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A Hybrid 2D-to-3D in vitro Differentiation Platform Improves Outcomes of Cerebral Cortical Organoid Generation in hiPSCs.
Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell-derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this "hybrid" approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC. Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs) Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs) Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids.
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Morphogen-Directed, High-Throughput Development of hiPSC-Derived Telencephalic Organoids for Comparative In Vitro Phenotyping.
Pluripotent stem cell (PSC)-derived telencephalic organoids have long been established as an in vitro cell model to study the molecular and cellular features of the developing cerebral cortex and associated brain structures. Numerous directed organoid differentiation protocols have been developed that recapitulate cell subtype specification, cytoarchitectural organization, and cellular function with a high degree of fidelity. Protocol development has focused on the scalability of this in vitro system, and most organoid culture platforms involve a population-based approach in large vessel sizes. This makes comparative study with multiple cell lines both costly and time-consuming, and lowers the throughput for phenotypic screening. We have established a high-throughput approach for in vitro generation of hiPSC-derived telencephalic organoids that can be used to develop comparative phenotypic assays involving high-content imaging platforms. This article first highlights the use of a scaled-down, miniaturized cell culture volume that is amenable to high-throughput production of multiple 384-well plates. Then, using controlled morphogen cues, 3D hiPSC aggregates are directed into an anterior telencephalic identity and further specified into dorsal pallium or ventral subpallium cell fates, which can additionally be combined into assembloids for modeling excitatory and inhibitory networks or left to develop separately as single organoid subtypes. Finally, we introduce a Support Protocol detailing an in vitro 3D cell painting assay for organoid morpho-phenotypic characterization. © 2026 Wiley Periodicals LLC. Basic Protocol 1: High-throughput 3D aggregation of hiPSCs in morphogen-rich media Basic Protocol 2: Recombinant inhibitory protein-directed induction of telencephalic organoids Basic Protocol 3: Morphogen-guided induction of dorsal telencephalic/pallial organoids Basic Protocol 4: Morphogen-guided induction of ventral telencephalic/subpallial organoids Support Protocol: 3D live cell painting for organoid morphometric phenotyping.
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Virus stamping for targeted single-cell infection in vitro and in vivo.
Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here we describe 'virus stamping', in which viruses are reversibly bound to a delivery vehicle-a functionalized glass pipette tip or magnetic nanoparticles in a pipette-that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces. Different single cells in the same tissue can be infected with different viruses and an individual cell can be simultaneously infected with different viruses. We use rabies, lenti, herpes simplex, and adeno-associated viruses to drive expression of fluorescent markers or a calcium indicator in target cells in cell culture, mouse retina, human brain organoid, and the brains of live mice. Virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types, both in vitro and in vivo.
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A proof of concept 'phase zero' study of neurodevelopment using brain organoid models with Vis/near-infrared spectroscopy and electrophysiology.
Homeostatic control of neuronal excitability by modulation of synaptic inhibition (I) and excitation (E) of the principal neurons is important during brain maturation. The fundamental features of in-utero brain development, including local synaptic E-I ratio and bioenergetics, can be modeled by cerebral organoids (CO) that have exhibited highly regular nested oscillatory network events. Therefore, we evaluated a 'Phase Zero' clinical study platform combining broadband Vis/near-infrared(NIR) spectroscopy and electrophysiology with studying E-I ratio based on the spectral exponent of local field potentials and bioenergetics based on the activity of mitochondrial Cytochrome-C Oxidase (CCO). We found a significant effect of the age of the healthy controls iPSC CO from 23 days to 3 months on the CCO activity (chi-square (2, N = 10) = 20, p = 4.5400e-05), and spectral exponent between 30-50 Hz (chi-square (2, N = 16) = 13.88, p = 0.001). Also, a significant effect of drugs, choline (CHO), idebenone (IDB), R-alpha-lipoic acid plus acetyl-L-carnitine (LCLA), was found on the CCO activity (chi-square (3, N = 10) = 25.44, p = 1.2492e-05), spectral exponent between 1 and 20 Hz (chi-square (3, N = 16) = 43.5, p = 1.9273e-09) and 30-50 Hz (chi-square (3, N = 16) = 23.47, p = 3.2148e-05) in 34 days old CO from schizophrenia (SCZ) patients iPSC. We present the feasibility of a multimodal approach, combining electrophysiology and broadband Vis-NIR spectroscopy, to monitor neurodevelopment in brain organoid models that can complement traditional drug design approaches to test clinically meaningful hypotheses.
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Breakthrough Moments: Yoshiki Sasai's Discoveries in the Third Dimension.
Yoshiki Sasai pioneered the organoid field with his idea of mimicking embryonic development in 3D. We shine a spotlight on his seminal work describing how the innate ability of embryonic stem cells to self-organize into layers and grow in a polarized fashion fosters their appropriate differentiation and response to morphogens.
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Temporal Control of Mammalian Cortical Neurogenesis by m6A Methylation.
