Generation of Blood Vascular Endothelial-Neural 3D Organoids by Serial Induction of Differentiation on Human iPSC-Derived Embryoid Bodies

The 3D brain organoids have been widely used as a tool to study human brain development and disorders. Although angiogenesis and blood vascular endothelial cells play important roles in brain development and pathogenesis in neurological disorders, most 3D brain organoids lack inherent endothelial cells and need either the addition of differentiated endothelial cells or to be transplanted to animals to reconstitute such vascular structures. However, these approaches could miss the developmental interactions between angiogenesis and neurogenesis in the human brain. To reconstitute a 3D organoid mimicking the in vivo development of neural and vascular endothelial cells, we cultured iPSC-derived embryoid bodies and sequentially applied endothelial and neuronal induction media along with Matrigel embedding. The resulting 3D organoid consists of both neural cells and endothelial cells with vascular-like structures, as determined by immunostaining. With scRNA-Seq analysis, the organoid was confirmed to contain neural cell types similar to human brains, including a variety of excitatory and inhibitory neurons and glia. Furthermore, when compared with conventionally generated cerebral organoids without endothelial cells using RNA-Seq analysis, the vascular endothelial-containing neural organoids (EC-neural organoids) showed different gene profiles and favored angiogenesis and vasculogenesis. Of the differentially expressed genes, KRBA2 expression was found to be higher in neural cells and its inhibition by siRNA treatment resulted in decreased transcriptions of a variety of genes specific to neuronal differentiation but not genes specific to pluripotent stem cells such as OCT4. The EC-neural organoids also expressed receptors to SARS-CoV-2 at levels similar to human brains. This 3D EC-neural model provides a useful tool to study the interactions between vascular endothelial cells and neural cells in brain development and potentially for the study of neural infectious disorders where vascular endothelial cells are targets for infection and mediators for neural damage.