ArticleBio-protocol2025
Generation of 3D Human iPSC-Derived Multi-Cell Type Neurospheres for Studying Neuron, Astrocyte, and Microglia Crosstalk.
Article in Bio-protocol, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
What it found
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Who cites it
5 citing papers in PubMed.
- Article
- Astrocyte-driven multicellular mechanisms of CNS repair and cerebroprotection.Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism · 2026Review
- Structure-Property-Function Relationships in Stimuli-Responsive Hydrogels for Brain Organoid Vascularization.Gels (Basel, Switzerland) · 2026Review
- The complexity of dementia development and its comorbidities: The collaborative cross-mouse population for multivarious tasks approach.Animal models and experimental medicine · 2026Review
- Focal cortical dysplasias: modeling pediatric drug-resistant epilepsy using human brain organoids.Frontiers in cellular neuroscience · 2026Review
Corrections and comments
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Authors and funding
10 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Three-dimensional (3D) human brain tissue models derived from induced pluripotent stem cells (iPSCs) have transformed the study of neural development and disease in vitro. While cerebral organoids offer high structural complexity, their large size often leads to necrotic core formation, limiting reproducibility and challenging the integration of microglia. Here, we present a detailed, reproducible protocol for generating multi-cell type 3D neurospheres that incorporate neurons, astrocytes, and optionally microglia, all derived from the same iPSCs. While neurons and astrocytes differentiate spontaneously from neural precursor cells, generated by dual SMAD-inhibition (blocking BMP and TGF-b signaling), microglia are generated in parallel and can infiltrate the mature neurosphere tissue after plating neurospheres into 48-well plates. The system supports a range of downstream applications, including functional confocal live imaging of GCaMP6f after adeno-associated virus (AAV) transduction of neurospheres or immunofluorescence staining after fixation. Our approach has been successfully implemented across multiple laboratories, demonstrating its robustness and translational potential for studying neuron-glia interactions and modeling neurodegenerative processes. Key features • Reproducible human iPSC-derived 3D neurosphere multi-cell type tissue culture system. • Optional addition of microglia allows for studying neuron-microglia interaction in vitro in 3D. • Reliable spontaneous activity offers functional tissue culture readouts of neural firing. • System allows modeling of human brain diseases, such as Alzheimer's disease.
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Registered trials
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