Abstracts

Rationale: Human brain organoids are a 3D-culture system where brain-like structures are created from human embryonic or induced pluripotent stem cells (hESCs or hiPSCs). This compelling new platform recapitulates unique aspects of human brain development and cytoarchitecture and has already provided novel insights into human neurological disease. However, much of the current literature on brain organoids has focused on utilizing the anatomical and cytoarchitectural characteristics of the organoid to model brain disease and has not focused on the physiological activity or network architecture of these remarkable structures. In the following studies we leveraged recent advances in organoid culture that have permitted the formation of cerebral cortex-ganglionic eminence (Cx-GE) “fusion” organoids in which excitatory and inhibitory neuron populations integrate to generate organoids with complex oscillatory and network events. Methods: Brain organoids were generated from the H9 stem cell line or Rett patient hiPSCs using established laboratory protocols. Cx-GE brain organoids were fused at age day 56. Fused organoids were infected with AAV-1-GcAMP at age day ~84 and subject to two-photon microscopy at age ~100 days. Ca2+ data were analyzed using MATLAB algorithms. Extracellular recordings were performed on day 100 fused organoids and oscillatory activity was quantified using Igor Pro. Immunohistochemical analyses were performed using standard laboratory protocols to establish cell-type expression and organoid cytoarchitecture. Results: Fusion organoids demonstrated complex oscillations and Ca2+ network activities not seen in un-fused GE or Cx organoids. Rett patient-derived fusion organoids did not exhibit gross morphological differences from isogenically matched controls, but demonstrated spontaneously synchronized Ca2+ networks distinct from control fusion organoids. Extracellular recordings from Rett fusion organoids revealed epileptiform-like spikes and high-frequency oscillations that can be clinically associated with epileptogenic tissue. Isogenically matched control fusion organoids displayed sustained lower frequency oscillations, but no epileptiform changes. The epileptiform features of Rett were found to be largely interneuron dependent, and were partially responsive to pharmacological treatment with sodium valproate or the the p53 inhibitor pifithrin-α. Conclusions: These data suggest that excitatory-inhibitory fusion organoids have unique and complex neural ciruit activities that can be leveraged to recapitulate human disease states such as the ictogenic changes in Rett. Further understanding of the underlying mechanism of these physiological activities may allow fusion organoids to provide novel insights into human epilepsies. Funding: No funding