3D Brain Organoids Research Articles

A human-derived 3D brain organoid model to study JC virus infection.

Progressive multifocal leukoencephalopathy (PML) is a frequent neurological complication in immunosuppressed patients. PML is caused by the JC virus (JCV), a neurotropic DNA polyomavirus that infects oligodendrocytes and astrocytes, causing inflammation and demyelination which lead to neurological dysfunction. The pathogenesis of PML is poorly understood due to the lack of in vitro or animal models to study mechanisms of disease as the virus most efficiently infects only human cells. We developed a human-derived brain organotypic system (also called brain organoid) to model JCV infection. The model was developed by using human-induced pluripotent stem cells (iPSC) and culturing them in 3D to generate an organotypic model containing neurons, astrocytes, and oligodendrocytes which recapitulates aspects of the environment of the human brain. We infected the brain organoids with the JCV MAD4 strain or cerebrospinal fluid of a patient with PML. The organoids were assessed for evidence of infection by qPCR, immunofluorescence, and electron microscopy at 1, 2, and 3 weeks post-exposure. JCV infection in both JCV MAD4 strain and PML CSF-exposed brain organoids was confirmed by immunocytochemical studies demonstrating viral antigens and electron microscopy showing virion particles in the nuclear compartment of oligodendrocytes and astrocytes. No evidence of neuronal infection was visualized. Infection was also demonstrated by JCV qPCR in the virus-exposed organoids and their media. In conclusion, the brain organoid model of JCV infection establishes a human model suitable for studying the mechanisms of JCV infection and pathogenesis of PML and may facilitate the exploration of therapeutic approaches.

Central nervous system organoids for modeling neurodegenerative diseases.

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed researchers to generate neurodegenerative disease-specific iPSCs and use the cells to derive a variety of relevant cell populations for laboratory modeling and drug testing. Nevertheless, these efforts have faced challenges related to immaturity and lack of complex developmental niches in the derived cell populations, limiting the utility of these in vitro models of neurodegenerative disease. Such limitations may be overcome by using human iPSC technology to generate three-dimensional (3D) brain organoids, which better recapitulate in vivo tissue architecture than traditional neuronal cultures to provide more complex and representative disease models and drug testing systems. In this review, we focus on the application of pluripotent stem cell-derived central nervous system (CNS) organoids to model neurodegenerative diseases. We first summarize recent progress in generating and characterizing various CNS organoids from pluripotent stem cells. We then review the application of CNS organoids for modeling several different human neurodegenerative diseases. We also describe several novel pathological mechanisms and drugs that were studied using patient iPSC-derived CNS organoids. Finally, we discuss remaining challenges and emerging opportunities for the use of 3D brain organoids for in vitro modeling of CNS development and neurodegeneration.

Reaching into the toolbox: Stem cell models to study neuropsychiatric disorders.

SummaryRecent advances in genetics, molecular biology, and stem cell biology have accelerated our understanding of neuropsychiatric disorders, like autism spectrum disorder (ASD), major depressive disorder (MDD), bipolar disorder (BD), and schizophrenia (SZ). This progress highlights the incredible complexity of both the human brain and mental illnesses from the biochemical to the cellular level. Contributing to the complexity of neuropsychiatric disorders are their polygenic nature, cellular and brain region interconnectivity, and dysregulation of human-specific neurodevelopmental processes. Here, we discuss available tools, including CRISPR-Cas9, and the applications of these tools to develop cell-based two-dimensional (2D) models and 3D brain organoid models that better represent and unravel the intricacies of neuropsychiatric disorder pathophysiology.

Generation of iPSC-Derived Brain Organoids for Drug Testing and Toxicological Evaluation.

The road to discover novel therapeutics for mental and neurological disorders has been severely hampered by the lack of access to relevant testing platforms. Currently, roughly 0.1% of drugs that show promise in preclinical testing make it to Phase I clinical trials, and 90% of those drugs go on to fail FDA approval. One of the reasons responsible for this low success rate is that conventional two-dimensional (2D) cell culture models are not accurate enough predictors of how drugs will work in humans. Three-dimensional (3D) brain organoids differentiated from induced pluripotent stem cells (iPSCs) to resemble specific parts of the human brain, which include architecture composition and physiology, can provide an alternative system that may lead to breakthroughs in key areas of drug testing and toxicological evaluation. Having reliable and scalable iPSC-derived brain organoid models that can much more accurately predict human drug responses will significantly increase success rate in developing treatments for brain-related disorders.

Transcriptomic Mapping of Neural Diversity, Differentiation and Functional Trajectory in iPSC-Derived 3D Brain Organoid Models.

