Exposure to PFOA, PFOS, and PFHxS Induces Alzheimer's Disease-like Neuropathology in Cerebral Organoids.

Per- and polyfluoroalkyl substances (PFASs), a class of ubiquitous synthetic organic chemicals, are widely utilized across various industrial applications. However, the long-term neurological health effects of PFAS mixture exposure in humans remain poorly understood. To address this gap, we have designed a comprehensive study to predict and validate cell-type-specific neurotoxicity of PFASs using single-cell RNA sequencing (scRNA-seq) and cerebral organoids. Cerebral organoids were exposed to a PFAS mixture at concentrations of 1× (10 ng/ml PFOS and PFOA, and 1 ng/ml PFHxS), 30×, and 900× over 35 days, with a follow-up analysis at day 70. Pathological alterations and lipidomic profiles were analyzed to identify disrupted molecular pathways and mechanisms. The scRNA-seq data revealed a significant impact of PFASs on neurons, suggesting a potential role in Alzheimer's Disease (AD) pathology, as well as intellectual and cognitive impairments. PFAS-treated cerebral organoids exhibited Aß accumulation and tau phosphorylation. Lipidomic analyses further revealed lipid disturbances in response to PFAS mixture exposure, linking PFAS-induced AD-like neuropathology to sphingolipid metabolism disruption. Collectively, our findings provide novel insights into the PFAS-induced neurotoxicity, highlighting the significance of sphingolipid metabolism in the development of AD-like neuropathology. The use of cerebral organoids and scRNA-seq offers a powerful methodology for evaluating the health risks associated with environmental contaminants, particularly those with neurotoxic potential.

The WFS1-ZnT3-Zn2+ Axis Regulates the Vicious Cycle of Obesity and Depression.

Obesity, a growing global health concern, is closely linked to depression. However, the neural mechanism of association between obesity and depression remains poorly understood. In this study, neural-specific WFS1 deficiency exacerbates the vicious cycle of obesity and depression in mice fed a high-fat diet (HFD), positioning WFS1 as a crucial factor in this cycle. Through human pluripotent stem cells (hESCs) neural differentiation, it is demonstrated that WFS1 regulates Zn2+ homeostasis and the apoptosis of neural progenitor cells (NPCs) and cerebral organoids by inhibiting the zinc transporter ZnT3 under the situation of dysregulated lipid metabolism. Notably, riluzole regulates ZnT3 expression to maintain zinc homeostasis and protect NPCs from lipotoxicity-induced cell death. Importantly, riluzole, a therapeutic molecule targeting the nervous system, in vivo administration prevents HFD-induced obesity and associated depression. Thus, a WFS1-ZnT3-Zn2+ axis critical is demonstrated for the vicious cycle of obesity and depression and that riluzole may have the potential to reverse this process against obesity and depression.

Molecular and network disruptions in neurodevelopment uncovered by single cell transcriptomics analysis of CHD8 heterozygous cerebral organoids.

More than 100 genes have been associated with significantly increased risks of autism spectrum disorders (ASD) with an estimate of ∼1000 genes that may contribute. The new challenge is to investigate the molecular and cellular functions of these genes during neural and brain development, and then even more challenging, to link the altered molecular and cellular phenotypes to the ASD clinical manifestations. In this study, we used single-cell RNA-seq analysis to study one of the top risk genes, CHD8, in cerebral organoids, which models early neural development. We identified 21 cell clusters in the organoid samples, representing non-neuronal cells, neural progenitors, and early differentiating neurons at the start of neural cell fate commitment. Comparisons of the cells with one copy of a CHD8 knockout allele, generated by CRISPR/Cas9 editing, and their isogenic controls uncovered thousands of differentially expressed genes, which were enriched with functions related to neural and brain development, cilium organization, and extracellular matrix organization. The affected genes were also enriched with genes and pathways previously implicated in ASD, but surprisingly not for schizophrenia and intellectual disability risk genes. The comparisons also uncovered cell composition changes, indicating potentially altered neural differential trajectories upon CHD8 reduction. Moreover, we found that cell-cell communications were affected in the CHD8 knockout organoids, including the interactions between neural and glial cells. Taken together, our results provide new data and information for understanding CHD8 functions in the early stages of neural lineage development and interaction.

3D Spheroid and Organoid Models to Study Neuroinfection of RNA Viruses.

