Establishing dorsal-ventral patterning in human neural tube organoids with synthetic organizers

Precise dorsal-ventral (D-V) patterning of the neural tube (NT) is essential for the development and function of the central nervous system. However, existing models for studying NT D-V patterning and related human diseases remain inadequate. Here, we present organizers derived from pluripotent stem cell aggregate fusion ("ORDER"), a method that establishes opposing BMP and SHH gradients within neural ectodermal cell aggregates. Using this approach, we generated NT organoids with ordered D-V patterning from both zebrafish and human pluripotent stem cells (hPSCs). Single-cell transcriptomic analysis revealed that the synthetic human NT organoids (hNTOs) closely resemble the human embryonic spinal cord at Carnegie stage 12 (CS12) and exhibit greater similarity to human NT than to mouse models. Furthermore, using the hNTO model, we demonstrated the critical role of WNT signaling in regulating intermediate progenitors, modeled TCTN2-related D-V patterning defects, and identified a rescue strategy.