Experimental models of the central nervous system (CNS) are imperative for developmental and pathophysiological studies of neurological diseases. Among these models, three-dimensional (3D) induced pluripotent stem cell (iPSC)-derived brain organoid models have been successful in mitigating some of the drawbacks of 2D models; however, they are plagued by high organoid-to-organoid variability, making it difficult to compare specific gene regulatory pathways across 3D organoids with those of the native brain. Single-cell RNA sequencing (scRNA-seq) transcriptome datasets have recently emerged as powerful tools to perform integrative analyses and compare variability across organoids. However, transcriptome studies focusing on late-stage neural functionality development have been underexplored. Here, we combine and analyze 8 brain organoid transcriptome databases to study the correlation between differentiation protocols and their resulting cellular functionality across various 3D organoid and exogenous brain models. We utilize dimensionality reduction methods including principal component analysis (PCA) and uniform manifold approximation projection (UMAP) to identify and visualize cellular diversity among 3D models and subsequently use gene set enrichment analysis (GSEA) and developmental trajectory inference to quantify neuronal behaviors such as axon guidance, synapse transmission and action potential. We showed high similarity in cellular composition, cellular differentiation pathways and expression of functional genes in human brain organoids during induction and differentiation phases, i.e., up to 3 months in culture. However, during the maturation phase, i.e., 6-month timepoint, we observed significant developmental deficits and depletion of neuronal and astrocytes functional genes as indicated by our GSEA results. Our results caution against use of organoids to model pathophysiology and drug response at this advanced time point and provide insights to tune in vitro iPSC differentiation protocols to achieve desired neuronal functionality and improve current protocols.

The cytotoxicity of zinc oxide nanoparticles to 3D brain organoids results from excessive intracellular zinc ions and defective autophagy.

Although the neurotoxicity of ZnO nanoparticles (NPs) has been evaluated in animal and nerve cell culture models, these models cannot accurately mimic human brains. Three-dimensional (3D) brain organoids based on human-induced pluripotent stem cells have been developed to study the human brains, but this model has rarely been used to evaluate NP neurotoxicity. We used 3D brain organoids that express cortical layer proteins to investigate the mechanisms of ZnO NP-induced neurotoxicity. Cytotoxicity caused by high levels of ZnO NPs (64μg/mL) correlated with high intracellular Zn ion levels but not superoxide levels. Exposure to a non-cytotoxic concentration of ZnO NPs (16μg/mL) increased the autophagy-marker proteins LC3B-II/I but decreased p62 accumulation, whereas a cytotoxic concentration of ZnO NPs (64μg/mL) decreased LC3B-II/I proteins but did not affect p62 accumulation. Fluorescence micro-optical sectioning tomography revealed that 64μg/mL ZnO NPs led to decreases in LC3B proteins that were more obvious at the outer layers of the organoids, which were directly exposed to the ZnO NPs. In addition to reducing LC3B proteins in the outer layers, ZnO NPs increased the number of micronuclei in the outer layers but not the inner layers (where LC3B proteins were still expressed). Adding the autophagy flux inhibitor bafilomycin A1 to ZnO NPs increased cytotoxicity and intracellular Zn ion levels, but adding the autophagy inducer rapamycin only slightly decreased cellular Zn ion levels. We conclude that high concentrations of ZnO NPs are cytotoxic to 3D brain organoids via defective autophagy and intracellular accumulation of Zn ions.

Generating Cerebral Organoids from Human Pluripotent Stem Cells.

3D brain organoids derived from human pluripotent stem cells (hPSCs) possess the remarkable ability to self-organize and differentiate into tissue resembling the early human fetal brain. Brain organoids provide a powerful tool for studying human brain development and disease in an in vitro system. Here we describe a protocol for the differentiation of hPSCs to human cerebral organoids using a commercially available kit (STEMdiff™ Cerebral Organoid Kit) and discuss methods to scale up the protocol in a high-throughput manner.

Human IPSC-Derived Model to Study Myelin Disruption.

