Cortical Versus Hippocampal Organoids: A Tale of Two Cities

Abstract

Cortical Versus Hippocampal Network Dysfunction in a Human Brain Assembloid Model of Epilepsy and Intellectual Disability

McCrimmon CM, Toker D, Pahos M, Cao Q, Lozano K, Lin JJ, Parent JM, Tidball A, Zheng J, Molnár L, Mody I, Novitch BG, Samarasinghe RA. Cell Rep. 2025 Sep 23;44(9):116217. PMID: 40925365 PMCID: PMC12674601 DOI: 10.1016/j.celrep.2025.116217

Neurodevelopmental disorders often impair multiple cognitive domains. For instance, a genetic epilepsy syndrome might cause seizures due to cortical hyperexcitability and present with memory impairments arising from hippocampal dysfunction. This study examines how a single disorder differentially affects distinct brain regions using induced pluripotent stem cell (iPSC)-derived cortical- and hippocampal-ganglionic eminence assembloids to model developmental and epileptic encephalopathy 13, a condition arising from gain-of-function mutations in the SCN8A gene encoding the sodium channel Nav1.6. While cortical assembloids showed network hyperexcitability akin to epileptogenic tissue, hippocampal assembloids did not, and instead displayed network dysregulation patterns similar to in vivo hippocampal recordings from epilepsy patients. Predictive computational modeling, immunohistochemistry, and single-nucleus RNA sequencing revealed changes in excitatory and inhibitory neuron organization that were specific to hippocampal assembloids. These findings highlight the unique impacts of a single pathogenic variant across brain regions and establish hippocampal assembloids as a platform for studying neurodevelopmental disorders.

Commentary

Epilepsy is a heterogeneous disorder encompassing numerous distinct syndromes. Among these, developmental and epileptic encephalopathies (DEEs) represent the most severe group, typically beginning in infancy and characterized by a developmental encephalopathy coupled with an epileptic encephalopathy. ¹ In these disorders, seizure control alone does not normalize cognition, and cognitive or behavioral deficits can precede seizure onset—challenging the long-held assumption that epilepsy severity is the sole determinant of neurodevelopmental outcomes. Instead, DEEs highlight the dual impact of the underlying etiology—often genetic—and ongoing epileptic activity. Both may independently and synergistically contribute to developmental and cognitive impairment. ¹ Disentangling the mechanisms that drive epileptogenesis from those responsible for cognitive comorbidities remains a major challenge and a critical goal toward precision therapies that directly address the developmental encephalopathy. ²

An important but poorly understood dimension of phenotypic diversity in epilepsy and neurodevelopmental disorders is how the same pathogenic variant exerts brain region-specific effects. This becomes increasingly important with the recognition that in addition to the usual suspects—hippocampus, cortex, thalamus—epilepsy involves widespread and diverse brain regions. For example, the cerebellum—long discounted as being important in epilepsy—is now recognized as a seizure modulator, ³ and recent work implicating the brainstem in SUDEP highlights the potential importance of understudied structures in epilepsy comorbidities. ⁴ Posing a challenge for the field, the brain's highly interconnected nature complicates efforts to localize seizure onset zones and epileptogenic networks. Even comprehensive stereo-EEG studies leave large regions unexplored. Moreover, the seizure onset zone is only part of the story, as non-ictogenic impairment of other brain regions may underlie epilepsy comorbidities like cognitive dysfunction or mood disorders. Identifying regions driving comorbidities is even more challenging without clear biomarkers. At the other end of the spectrum, dissociated cell and slice culture systems have been invaluable for revealing some critical cell-specific effects, such as the greater impact of pathogenic variants in SCN1A, another cause of DEE, on sodium channel function of interneurons. ⁵ These simpler models, however, fail to capture developmental and brain region-specific differences that may drive disease phenotypes.

