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). 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 structures. To enhance physiological activity, we generated “fusion” organoids in which cortex-like organoids predominantly containing excitatory neurons and ganglionic eminence (GE)-like organoids primarily with inhibitory neurons integrate. These fusions resulted in organoids with complex network activity including neural oscillations with similar frequencies as observed in human cortex in vivo. In order to further characterize brain region specific differences in neural network activity, we expanded on this approach and also generated hippocampus-GE fusions.

Methods: Brain organoids were generated from SCN8A mutant or isogenic control hiPSCs using established laboratory protocols. Organoids were fused at age day 56. Fused organoids were infected with AAV-1-GcAMP at age day ~110 and subject to two-photon microscopy at age ~124 days. Ca2+ data were analyzed using MATLAB algorithms. Extracellular recordings were performed on day 120-130 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: As previously seen with cortex, non-mutant hippocampus fusions generated neural oscillations at multiple frequencies, but additionally generated sharp wave ripple complexes. The latter are a unique class of oscillation associated with hippocampal memory consolidation. We next generated organoids from the hiPSCs of a patient with developmental epileptic encephalopathy-13 (DEE-13) due to a pathogenic gain of function mutation in the SCN8A sodium channel. Calcium indicator imaging and extracellular recordings of local field potentials showed substantial hyperexcitability as well as a loss of sustained oscillatory activity in the cortex-GE fusions compared to isogenic controls. In contrast, in DEE-13 hippocampus fusion organoids we did not observe overt hyperexcitability. Instead, we found more subtle changes including a loss of theta and alpha oscillations with relative preservation of gamma activity. In addition, in SCN8A mutant hippocampus we observed fewer instances of spontaneous sharp wave ripple complexes.

Conclusions: These data suggest that (1) hippocampus and cortex fusion organoids generate complex and distinct network activities, and (2) that human brain organoids may provide unique insights into brain-region specific changes that result from the same pathogenic SCN8A mutation.

Funding: Please list any funding that was received in support of this abstract.: CURE -Taking Flight
Simons Foundation- Bridge to Independence; American Epilepsy Society.