Myelin is of vital importance to the central nervous system and its disruption is related to a large number of both neurodevelopmental and neurodegenerative diseases. The differences observed between human and rodent oligodendrocytes make animals inadequate for modeling these diseases. Although developing human in vitro models for oligodendrocytes and myelinated axons has been a great challenge, 3D cell cultures derived from iPSC are now available and able to partially reproduce the myelination process. We have previously developed a human iPSC-derived 3D brain organoid model (also called BrainSpheres) that contains a high percentage of myelinated axons and is highly reproducible. Here, we have further refined this technology by applying multiple readouts to study myelination disruption. Myelin was assessed by quantifying immunostaining/confocal microscopy of co-localized myelin basic protein (MBP) with neurofilament proteins as well as proteolipid protein 1 (PLP1). Levels of PLP1 were also assessed by Western blot. We identified compounds capable of inducing developmental neurotoxicity by disrupting myelin in a systematic review to evaluate the relevance of our BrainSphere model for the study of the myelination/demyelination processes. Results demonstrated that the positive reference compound (cuprizone) and two of the three potential myelin disruptors tested (Bisphenol A, Tris(1,3-dichloro-2-propyl) phosphate, but not methyl mercury) decreased myelination, while ibuprofen (negative control) had no effect. Here, we define a methodology that allows quantification of myelin disruption and provides reference compounds for chemical-induced myelin disruption.

Modeling Sporadic Alzheimer's Disease in Human Brain Organoids under Serum Exposure.

Alzheimer's disease (AD) is a progressive neurodegenerative disease with no cure. Huge efforts have been made to develop anti‐AD drugs in the past decades. However, all drug development programs for disease‐modifying therapies have failed. Possible reasons for the high failure rate include incomplete understanding of complex pathophysiology of AD, especially sporadic AD (sAD), and species difference between humans and animal models used in preclinical studies. In this study, sAD is modeled using human induced pluripotent stem cell (hiPSC)‐derived 3D brain organoids. Because the blood–brain barrier (BBB) leakage is a well‐known risk factor for AD, brain organoids are exposed to human serum to mimic the serum exposure consequence of BBB breakdown in AD patient brains. The serum‐exposed brain organoids are able to recapitulate AD‐like pathologies, including increased amyloid beta (Aβ) aggregates and phosphorylated microtubule‐associated tau protein (p‐Tau) level, synaptic loss, and impaired neural network. Serum exposure increases Aβ and p‐Tau levels through inducing beta‐secretase 1 (BACE) and glycogen synthase kinase‐3 alpha / beta (GSK3α/β) levels, respectively. In addition, single‐cell transcriptomic analysis of brain organoids reveals that serum exposure reduced synaptic function in both neurons and astrocytes and induced immune response in astrocytes. The human brain organoid‐based sAD model established in this study can provide a powerful platform for both mechanistic study and therapeutic development in the future.

Stem cell-based 3D brain organoids for mimicking, investigating, and challenging Alzheimer's diseases.

Stem cell-based 3D brain organoids for mimicking, investigating, and challenging Alzheimer's diseases.

Gene-Environment Interactions in Developmental Neurotoxicity: a Case Study of Synergy between Chlorpyrifos and CHD8 Knockout in Human BrainSpheres.

Background:Autism spectrum disorder (ASD) is a major public health concern caused by complex genetic and environmental components. Mechanisms of gene–environment () interactions and reliable biomarkers associated with ASD are mostly unknown or controversial. Induced pluripotent stem cells (iPSCs) from patients or with clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9)-introduced mutations in candidate ASD genes provide an opportunity to study () interactions.Objectives:In this study, we aimed to identify a potential synergy between mutation in the high-risk autism gene encoding chromodomain helicase DNA binding protein 8 (CHD8) and environmental exposure to an organophosphate pesticide (chlorpyrifos; CPF) in an iPSC-derived human three-dimensional (3D) brain model.Methods:This study employed human iPSC-derived 3D brain organoids (BrainSpheres) carrying a heterozygote CRISPR/Cas9-introduced inactivating mutation in CHD8 and exposed to CPF or its oxon-metabolite (CPO). Neural differentiation, viability, oxidative stress, and neurite outgrowth were assessed, and levels of main neurotransmitters and selected metabolites were validated against human data on ASD metabolic derangements.Results:Expression of CHD8 protein was significantly lower in CHD8 heterozygous knockout () BrainSpheres compared with ones. Exposure to CPF/CPO treatment further reduced CHD8 protein levels, showing the potential () interaction synergy. A novel approach for validation of the model was chosen: from the literature, we identified a panel of metabolic biomarkers in patients and assessed them by targeted metabolomics in vitro. A synergistic effect was observed on the cholinergic system, S-adenosylmethionine, S-adenosylhomocysteine, lactic acid, tryptophan, kynurenic acid, and acid levels. Neurite outgrowth was perturbed by CPF/CPO exposure. Heterozygous knockout of CHD8 in BrainSpheres led to an imbalance of excitatory/inhibitory neurotransmitters and lower levels of dopamine.Discussion:This study pioneered () interaction in iPSC-derived organoids. The experimental strategy enables biomonitoring and environmental risk assessment for ASD. Our findings reflected some metabolic perturbations and disruption of neurotransmitter systems involved in ASD. The increased susceptibility of BrainSpheres to chemical insult establishes a possibly broader role of () interaction in ASD. https://doi.org/10.1289/EHP8580

Generation of Human Brain Organoids for Mitochondrial Disease Modeling.