Three-dimensional culture models of the brain enable the study of neuroinfection in the context of a complex interconnected cell matrix. Depending on the differentiation status of the neural cells, two models exist: 3D spheroids also called neurospheres and cerebral organoids. Here, we describe the preparation of 3D spheroids and cerebral organoids and give an outlook on their usage to study Rift Valley fever virus and other neurotropic viruses.

Generation of Alzheimer's Disease Model Derived from Induced Pluripotent Stem Cells with APP Gene Mutation.

Alzheimer's disease (AD), the most common cause of dementia, is characterized by disruptions in memory, cognition, and personality, significantly impacting morbidity and mortality rates among older adults. However, the exact pathophysiological mechanism of AD remains unknown, and effective treatment options for AD are still lacking. Human induced pluripotent stem cells (iPSC) are emerging as promising platforms for disease research, offering the ability to model the genetic mutations associated with various conditions. Patient-derived iPSCs are useful for modeling neurodegenerative and neurodevelopmental disorders. In this study, we generated AD iPSCs from peripheral blood mononuclear cells obtained from a 65-year-old patient with AD carrying the E682K mutation in the gene encoding the amyloid precursor protein. Cerebral organoids derived from AD iPSCs recapitulated the AD phenotype, exhibiting significantly increased levels of tau protein. Our analysis revealed that an iPSC disease model of AD is a valuable assessment tool for pathophysiological research and drug screening.

A microglia-containing cerebral organoid model to study early life immune challenges.

Prenatal infections and activation of the maternal immune system have been proposed to contribute to causing neurodevelopmental disorders (NDDs), chronic conditions often linked to brain abnormalities. Microglia are the resident immune cells of the brain and play a key role in neurodevelopment. Disruption of microglial functions can lead to brain abnormalities and increase the risk of developing NDDs. How the maternal as well as the fetal immune system affect human neurodevelopment and contribute to NDDs remains unclear. An important reason for this knowledge gap is the fact that the impact of exposure to prenatal risk factors has been challenging to study in the human context. Here, we characterized a model of cerebral organoids (CO) with integrated microglia (COiMg). These organoids express typical microglial markers and respond to inflammatory stimuli. The presence of microglia influences cerebral organoid development, including cell density and neural differentiation, and regulates the expression of several ciliated mesenchymal cell markers. Moreover, COiMg and organoids without microglia show similar but also distinct responses to inflammatory stimuli. Additionally, IFN-γ induced significant transcriptional and structural changes in the cerebral organoids, that appear to be regulated by the presence of microglia. Specifically, interferon-gamma (IFN-γ) was found to alter the expression of genes linked to autism. This model provides a valuable tool to study how inflammatory perturbations and microglial presence affect neurodevelopmental processes.

Trans-omic profiling uncovers molecular controls of early human cerebral organoid formation.

Defining the molecular networks orchestrating human brain formation is crucial for understanding neurodevelopment and neurological disorders. Challenges in acquiring early brain tissue have incentivized the use of three-dimensional human pluripotent stem cell (hPSC)-derived neural organoids to recapitulate neurodevelopment. To elucidate the molecular programs that drive this highly dynamic process, here, we generate a comprehensive trans-omic map of the phosphoproteome, proteome, and transcriptome of the exit of pluripotency and neural differentiation toward human cerebral organoids (hCOs). These data reveal key phospho-signaling events and their convergence on transcriptional factors to regulate hCO formation. Comparative analysis with developing human and mouse embryos demonstrates the fidelity of our hCOs in modeling embryonic brain development. Finally, we demonstrate that biochemical modulation of AKT signaling can control hCO differentiation. Together, our data provide a comprehensive resource to study molecular controls in human embryonic brain development and provide a guide for the future development of hCO differentiation protocols.

Lack of Transmission of Chronic Wasting Disease Prions to Human Cerebral Organoids.

Chronic wasting disease (CWD) is a cervid prion disease with unknown zoonotic potential that might pose a risk to humans who are exposed. To assess the potential of CWD to infect human neural tissue, we used human cerebral organoids with 2 different prion genotypes, 1 of which has previously been associated with susceptibility to zoonotic prion disease. We exposed organoids from both genotypes to high concentrations of CWD inocula from 3 different sources for 7 days, then screened for infection periodically for up to 180 days. No de novo CWD propagation or deposition of protease-resistant forms of human prions was evident in CWD-exposed organoids. Some persistence of the original inoculum was detected, which was equivalent in prion gene knockout organoids and thus not attributable to human prion propagation. Overall, the unsuccessful propagation of CWD in cerebral organoids supports a strong species barrier to transmission of CWD prions to humans.

