Patterning ganglionic eminences in developing human brain organoids using morphogen gradient inducing device

In early neurodevelopment, the central nervous system is established through the coordination of various neural organizers directing tissue patterning and cell differentiation. Better recapitulation of morphogen gradient production and signaling will be crucial for establishing improved developmental models of the brainin vitro. Here, we developed a method by assembling polydimethylsiloxane (PDMS) devices capable of generating a sustained chemical gradient to produce patterned brain organoids, which we termedMorphogen-gradientInducedBrainOrganoids (MIBOs). At 3.5 weeks, MIBOs replicated Dorsal-Ventral patterning observed in the Ganglionic Eminence (GE). Analysis of matured MIBOs through single-cell RNA sequencing revealed distinct Dorsal GE derived CALB2+ interneurons (INs), Medium Spiny Neurons (MSNs), and MGE derived cell types. Finally, we demonstrate long term culturing capabilities with MIBOs maintaining stable neural activity in cultures grown up to 5.5 months. MIBOs demonstrate a versatile approach for generating spatially patterned brain organoids for embryonic development and disease modeling. Overall design: Gradient patterned forebrain organoid was dissociated into single cells using papain digestion methods. We used singlecell RNA sequencing (scRNA-Seq) to analyze