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

Modeling Rett Syndrome with Human Pluripotent Stem Cells: Mechanistic Outcomes and Future Clinical Perspectives.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Among many different roles, MeCP2 has a high phenotypic impact during the different stages of brain development. Thus, it is essential to intensively investigate the function of MeCP2, and its regulated targets, to better understand the mechanisms of the disease and inspire the development of possible therapeutic strategies. Several animal models have greatly contributed to these studies, but more recently human pluripotent stem cells (hPSCs) have been providing a promising alternative for the study of RTT. The rapid evolution in the field of hPSC culture allowed first the development of 2D-based neuronal differentiation protocols, and more recently the generation of 3D human brain organoid models, a more complex approach that better recapitulates human neurodevelopment in vitro. Modeling RTT using these culture platforms, either with patient-specific human induced pluripotent stem cells (hiPSCs) or genetically-modified hPSCs, has certainly contributed to a better understanding of the onset of RTT and the disease phenotype, ultimately allowing the development of high throughput drugs screening tests for potential clinical translation. In this review, we first provide a brief summary of the main neurological features of RTT and the impact of MeCP2 mutations in the neuropathophysiology of this disease. Then, we provide a thorough revision of the more recent advances and future prospects of RTT modeling with human neural cells derived from hPSCs, obtained using both 2D and organoids culture systems, and its contribution for the current and future clinical trials for RTT.

Evolutionary conservation and divergence of the human brain transcriptome

BackgroundMouse models have allowed for the direct interrogation of genetic effects on molecular, physiological, and behavioral brain phenotypes. However, it is unknown to what extent neurological or psychiatric traits may be human- or primate-specific and therefore which components can be faithfully recapitulated in mouse models.ResultsWe compare conservation of co-expression in 116 independent data sets derived from human, mouse, and non-human primate representing more than 15,000 total samples. We observe greater changes occurring on the human lineage than mouse, and substantial regional variation that highlights cerebral cortex as the most diverged region. Glia, notably microglia, astrocytes, and oligodendrocytes are the most divergent cell type, three times more on average than neurons. We show that cis-regulatory sequence divergence explains a significant fraction of co-expression divergence. Moreover, protein coding sequence constraint parallels co-expression conservation, such that genes with loss of function intolerance are enriched in neuronal, rather than glial modules. We identify dozens of human neuropsychiatric and neurodegenerative disease risk genes, such as COMT, PSEN-1, LRRK2, SHANK3, and SNCA, with highly divergent co-expression between mouse and human and show that 3D human brain organoids recapitulate in vivo co-expression modules representing several human cell types.ConclusionsWe identify robust co-expression modules reflecting whole-brain and regional patterns of gene expression. Compared with those that represent basic metabolic processes, cell-type-specific modules, most prominently glial modules, are the most divergent between species. These data and analyses serve as a foundational resource to guide human disease modeling and its interpretation.

Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids

BackgroundThe inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes—in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points.ResultsConventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes.ConclusionWe present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.

Enhanced Neuronal Activity and Asynchronous Calcium Transients Revealed in a 3D Organoid Model of Alzheimer’s Disease

Advances in the development of three-dimensional (3D) brain organoids maintained in vitro have provided excellent opportunities to study brain development and neurodegenerative disorders, including Alzheimer's disease (AD). However, there remains a need to generate AD organoids bearing patient-specific genomic backgrounds that can functionally recapitulate the key features observed in the AD patient's brain. To address this need, we described a strategy to generate self-organizing 3D cerebral organoids which develop a functional neuronal network connectivity. This was achieved by neuroectoderm induction of human pluripotent stem cell (hPSCs) aggregates and subsequent differentiation into desired neuroepithelia and mature neurons in a 3D Matrigel matrix. Using this approach, we successfully generated AD cerebral organoids from human pluripotent stem cells (hPSCs) derived from a familial AD patient with a common mutation in presenilin 2 (PSEN2N141I). An isogenic control with an identical genetic background but wild-type PSEN2 was generated using CRISPR/Cas9 technology. Both control and AD organoids were characterized by analyzing their morphology, the Aβ42/Aβ40 ratio, functional neuronal network activity, drug sensitivity, and the extent of neural apoptosis. The spontaneous activity of the network and its synchronization was measured in the organoids via calcium imaging. We found that compared with the mutation-corrected control organoids, AD organoids had a higher Aβ42/Aβ40 ratio, asynchronous calcium transients, and enhanced neuronal hyperactivity, successfully recapitulating an AD-like pathology at the molecular, cellular, and network level in a human genetic context. Moreover, two drugs which increase neuronal activity, 4-aminopyridine (4-AP) and bicuculline methochloride, induced high-frequency synchronized network bursting to a similar extent in both organoids. Therefore, our study presents a promising organoid-based biosystem for the study of the pathophysiology of AD and a platform for AD drug development.