Isolation and Characterization of Cell-Free DNA from Cerebral Organoids.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and poses a high risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. In particular, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, wherein many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed associations with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.

Brain organoid protocols and limitations.

Stem cell-derived organoid technology is a powerful tool that revolutionizes the field of biomedical research and extends the scope of our understanding of human biology and diseases. Brain organoids especially open an opportunity for human brain research and modeling many human neurological diseases, which have lagged due to the inaccessibility of human brain samples and lack of similarity with other animal models. Brain organoids can be generated through various protocols and mimic whole brain or region-specific. To provide an overview of brain organoid technology, we summarize currently available protocols and list several factors to consider before choosing protocols. We also outline the limitations of current protocols and challenges that need to be solved in future investigation of brain development and pathobiology.

Weighing the moral status of brain organoids and research animals.

Recent advances in human brain organoid systems have raised serious worries about the possibility that these in vitro 'mini-brains' could develop sentience, and thus, moral status. This article considers the relative moral status of sentient human brain organoids and research animals, examining whether we have moral reasons to prefer using one over the other. It argues that, contrary to common intuitions, the wellbeing of sentient human brain organoids should not be granted greater moral consideration than the wellbeing of nonhuman research animals. It does so not by denying that typical humans have higher moral status than animals, but instead by arguing that none of the leading justifications for granting humans higher moral status than nonhuman animals apply to brain organoids. Additionally, it argues that there are no good reasons to be more concerned about the well-being of human brain organoids compared to those generated from other species.

Applications of multiphoton microscopy in imaging cerebral and retinal organoids.

Cerebral organoids, self-organizing structures with increased cellular diversity and longevity, have addressed shortcomings in mimicking human brain complexity and architecture. However, imaging intact organoids poses challenges due to size, cellular density, and light-scattering properties. Traditional one-photon microscopy faces limitations in resolution and contrast, especially for deep regions. Here, we first discuss the fundamentals of multiphoton microscopy (MPM) as a promising alternative, leveraging non-linear fluorophore excitation and longer wavelengths for improved imaging of live cerebral organoids. Then, we review recent applications of MPM in studying morphogenesis and differentiation, emphasizing its potential for overcoming limitations associated with other imaging techniques. Furthermore, our paper underscores the crucial role of cerebral organoids in providing insights into human-specific neurodevelopmental processes and neurological disorders, addressing the scarcity of human brain tissue for translational neuroscience. Ultimately, we envision using multimodal multiphoton microscopy for longitudinal imaging of intact cerebral organoids, propelling advancements in our understanding of neurodevelopment and related disorders.

Efficient generation of human cerebral organoids directly from adherent cultures of pluripotent stem cells.

Human cerebral organoids (hCOs) offer the possibility of deepening the knowledge of human brain development, as well as the pathologies that affect it. The method developed here describes the efficient generation of hCOs by going directly from two-dimensional (2D) pluripotent stem cell (PSC) cultures to three-dimensional (3D) neuroepithelial tissue, avoiding dissociation and aggregation steps. This has been achieved by subjecting 2D cultures, from the beginning of the neural induction step, to dual-SMAD inhibition in combination with CHIR99021. This is a simple and reproducible protocol in which the hCOs generated develop properly presenting proliferative ventricular zones (VZs) formed by neural precursor and radial glia (RG) that differentiate to give rise to mature neurons and glial cells. The hCOs present additional cell types such as oligodendrocyte precursors, astrocytes, microglia-like cells, and endothelial-like cells. This new approach could help to overcome some of the existing limitations in the field of organoid biotechnology, facilitating its execution in any laboratory setting.

Uniform cerebral organoid culture on a pillar plate by simple and reproducible spheroid transfer from an ultralow attachment well plate.

