Related Experiment Video
Updated: Jun 15, 2026
Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM
Published on: May 8, 2020
Defined human tri-lineage brain microtissues
Takeshi Uenaka1,2, Sascha Jung3, Ishan Kumar1,2
1Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA.
Abstract:
Microglia are the immune cells of the central nervous system and are thought to be key players in both physiological and disease conditions. Several microglial features are poorly conserved between mice and human, such as the function of the neurodegeneration-associated immune receptor Trem2. Induced pluripotent stem cell (iPSC)-derived microglia offer a powerful opportunity to generate and study human microglia. However, human iPSC-derived microglia often exhibit activated phenotypes in vitro, and assessing their impact on other brain cell types remains challenging due to limitations in current co-culture systems. Here, we developed fully defined brain microtissues, composed of human iPSC-derived neurons, astrocytes, and microglia, co-cultured in 2D or 3D formats. Our microtissues are stable and self-sufficient over time, requiring no exogenous cytokines or growth factors. All three cell types exhibit morphologies characteristic of their in vivo environment and show functional properties. Co-cultured microglia develop more homeostatic phenotypes compared to microglia exposed to exogenous cytokines. Hence, these tri-cultures provide a unique approach to investigate cell-cell interactions between brain cell types. We found that astrocytes and not neurons are sufficient for microglial survival and maturation, and that astrocyte-derived M-CSF is essential for microglial survival. Single-cell and single-nucleus RNA sequencing analyses nominated a network of reciprocal communication between cell types. Brain microtissues faithfully recapitulated pathogenic α-synuclein seeding and aggregation, suggesting their usefulness as human cell models to study not only normal but also pathological cell biological processes.
Insights
Human iPSC-derived microglia were co-cultured with neurons and astrocytes in novel brain microtissues. These microtissues promote homeostatic microglia phenotypes and model neurodegenerative diseases.
Area of Science:
- Neuroscience
- Stem Cell Biology
- Immunology
Background:
- Microglia are central nervous system immune cells crucial for brain health and disease.
- Human and mouse microglia differ significantly, necessitating human-specific models.
- Existing models struggle to replicate human microglia's in vivo environment and interactions.
Purpose of the Study:
- To develop a robust human iPSC-based brain microtissue model for studying microglia-neuron-astrocyte interactions.
- To establish a system that supports homeostatic microglial phenotypes in vitro.
- To investigate cell-cell communication networks and model neurodegenerative processes.
Main Methods:
- Co-culture of human induced pluripotent stem cell (iPSC)-derived neurons, astrocytes, and microglia in 2D and 3D microtissues.
- Cultivation in fully defined media without exogenous cytokines.
- Analysis of cell morphology, function, survival, maturation, and gene expression via scRNA-seq and snRNA-seq.
- Modeling of alpha-synuclein seeding and aggregation.
Main Results:
- Developed stable, self-sufficient brain microtissues with characteristic cell morphologies and functions.
- Co-cultured microglia exhibited homeostatic phenotypes, unlike those with exogenous cytokines.
- Astrocytes, not neurons, were sufficient for microglial survival and maturation, with M-CSF being essential.
- Identified reciprocal cell communication networks via multi-omic analyses.
- Successfully recapitulated alpha-synuclein pathology in the microtissues.
Conclusions:
- Human iPSC-derived brain microtissues provide a powerful platform for studying microglia in a homeostatic and disease context.
- Astrocytes play a critical role in supporting microglial development and function.
- These microtissues serve as valuable human cell models for neurodegenerative disease research.
Related Concept Videos
01:13Organization of the Nervous System
The CNS, comprising the brain and spinal cord, houses billions of neurons. The brain is housed in the skull, while the spinal cord is linked to the brain through the foramen magnum of the occipital bone and is surrounded by the protective structure of the vertebral column. It is responsible for processing various...
01:20Anatomy of the Brain: Major Regions
The cerebrum is the largest section of the brain and divides into left and right hemispheres, separated by a deep fissure. The cerebral outer layer of grey matter — the cerebral cortex — comprises elevations called gyri and shallow groves called sulci. The inner portion of white matter includes long nerve fibers known as axons, which connect various areas...
01:11Cerebrum: Anatomical Overview II
01:15Functional Brain Systems: Limbic System
01:13Functional Brain Systems: Reticular Formation
Within the reticular formation, there are several distinct nuclei that can be classified into three broad categories. The Raphe nuclei are located along the midline of the brainstem. They are primarily known for their role in synthesizing and releasing serotonin, a neurotransmitter involved in regulating mood, appetite, sleep, and circadian rhythms. The...
01:30Organization of the Brain
Hindbrain
The hindbrain, located at the base of the brain, plays a vital role in regulating automatic processes that sustain life. It includes the medulla oblongata, which is essential for...