Self-organized anteroposterior regionalization of early midbrain and hindbrain/spinal cord using micropatterned human pluripotent stem cells

Anterior-posterior (AP) spatial regionalization is crucial for central nervous system development. Previous studies suggest that morphogen gradients can induce AP patterning in animal and organoid models. While self-organization in early embryogenesis and neurogenesis has been found using geometrically confined microtissues, spontaneously induced AP patterning has not been reported. Here, we show that circularly micropatterned human pluripotent stem cells self-organize into spatially distinct FOXG1-FOXA1+OTX2+ midbrain-like and HOXB4+ hindbrain/spinal cord-like regions. Notably, the tissue then folds inwardly to form a 3D annular structure, maintaining a distinct boundary between OTX2+ and HOXB4+ zones. The reaction-diffusion of BMP/Noggin plays a key role in the mechanism of AP patterning. Our model is validated by its capability to distinguish the teratogenic effects of valproic acid and isotretinoin. Our work suggests a novel regulatory mechanism for AP patterning and provides a tool for fast screening of teratogens.