02641nas a2200325   4500000000100000000000100001008004100002260000900043653002300052653003200075653003300107653002500140100002300165700002200188700001700210700001300227700001400240700002200254700001400276700001400290700002300304700002100327700001900348245009400367856006700461300001100528490000700539520175500546022001402301       2025                            d  c202510aendothelial system10amulti-region brain organoid10aneurodevelopmental processes10aorganoid engineering1 aAnannya Kshirsagar1 aHayk Mnatsakanyan1 aSai Kulkarni1 aJohn Guo1 aKai Cheng1 aLuke Daniel Ofria1 aOce Bohra1 aRam Sagar1 aVasiliki Mahairaki1 aChristian E Badr1 aAnnie Kathuria00aMulti-Region Brain Organoids Integrating Cerebral, Mid-Hindbrain, and Endothelial Systems  uhttps://onlinelibrary.wiley.com/doi/abs/10.1002/advs.202503768  ae037680 v123 aBrain organoid technology has revolutionized the ability to model human neurodevelopment in vitro. However, current techniques remain limited by their reliance on simplified endothelial cell populations. Multi-Region Brain Organoids (MRBOs) are engineered that integrate cerebral, mid/hindbrain, and complex endothelial organoids into one structure. Unlike earlier approaches based on isolated Human Umbilical Vein Endothelial Cells, the endothelial organoids contain diverse vascular cell types, including progenitors, mature endothelial cells, pericytes, proliferating angiogenic cells, and stromal cells. The strategy employs sequential modulation of key developmental pathways to generate individual organoids, followed by optimized fusion conditions that maintain regional identities while supporting cellular integration. Single-nucleus RNA sequencing reveals that MRBOs develop discrete neural populations specific to each brain region alongside specialized endothelial populations that establish paracrine signaling networks. Integration analysis with human fetal brain data shows that MRBOs contribute to 80% of cellular clusters found in human fetal brain tissue (Carnegie stages 12–16). CellChat analysis identifies 13 previously uncharacterized endothelial-neural signaling interactions. Endothelial-derived factors are uncovered that support intermediate progenitor populations during hindbrain development, but not cerebral development, revealing a role for endothelial populations in regional brain patterning. This platform enables matching of multiple developmental regions while incorporating endothelial components, providing opportunities for studying neurodevelopmental disorders with disrupted neural-endothelial interactions.  a2198-3844