TY  - JOUR
TI  - Generation of 3D Human iPSC-Derived Multi-Cell Type Neurospheres for Studying Neuron, Astrocyte, and Microglia Crosstalk
AU  - Wendt, Stefan 
AU  - Lee, Christopher 
AU  - Cai, Wenji 
AU  - Lin, Ada J.
AU  - Huang, Jessica 
AU  - Poon, V. 
AU  - Xiang, Xianyuan 
AU  - Hong, Wei 
AU  - MacVicar, Brian A.
AU  - Nygaard, Haakon B.
VL  - 15
IS  - 21
PY  - 2025
DA  - 2025/11/05
SP  - e5493
C1  - Bio-protocol 2025;15:e5493
DO  - 10.21769/BioProtoc.5493
UR  - https://doi.org/10.21769/BioProtoc.5493
AB  - 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.
KW  - iPSC-derived cells
KW  - 3D neural tissue
KW  - Microglia
KW  - Neuro–glia interaction
KW  - In vitro disease modeling
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False