Brainstem Organoid Modeling Service

Introduction

The brainstem is essentially the body's mission control, handling vital functions like breathing, blood pressure, and heartbeat without us thinking about it. Because it's so critical, problems affecting this area – whether from neurodegenerative diseases, strokes, tumors, or developmental issues – can be incredibly serious, often life-threatening. This presents a huge challenge for researchers: how do you study disorders of the human brainstem when accessing the tissue directly is nearly impossible? Our specialized Brainstem Organoids Modeling Service was created specifically to address this gap, using modern methods to grow human brainstem organoids (hBSOs) from human pluripotent stem cells (hPSCs).

These hBSOs are complex 3D structures that develop in the lab. What's remarkable is how they manage to recreate key features of the developing human brainstem, including aspects resembling the midbrain, pons, and medulla oblongata. Inside, they develop a rich variety of cell types that belong there, including important progenitor cells, neurons that produce dopamine or norepinephrine/acetylcholine, and even cells derived from the neural crest. We use careful differentiation methods and confirm the results using advanced checks like protein analysis and electrical recordings. The aim is to provide researchers with a reliable in vitro model that reflects human brainstem biology, making it possible to study its development, understand disease mechanisms, and test potential treatments more effectively.

Key Advantages

• Comprehensive Cellular Diversity: A Faithful Reflection of Brainstem Complexity

Our hBSOs are designed to faithfully mirror the intricate cellular makeup of the human brainstem, encompassing a wide spectrum of functionally important cell populations. These include dopamine-secreting neurons (identified by TH expression), cholinergic neurons (marked by ChAT), noradrenergic neurons (indicated by DBH), and even neural crest-derived melanocytes. This rich cellular diversity allows for nuanced studies on the complex interplay between various neuronal subtypes and non-neuronal cells within the context of disease progression.

• Stringent Validation: Ensuring Reliability and Relevance
  Our brainstem organoids undergo a multi-faceted and rigorous quality control process to guarantee their scientific reliability and translational relevance:
  
  • scRNA-seq Profiling: Detailed transcriptional analysis is performed to confirm a high degree of molecular similarity between our organoids and the developing human fetal brainstem.
  • Proteomic Analysis: Comprehensive protein expression profiling is conducted to validate the presence and appropriate levels of key brainstem-specific protein markers (e.g., FOXA2, LMX1A, SOX9).
  • Electrophysiological Testing: Functional assessments are carried out to verify the neuronal maturity of the organoids, including their ability to generate action potentials and exhibit characteristic HCN channel activity.

• Powerful Tools for Disease Modeling & Drug Screening

Our hBSOs offer an exceptional platform for creating models of various brainstem-specific disorders, such as the neurodegeneration seen in Parkinson’s disease, the aggressive growth of brainstem gliomas, and the life-threatening breathing dysregulation of congenital central hypoventilation syndrome. Furthermore, they provide a human-relevant system for the preclinical screening of potential neuroprotective compounds. The inclusion of neural crest-derived cells also broadens the applicability of our model to the study of neurodevelopmental conditions like Waardenburg-Shah syndrome or DiGeorge syndrome.

• Customizable Protocols: Tailored Solutions for Specific Research Questions

We understand that each research project has unique needs. Therefore, we offer flexible and adaptable induction protocols that can be specifically tailored to enrich the generation of particular cell populations of interest (for example, midbrain dopaminergic neurons). We also provide the capability to utilize patient-derived iPSCs, enabling highly personalized approaches to disease modeling.

Our Comprehensive Brainstem Organoid Services

1. Brainstem Organoid Generation

• Flexible Cell Source Options: You can provide your own well-characterized iPSCs or ESCs, or leverage our rigorously validated in-house human pluripotent stem cell lines.
• Refined Differentiation Protocol: Our meticulously optimized protocol employs a precise combination of essential growth factors and key differentiation agents to ensure the consistent and reproducible generation of hBSOs with the desired cellular composition.
• Adjustable Timelines: Organoids typically reach a stage of significant maturity within 4 to 12 weeks of culture. We also offer the option for extended culture periods to facilitate the development of more complex and intricate neuronal networks.

Fig.1 We induced hBSOs following the shown schematic. One-month-old organoids underwent immunohistochemical analysis for cell identification.^1,3

2. Comprehensive Phenotypic & Functional Analysis

• Immunohistochemistry (IHC): Detailed spatial visualization and analysis of specific cell types and protein expression patterns using a carefully selected panel of established markers, including OTX2 (midbrain), TH (dopaminergic neurons), and SOX9 (neural crest).

Fig.2 Representative images of 1-month-old hBSOs.^1,3

• Electrophysiology: Precise measurement and analysis of neuronal electrical activity, synaptic connections, and the specific responses of neuronal circuits to various pharmacological compounds.

Fig.3 The voltage responses of individual cells within 1-month-old hBSOs to the delivery of current pulses were recorded and analyzed.^1,2

3. Cutting-Edge Drug Discovery & Toxicity Testing

• High-Throughput Screening Platforms: Efficient and rapid evaluation of the therapeutic potential or potential neurotoxic effects of various compounds within a physiologically relevant, three-dimensional human-based system.
• Mechanistic Investigations: In-depth studies focused on elucidating the roles of critical signaling pathways (e.g., Wnt, SHH) or the precise outcomes of gene-editing interventions (CRISPR/Cas9) within disease-relevant organoid models.
• Accelerated Drug Screening: Leverage the rapid induction of hBSOs (functional within 1.5 months) for efficient drug discovery. Electrophysiological validation confirms early expression of HCN channels, Na⁺/K⁺ channels, and heterogeneous neuronal populations, enabling robust assessment of compounds targeting brainstem neurodegenerative diseases.

4. Customized Disease Modeling Solutions

• Patient-Specific Organoid Generation: We can generate hBSOs directly from iPSCs derived from individuals affected by specific brainstem disorders, enabling highly personalized research approaches that can yield more translatable insights.
• Neural Crest Disorder Models: Utilize hBSOs containing neural crest cells to study diseases like DiGeorge syndrome and Waardenburg-Shah syndrome. Our organoids provide a platform to unravel pathologies caused by neural crest dysfunction and test therapeutic interventions.
• Precise Gene Editing Capabilities: We offer services to introduce specific disease-associated mutations (e.g., PHOX2B for congenital hypoventilation) or to create valuable reporter lines for advanced live-cell imaging studies of dynamic cellular processes.

5. Collaborative Research Support

• Open Data Access: We provide access to raw datasets (RNA-seq, proteomics) and user-friendly bioinformatics tools to facilitate comprehensive integrative analysis of your research findings.
• Joint Protocol Development: We actively collaborate with researchers to co-develop bespoke protocols tailored to novel and specialized applications, such as detailed studies of neural crest cell migration or the integration of blood-brain barrier components within the organoid system.

Our Workflow

References

1. Eura, Nobuyuki et al. “Brainstem Organoids From Human Pluripotent Stem Cells.” Frontiers in neuroscience vol. 14 538. 26 Jun. 2020, doi:10.3389/fnins.2020.00538.
2. Distributed under Open Access License CC BY 4.0 without modification.
3. Distributed under Open Access License CC BY 4.0. The original image was modified.