Human induced pluripotent stem cell (iPSC)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effectivein vitromodel for studying both normal brain development and disorders. However, current brain organoid culture methods face several challenges, including low throughput, high variability in organoid generation, and time-consuming, multiple transfer and encapsulation of cells in hydrogels throughout the culture. These limitations hinder the widespread application of brain organoids including high-throughput assessment of compounds in clinical and industrial lab settings. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies in an ultra-low attachment 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95%-100%. Using this approach, we robustly generated cerebral organoids on the pillar plate and demonstrated an intra-batch coefficient of variation below 9%-19% based on ATP-based cell viability and compound treatment. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowingin situorganoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

Transcriptomic and morphological maturation of human astrocytes in cerebral organoids.

Cerebral organoids (CerOrgs) derived from human induced pluripotent stem cells (iPSCs) are a valuable tool to study human astrocytes and their interaction with neurons and microglia. The timeline of astrocyte development and maturation in this model is currently unknown and this limits the value and applicability of the model. Therefore, we generated CerOrgs from three healthy individuals and assessed astrocyte maturation after 5, 11, 19, and 37 weeks in culture. At these four time points, the astrocyte lineage was isolated based on the expression of integrin subunit alpha 6 (ITGA6). Based on the transcriptome of the isolated ITGA6-positive cells, astrocyte development started between 5 and 11 weeks in culture and astrocyte maturation commenced after 11 weeks in culture. After 19 weeks in culture, the ITGA6-positive astrocytes had the highest expression of human mature astrocyte genes, and the predicted functional properties were related to brain homeostasis. After 37 weeks in culture, a subpopulation of ITGA6-negative astrocytes appeared, highlighting the heterogeneity within the astrocytes. The morphology shifted from an elongated progenitor-like morphology to the typical bushy astrocyte morphology. Based on the morphological properties, predicted functional properties, and the similarities with the human mature astrocyte transcriptome, we concluded that ITGA6-positive astrocytes have developed optimally in 19-week-old CerOrgs.

Neural cell engraftment therapy for sporadic Creutzfeldt-Jakob disease restores neuroelectrophysiological parameters in a cerebral organoid model.

BACKGROUND: Sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is a fatal neurodegenerative disease with currently no treatment options. Stem cell therapy for neurodegenerative diseases is emerging as a possible treatment option. However, while there are a few clinical trials for other neurodegenerative disorders such as Parkinson's disease, prion disease cell therapy research has so far been confined to animal models. METHODS: Here, we use a novel approach to study cell therapies in sCJD using a human cerebral organoid model. Cerebral organoids can be infected with sCJD prions allowing us to assess how neural precursor cell (NPC) therapy impacts the progression of sCJD. After 90 days of sCJD or mock infection, organoids were either seeded with NPCs or left unseeded and monitored for cellular composition changes, prion infection parameters and neuroelectrophysiological function at 180 days post-infection. RESULTS: Our results showed NPCs integrated into organoids leading to an increase in neuronal markers and changes in cell signaling irrespective of sCJD infection. Although a small, but significant, decrease in protease-resistant PrP deposition was observed in the CJD-infected organoids that received the NPCs, other disease-associated parameters showed minimal changes. However, the NPCs had a beneficial impact on organoid function following infection. sCJD infection caused reduction in neuronal spike rate and mean burst spike rate, indicative of reduced action potentials. NPC seeding restored these electrophysiological parameters to the uninfected control level. CONCLUSIONS: Together with the previous animal studies, our results support that cell therapy may have some functional benefit for the treatment of human prion diseases.

Molecular and network disruptions in neurodevelopment uncovered by single cell transcriptomics analysis of CHD8 heterozygous cerebral organoids.

About 100 genes have been associated with significantly increased risks of autism spectrum disorders (ASD) with an estimate of ~1000 genes that may be involved. The new challenge now is to investigate the molecular and cellular functions of these genes during neural and brain development, and then even more challenging, to link the altered molecular and cellular phenotypes to the ASD clinical manifestations. In this study, we use single cell RNA-seq analysis to study one of the top risk gene, CHD8, in cerebral organoids, which models early neural development. We identify 21 cell clusters in the organoid samples, representing non-neuronal cells, neural progenitors, and early differentiating neurons at the start of neural cell fate commitment. Comparisons of the cells with one copy of the CHD8 knockout and their isogenic controls uncover thousands of differentially expressed genes, which are enriched with function related to neural and brain development, with genes and pathways previously implicated in ASD, but surprisingly not for Schizophrenia and intellectual disability risk genes. The comparisons also find cell composition changes, indicating potential altered neural differential trajectories upon CHD8 reduction. Moreover, we find that cell-cell communications are affected in the CHD8 knockout organoids, including the interactions between neural and glial cells. Taken together, our results provide new data for understanding CHD8 functions in the early stages of neural lineage development and interaction.