In this context, the recent work by McCrimmon and colleagues meets in the middle, using a brain organoid approach to compare cortical and hippocampal tissues. ⁶ The investigators focused on developmental and epileptic encephalopathy 13 (DEE-13), which is caused by gain-of-function mutations in SCN8A, coding for the sodium channel Nav1.6. Cortical and hippocampal organoids were generated using patient-derived iPSCs and CRISPR-corrected control iPSCs. SCN8A is particularly important for regulating interneuron excitability, and accordingly, interneuron dysfunction is implicated in driving seizures. ⁷ To model this important cellular component of the disease, the investigators generated interneuron organoids, which were then fused with cortical or hippocampal organoids, creating “assembloids.” ⁸ The team then used calcium imaging and field potential recordings to assess excitability. Cortical assembloids showed clear evidence of hyperexcitability, including increased rates of high amplitude field potential bursts, spikes, and periods of synchronized neuronal activity relative to controls. By contrast, hippocampal assembloids did not show evidence of increased excitability. Hippocampal assembloids did, however, show a reduction in high gamma (80-160 Hz) activity, and a change in theta-gamma coupling, with gamma amplitudes showing more variability in whether they locked with theta rising or falling phases. Gamma in control assembloids tended to lock with the theta peak. Providing more clinical relevance to the latter finding, a similar change in theta-gamma coupling was observed in patients with temporal lobe epilepsy. Finally, immunohistochemistry and single-nucleus RNA sequencing studies were conducted, demonstrating an increase in excitatory neurons and a reduction in somatostatin-expressing inhibitory neurons in mutant hippocampal assembloids. Modeling studies conducted by the group suggest that these changes in cell numbers could contribute to altered theta-gamma coupling. Similar studies in cortical assembloids revealed an increase in excitatory neurons, with no change in interneurons, providing one possible mechanism of divergence. Together, findings suggest that different brain regions might be responsible for different aspects of the patient phenotype in DEE-13, with cortical hyperexcitability potentially driving seizures, while hippocampal dysfunction might underlie learning and memory deficits.

The work by McCrimmon and colleagues fully capitalizes on the organoid model to address a critical question in the field: specifically, how different brain regions respond to the same pathogenic variant to produce the full constellation of disease symptoms. Use of patient iPSCs provides strong construct validity, and the organoid/assembloid approach provides a rigorous system to compare different tissue types starting from identical iPSCs. Findings give compelling evidence that cortical and hippocampal tissues respond differently to SCN8A gain-of-function pathogenic variants in ways that may be highly relevant to patient phenotypes. The results also deliver some clear direction for follow-up studies in patients, including better characterization of seizure onset and guidance on potential neuronal populations that may be particularly vulnerable. SCN8A pathogenic variants are rare (1:56,000), so clear guidance on how to best utilize valuable patient samples is particularly important. ⁹

Follow-up studies will be necessary to validate in vitro findings. Organoids do not accurately reproduce all aspects of brain development, so it is conceivable that some findings could be model-specific. This might be particularly relevant for more complex circuit outputs, such as gamma oscillations and ripples. Whether the cellular mechanisms producing these features in organoids are identical to the intact brain remains unclear. In addition, while organoid technology has advanced tremendously, developmental processes are very different from the intact brain, and keeping organoids healthy can be challenging. Neurons might be more vulnerable to death or disruption under the in vitro conditions.

Why would the same pathogenic variant produce such different effects in hippocampal versus cortical assembloids? Like Paris and London from Dickens’ classic novel, the differences between the brain regions are extensive. Cortex and hippocampus vary substantially in gene expression, developmental processes, cellular composition, and circuitry. It is easy to imagine that the distinct features of each region provide unique vulnerabilities and resilience. Perhaps more importantly, the assembloid model provides a tool to begin to address these questions. For example, the interneurons merged with cortical or hippocampal organoids were initially identical, so what differs between the cortical or hippocampal tissues that leads to reduced numbers in the latter? The accessibility of the assembloid system provides real-time imaging possibilities, and the flexibility allows many interesting combinations. Merging control interneurons into mutant organoids and vice versa, for example, would provide insights into whether interneuron loss is an autocrine or paracrine process.

With caveats in mind, this work represents a technological advance that offers important new insights into epilepsy within the context of neurodevelopmental disorders such as DEE. In such conditions, cognitive deficits likely arise not only from seizure-related network disruption but also from variant-driven effects on specific circuits, leading to widespread network dysfunction and drug-resistant epilepsy. Supporting the concept of encephalopathies, quantitative EEG analysis in STXBP1-related DEE revealed a pattern of frontal network dysfunction that correlates with phenotypic severity in epilepsy and neurodevelopmental impairment. ¹⁰ For DEEs broadly, seizure management alone has proven insufficient to mitigate diffuse network abnormalities. The findings by McCrimmon underscore the need to disentangle mechanisms underlying seizures from those driving cognitive impairment. Early interventions targeting vulnerable networks implicated in neurocognitive deficits—alongside strategies to suppress epileptogenesis—may be critical to improving developmental outcomes and reducing comorbidities.