Expanding the genetic code to generate human brain organoids with vasculature and microglia

Genetic code expansion technology (GCE-T) has been applied to precisely manipulate protein structure and function in bacteria, virus, yeast, mammalian cells, and even in animals. It has not been fully explored in 3D tissues or organoids. Here, we presented a new strategy leveraging GCE-T to generate vascularized human cerebral organoids (vhCOs) containing resident microglia. By reassigning a premature termination codon in ETV2, a targeted noncanonical amino acid was incorporated to drive endothelial differentiation in hPSCs at the translational level. Strikingly, the generated vhCOs exhibited coordinated differentiation of functional vasculature, blood brain barrier feature and microglia. single-nucleus transcriptomic analysis revealed multiple brain regional distribution, marked neurogenesis, and neuro-vascular-immune network in vhCOs, resembling human fetal brain. Upon Zika virus infection, vhCOs exhibited impaired vasculature, disrupted neurogenesis and microglial development. This strategy offers a unique platform for producing high-fidelity cerebral organoids integrated with vasculature and immune components, opening a new avenue to advance organoid research and applications.