Modelling hyperexcitability in human cerebral cortical organoids: Oxygen/glucose deprivation most effective stimulant.

Epilepsy is a common neurological disorder that affects 1% of the global population. The neonatal period constitutes the highest incidence of seizures. Despite the continual developments in seizure modelling and anti-epileptic drug development, the mechanisms involved in neonatal seizures remain poorly understood. This leaves infants with neonatal seizures at a high risk of death, poor prognosis of recovery and risk of developing neurological disorders later in life. Current in vitro platforms for modelling adult and neonatal epilepsies - namely acute cerebral brain slices or cell-derived cultures, both derived from animals-either lack a complex cytoarchitecture, high-throughput capabilities or physiological similarities to the neonatal human brain. Cerebral organoids, derived from human embryonic stem cells (hESCs), are an emerging technology that could better model neurodevelopmental disorders in the developing human brain. Herein, we study induced hyperexcitability in human cerebral cortical organoids - setting the groundwork for neonatal seizure modelling - using electrophysiological techniques and pharmacological manipulations. In neonatal seizures, energy failure - specifically due to deprivation of oxygen and glucose - is a consistent and reliable seizure induction method that has been used to study the underlying cellular and molecular mechanisms. Here, we applied oxygen-glucose deprivation (OGD) as well as common chemoconvulsants in 3-7-month-old cerebral organoids. Remarkably, OGD resulted in hyperexcitability, with increased power and spontaneous events compared to other common convulsants tested at the population level. These findings characterize OGD as the stimulus most capable of inducing hyperexcitable changes in cerebral organoid tissue, which could be extended to future modelling of neonatal epilepsies in cerebral organoids.

Proneural genes define ground-state rules to regulate neurogenic patterning and cortical folding.

Asymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds.

Production of Phenotypically Uniform Human Cerebral Organoids from Pluripotent Stem Cells.

Recent advances in stem cell technology have allowed researchers to generate 3D cerebral organoids (COs) from human pluripotent stem cells (hPSCs). Indeed, COs have provided an unprecedented opportunity to model the developing human brain in a 3D context, and in turn, are suitable for addressing complex neurological questions by leveraging advancements in genetic engineering, high resolution microscopy, and tissue transcriptomics. However, the use of this model is limited by substantial variations in the overall morphology and cellular composition of organoids derived from the same pluripotent cell line. To address these limitations, we established a robust, high-efficiency protocol for the production of consistent COs by optimizing the initial phase of embryoid body (EB) formation and neural induction. Using this protocol, COs can be reproducibly generated with a uniform size, shape, and cellular composition across multiple batches. Furthermore, organoids that developed over extended periods of time (3-6 months) showed the establishment of relatively mature features, including electrophysiologically active neurons, and the emergence of oligodendrocyte progenitors. Thus, this platform provides a robust experimental model that can be used to study human brain development and associated disorders. Graphic abstract: Overview of cerebral organoid development from pluripotent stem cells.

Characterization of mitochondrial health from human peripheral blood mononuclear cells to cerebral organoids derived from induced pluripotent stem cells.

Mitochondrial health plays a crucial role in human brain development and diseases. However, the evaluation of mitochondrial health in the brain is not incorporated into clinical practice due to ethical and logistical concerns. As a result, the development of targeted mitochondrial therapeutics remains a significant challenge due to the lack of appropriate patient-derived brain tissues. To address these unmet needs, we developed cerebral organoids (COs) from induced pluripotent stem cells (iPSCs) derived from human peripheral blood mononuclear cells (PBMCs) and monitored mitochondrial health from the primary, reprogrammed and differentiated stages. Our results show preserved mitochondrial genetics, function and treatment responses across PBMCs to iPSCs to COs, and measurable neuronal activity in the COs. We expect our approach will serve as a model for more widespread evaluation of mitochondrial health relevant to a wide range of human diseases using readily accessible patient peripheral (PBMCs) and stem-cell derived brain tissue samples.

Robust production of uniform human cerebral organoids from pluripotent stem cells.

Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human-specific cortical development and pathophysiology in an experimentally tractable system. Although diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we have directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single-cell profiling that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.