Human iNSC-derived brain organoid model of lysosomal storage disorder in Niemann\u2013Pick disease type C

Recent studies on developing three-dimensional (3D) brain organoids from stem cells have allowed the generation of in vitro models of neural disease and have enabled the screening of drugs because these organoids mimic the complexity of neural tissue. Niemann-Pick disease, type C (NPC) is a neurodegenerative lysosomal storage disorder caused by mutations in the NPC1 or NPC2. The pathological features underlying NPC are characterized by the abnormal accumulation of cholesterol in acidic compartments, including late endosomes and lysosomes. Due to the inaccessibility of brain tissues from human NPC patients, we developed NPC brain organoids with induced neural stem cells from NPC patient-derived fibroblasts. NPC organoids exhibit significantly reduced size and proliferative ability, which are accompanied by accumulation of cholesterol, impairment in neuronal differentiation, and autophagic flux and dysfunction of lysosomes; therefore, NPC organoids can recapitulate the main phenotypes of NPC patients. Furthermore, these pathological phenotypes observed in NPC organoids were reversed by treatment with valproic acid and HPBCD, which are known to be an effective treatment for several neurodegenerative diseases. Our data present patient-specific phenotypes in 3D organoid-based models of NPC and highlight the application of this model to drug screening in vitro.

Development of a functional 3D brain organoid with microvasculature and a blood‐brain barrier

AbstractBackgroundBrain organoids are self‐assembled 3D cell aggregates developed from pluripotent stem cells with various cell types and extracellular structures that resemble the embryonic human brain. Induced pluripotent stem cells (iPSC) based organoids show great promise in disease modeling applications. Our previous work has validated the use of novel polyphenolic compounds to combat neurodegenerative diseases in animal and traditional cell culture models. The goal of the present study is to continue to explore the development of a functional brain organoid system with microvasculature and a blood‐brain barrier using iPSCs.MethodiPSCs were differentiated into neurons, astrocytes, pericytes, oligodendrocytes, brain microvascular endothelial cells, and other relevant cell types. The organoid was developed using previously published protocols. The blood‐brain barrier was constructed by co‐culturing the different cell types.ResultIn this ongoing study, we demonstrate the development of a more accurate and functional brain organoid system with microvasculature and a blood‐brain barrier using iPSCs.ConclusionOur organoid will serve as a model for neurodegenerative diseases, drug discovery and the screening of novel polyphenolic compounds from dietary flavanols which have been recently associated with reduced risk of developing Alzheimer’s Dementia.

SARS‐CoV‐2 targets neurons of 3D human brain organoids

COVID‐19 pandemic caused by SARS‐CoV‐2 infection is a public health emergency. COVID‐19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS‐CoV‐2 on the central nervous system (CNS). Here, we show that a Düsseldorf isolate of SARS‐CoV‐2 enters 3D human brain organoids within 2 days of exposure. We identified that SARS‐CoV‐2 preferably targets neurons of brain organoids. Imaging neurons of organoids reveal that SARS‐CoV‐2 exposure is associated with altered distribution of Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Our studies, therefore, provide initial insights into the potential neurotoxic effect of SARS‐CoV‐2 and emphasize that brain organoids could model CNS pathologies of COVID‐19.

Challenges in Physiological Phenotyping of hiPSC-Derived Neurons: From 2D Cultures to 3D Brain Organoids.

Neurons derived from human induced pluripotent stem cells (hiPSC-derived neurons) offer novel opportunities for the development of preclinical models of human neurodegenerative diseases (NDDs). Recent advances in the past few years have increased substantially the potential of these techniques and have uncovered new challenges that the field is facing. Here, we outline and discuss challenges related to the functional characterization of hiPSC-derived neurons and propose ways to overcome current difficulties. In particular, the enormous variability among studies in the electrical properties of hiPSC-derived neurons and broad differences in cell maturation are factors that impair reproducibility. Furthermore, we discuss how the use of 3D brain organoids are of help in resolving some difficulties posed by 2D cultures. Finally, we elaborate on recent and future advances that may help to overcome the discussed challenges and speed-up progress in the field.