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.