Expanding Genetic Code to Generate Human Brain Organoids with Both Vasculature and Microglia‐Like Cells

Brain organoids offer an invaluable model system for studying human brain development and disease. However, the establishment of high-fidelity brain organoids with multiple cell lineages, including vasculature and immune cells, remains a huge challenge. Here, we present a new strategy to generate human cerebral organoids with vasculature and microglia-like cells using genetic code expansion technology via site-specific protein engineering. The strategy integrates orthogonal genetic translation machinery in hPSCs, enabling temporal control of ETV2 expression and endothelial differentiation in hPSC-derived cerebral organoids. The vascularized human cerebral organoids (vhCOs) exhibit coordinated development of multiple cell lineages and blood-brain barrier features, and form a perfusable vascular network after being transplanted into immune-deficient mice. Single-nucleus RNA sequencing reveals enhanced neurovascular interactions, multi-brain-regional identities, diverse neuronal subtypes, and specialized endothelial subclusters in vhCOs, closely resembling the human fetal brain. Strikingly, we identify enriched microglia-like cells comprising three distinct subtypes in vhCOs, which contribute to microglia-vascular interactions and synergistically modulate vascular development. Upon Zika virus infection, vhCOs show neurovascular dysfunction and impaired microglia development, offering new insights into viral-induced neurodevelopmental disorders. This study offers a unique platform for producing more valuable brain organoids with vasculature and immune components, opening a new avenue to advance organoid research and applications.