Volumetric compression by heterogeneous scaffold embedding promotes cerebral organoid maturation and does not impede growth.

Although biochemical regulation has been extensively studied in organoid modeling protocols, the role of mechanoregulation in directing stem cell fate and organoid development has been relatively unexplored. To accurately replicate the dynamic organoid development observed in nature, in this study, we present a method of heterogeneous embedding using an alginate-shell-Matrigel-core system. This approach allows for cell-Matrigel remodeling by the inner layer and provides short-term moderate-normal compression through the soft alginate outer layer. Our results show that the time-limited confinement contributes to increased expression of neuronal markers such as neurofilament (NF) and microtubule-associated protein 2 (MAP2). Compared with non-alginate embedding and alginate compression groups, volume growth remains unimpeded. Our findings demonstrate the temporary mechanical regulation of cerebral organoid growth, which exhibits a regular growth profile with enhanced maturation. These results highlight the importance and potential practical applications of mechanoregulation in the establishment of brain organoids. A record of this paper's transparent peer review process is included in the supplemental information.

Bioactive and chemically defined hydrogels with tunable stiffness guide cerebral organoid formation and modulate multi-omics plasticity in cerebral organoids.

Organoids are an emerging technology with great potential in human disease modelling, drug development, diagnosis, tissue engineering, and regenerative medicine. Organoids as 3D-tissue culture systems have gained special attention in the past decades due to their ability to faithfully recapitulate the complexity of organ-specific tissues. Despite considerable successes in culturing physiologically relevant organoids, their real-life applications are currently limited by challenges such as scarcity of an appropriate biomimetic matrix. Peptide amphiphiles (PAs) due to their well-defined chemistry, tunable bioactivity, and extracellular matrix (ECM)-like nanofibrous architecture represent an attractive material scaffold for organoids development. Using cerebral organoids (COs) as exemplar, we demonstrate the possibility to create bio-instructive hydrogels with tunable stiffness ranging from 0.69 kPa to 2.24 kPa to culture and induce COs growth. We used orthogonal chemistry involving oxidative coupling and supramolecular interactions to create two-component hydrogels integrating the bio-instructive activity and ECM-like nanofibrous architecture of a laminin-mimetic PAs (IKVAV-PA) and tunable crosslinking density of hyaluronic acid functionalized with tyramine (HA-Try). Multi-omics technology including transcriptomics, proteomics, and metabolomics reveals the induction and growth of COs in soft HA-Tyr hydrogels containing PA-IKVAV such that the COs display morphology and biomolecular signatures similar to those grown in Matrigel scaffolds. Our materials hold great promise as a safe synthetic ECM for COs induction and growth. Our approach represents a well-defined alternative to animal-derived matrices for the culture of COs and might expand the applicability of organoids in basic and clinical research. STATEMENT OF SIGNIFICANCE: Synthetic bio-instructive materials which display tissue-specific functionality and nanoscale architecture of the native extracellular matrix are attractive matrices for organoids development. These synthetic matrices are chemically defined and animal-free compared to current gold standard matrices such as Matrigel. Here, we developed hydrogel matrices with tunable stiffness, which incorporate laminin-mimetic peptide amphiphiles to grow and expand cerebral organoids. Using multi-omics tools, the present study provides exciting data on the effects of neuro-inductive cues on the biomolecular profiles of brain organoids.

Uniform cerebral organoid culture on a pillar plate by simple and reproducible spheroid transfer from an ultralow attachment well plate.

Human induced pluripotent stem cell (iPSCs)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effective in vitro model for studying both normal brain development and disorders. However, current brain organoid culture methods face several challenges, including low throughput, high variability in organoid generation, and time-consuming, multiple transfer and encapsulation of cells in hydrogels throughout the culture. These limitations hinder the widespread application of brain organoids including high-throughput assessment of compounds in clinical and industrial lab settings. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies (EBs) in an ultra-low attachment (ULA) 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95 - 100%. By differentiating the EBs on the pillar plate, we achieved robust generation of cerebral organoids with a coefficient of variation (CV) below 19%. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowing in situ organoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.