receipt. These live cultures must be processed immediately to ensure viability and optimal functionality. Proper handling is crucial to preserve the cellular and structural integrity of the organoids, and detailed instructions for unpacking, centrifugation, and transfer are provided with each shipment.

For more detailed instructions on how to handle and culture your midbrain organoids, you can view our instructional video, "How to Unpack and Process the Shipment of Human iPSC-Derived Midbrain Organoids."

Figure 6. Example of 1000 μL Pipette Tips Required to Transfer Organoids

(A) Standard 1000 μL pipette tips (bore size of < 1 mm) and (B) commercially-available wide-bore 1000 μL pipette tips (bore size of ~1.5 mm) are not suitable for handling Differentiated or Mature Human iPSC-Derived Midbrain Organoids. (C) Standard 1000 μL pipette tips cut 1.5 cm from the end (bore size of ~3 mm) are suitable for handling Differentiated Organoids. (D) Standard 1000 μL pipette tips cut 2 cm from the end (bore size of ~4 - 5 mm) are suitable for handling Mature Organoids.

Summary

STEMCELL Technologies has developed Human iPSC-Derived Midbrain Organoids (Catalog #200-0790, #200-0791, #200-0792, and #200-0793) designed for academic and commercial research, optimized for studies on midbrain development and neurodegenerative diseases such as Parkinson’s.

• High-Quality Midbrain Organoids: Derived from the well-characterized iPSC control line, SCTi003-A, ensuring consistency and reliability for research applications. These organoids undergo strict quality control to guarantee high reproducibility and performance across experiments.
• Robust Midbrain Marker Expression: These organoids exhibit strong expression of midbrain-specific markers such as FOXA2, LMX1A, TH, and NURR1, confirming their commitment to a midbrain identity. The presence of dopaminergic neurons makes them highly relevant for neurodegenerative disease modeling.
• Functional Neuronal Activity: Achieve physiologically relevant results with validated neuronal activity, including dopaminergic neuron function, as demonstrated using the MEA assay. These organoids provide a realistic model for studying midbrain-related diseases and functions.
• Long-Term Culture and Maintenance: Maintain midbrain organoids for extended periods using the STEMdiff? Neural Organoid Maintenance Kit (Catalog #100-0120), supporting long-term studies and experiments. This ensures comprehensive analysis of neuronal development, synaptic functionality, and disease modeling.
• Live-Shipped and Ready for Use: Midbrain organoids are shipped live at ambient temperature, ready for immediate use upon receipt. This enables researchers to begin experiments faster, with organoids already differentiated and functionally validated.

Human iPSC-Derived Midbrain Organoids are available now at various stages of differentiation, providing flexibility for diverse research applications. These organoids can be purchased as Differentiated Organoids, harvested at Day 42-49, or as Mature Organoids, harvested at Day 90-97. Differentiated Organoids are available in two formats: 48 organoids (half plate) for $5,495 USD or 96 organoids (full plate) for $8,895 USD. Similarly, Mature Organoids are offered as 48 organoids (half plate) for $10,995 USD or 96 organoids (full plate) for $15,995 USD. This variety allows researchers to select the most suitable option for their experimental needs.

For more information about STEMCELL's iPSC lines, differentiated cells, and organoids, refer to our Frequently Asked Questions on iPSCs.

For any other queries, click here to contact STEMCELL's iPSC Team or email us directly at [email protected].