ASTROCYTE ENRICHMENT AND MECHANOSENSORY STIMULATION INCREASED THE MATURATION OF HUMAN BRAIN ORGANOIDS

Brain organoids (BOs) derived from human pluripotent stem cells (hiPSCs) are promising for studying human brain development and disease. However, current BOs lack mature glial cells. While astrocytes emerge at late stages of organoid maturation¹, microglia do not differentiate spontaneously.
To recapitulate the physiological astrocyte–neuron ratio, we developed an astrocyte enrichment protocol based on the fusion of hiPSC-derived astrocytes and neural progenitor cells during neurosphere formation. Using LC–MS/MS proteomics, single-cell RNA sequencing, and immunohistochemistry, we show that astrocytes integrate and mature in the organoid microenvironment. Additionally, astrocyte enrichment significantly promotes the expression of extracellular matrix (ECM) and synaptic markers.
When exposed to a physiological heartbeat-like sound that recapitulates the embryonic acoustic environment, astrocyte-enriched BOs showed increased neuronal and astrocytic differentiation. Astrocytic mechanosensory stimulation further promoted ECM and synaptic protein expression. Shadow imaging² combined with two-photon and super-resolution STED microscopy in living BOs demonstrated that upregulated ECM expression increased the extracellular space in heartbeat-stimulated BOs. These results highlight a functional role of astrocytes in mechanotransduction-mediated tissue maturation³.
To overcome the absence of microglial cells, we co-cultivated hiPSC-derived microglia with BOs. Microglia efficiently infiltrated the organoid parenchyma after 60 days of cultivation and displayed characteristic ramified morphology under control conditions. Ischemia-reperfusion injury induced a transition towards an amoeboid-like hyperactivated phenotype, closely resembling microglial responses observed in experimental stroke models in mice⁴.
These results establish astrocyte-enriched brain organoids as a human model to study neuron-glia interactions, ECM remodelling, and glia-mediated responses to injury.
¹ doi:10.1016/j.neuron.2017.07.035
² doi:10.1016/j.cell.2018.02.007
³ doi:10.1101/2025.10.21.683816
⁴ doi:10.1016/j.bbi.2020.10.016