N6-methyladenosine (m6A), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether m6A regulates mammalian brain development is unknown. Here, we show that m6A depletion by Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages. m6A depletion by Mettl3 knockdown also leads to a prolonged cell cycle and maintenance of radial glia cells. m6A sequencing of embryonic mouse cortex reveals enrichment of mRNAs related to transcription factors, neurogenesis, the cell cycle, and neuronal differentiation, and m6A tagging promotes their decay. Further analysis uncovers previously unappreciated transcriptional prepatterning in cortical neural stem cells. m6A signaling also regulates human cortical neurogenesis in forebrain organoids. Comparison of m6A-mRNA landscapes between mouse and human cortical neurogenesis reveals enrichment of human-specific m6A tagging of transcripts related to brain-disorder risk genes. Our study identifies an epitranscriptomic mechanism in heightened transcriptional coordination during mammalian cortical neurogenesis.
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Single molecule nanopore counting assay targeting small extracellular vesicle cargo for non-invasive monitoring of cerebral organoid development and health.
Organoids are three-dimensional tissue cultures intended to replicate in vivo organs such that their function can be analyzed for applications in drug discovery, diagnostics, and research. This requires the ability to assess organoid health, development, and function on the cellular and molecular level, possibly frequently and over long periods of time. Here, we report an assay for monitoring organoid development and health by tracking the molecular cargo of small extracellular vesicles (sEV) with an integrated nanopore sensor chip. Specifically, we implement amplification-free and label-free quantification of the organoid stress marker ENO1 produced by cerebral organoid tissue. We demonstrate that mRNA levels measured non-invasively in sEVs are representative of the amounts measured by PCR measurements of the tissue cells. We also quantify the ENO1 RNA load in sEVs over the course of 15 weeks and show that ENO1 expression levels are correlated with other physiological parameters such as organoid glucose consumption. These results illustrate the capability of single molecule nanopore sensors for providing simple, continuous, quantitative assessment of organoids' phenotypes on the molecular level. This approach can be expanded to other molecular biomarkers such as protein transcripts, multiplexed analysis, and fully integrated in-line analysis in an automated tissue culture platform.
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Protocol for the establishment and morphological characterization of long-term cultivated murine cerebral organoids.
Murine cerebral organoids provide a rapid and reproducible in vitro system that recapitulates key aspects of neurogenesis. While human cerebral organoid protocols are well established, methods for non-human models remain limited. Here, we present a protocol for generating long-term cultured murine cerebral organoids from E14.5 embryonic stem cells (ESCs). We describe steps for generating mature organoids, followed by histological processing including paraffin embedding and microtome sectioning. We then detail procedures for characterizing murine cerebral organoids through H&E staining and immunofluorescence techniques.
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OLIG2 Drives Abnormal Neurodevelopmental Phenotypes in Human iPSC-Based Organoid and Chimeric Mouse Models of Down Syndrome.
Down syndrome (DS) is a common neurodevelopmental disorder, and cognitive defects in DS patients may arise from imbalances in excitatory and inhibitory neurotransmission. Understanding the mechanisms underlying such imbalances may provide opportunities for therapeutic intervention. Here, we show that human induced pluripotent stem cells (hiPSCs) derived from DS patients overproduce OLIG2+ ventral forebrain neural progenitors. As a result, DS hiPSC-derived cerebral organoids excessively produce specific subclasses of GABAergic interneurons and cause impaired recognition memory in neuronal chimeric mice. Increased OLIG2 expression in DS cells directly upregulates interneuron lineage-determining transcription factors. shRNA-mediated knockdown of OLIG2 largely reverses abnormal gene expression in early-stage DS neural progenitors, reduces interneuron production in DS organoids and chimeric mouse brains, and improves behavioral deficits in DS chimeric mice. Thus, altered OLIG2 expression may underlie neurodevelopmental abnormalities and cognitive defects in DS patients.
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Electrodeposition-Optimized PEDOT Interfaces on Printed Circuit Boards for Stable, Low-Impedance Organoid Electrophysiology.
Reliabl electrophysiological sensing in brain organoids is limited by the high interfacial impedance and noise of microscale electrodes, particularly when scalable and reusable platforms are required. In this work, we introduce PEDOT-coated printed circuit board (PCB) electrodes as a biosensing platform for low-noise recording of organoid electrical activity. Conducting polymer coatings were electrodeposited directly onto PCB-integrated electrodes and systematically optimized to maximize interfacial capacitance while preserving coating adhesion and durability. The optimized PEDOT interfaces reduced electrode impedance to 3-5 kΩ at 1 kHz, corresponding to about three-order-of-magnitude decrease compared to Au-coated electrodes. The low-impedance response was retained after repeated autoclave sterilization and sonication, demonstrating robustness under conditions relevant to routine biological use. When applied to human brain organoids, the PEDOT-coated electrodes exhibited markedly reduced background noise and significantly enhanced signal-to-noise ratios (SNR), enabling reliable detection of extracellular spikes and synchronized burst activity across multiple channels. Relative to gold electrodes, the PEDOT-modified PCB platform recorded higher-amplitude signals and increased spike counts, indicating improved electrode-tissue coupling. These results establish PEDOT-coated PCB electrodes as a scalable and reusable biosensing interface for electrophysiological interrogation of 3D neural tissues.