Stem Cell-derived Brain Organoids Research Articles

Dolutegravir induces FOLR1 expression during brain organoid development.

During the first month of pregnancy, the brain and spinal cord are formed through a process called neurulation. However, this process can be altered by low serum levels of folic acid, environmental factors, or genetic predispositions. In 2018, a surveillance study in Botswana, a country with a high incidence of human immunodeficiency virus (HIV) and lacking mandatory food folate fortification programs, found that newborns whose mothers were taking dolutegravir (DTG) during the first trimester of pregnancy had an increased risk of neural tube defects (NTDs). As a result, the World Health Organization and the U.S. Food and Drug Administration have issued guidelines emphasizing the potential risks associated with the use of DTG-based antiretroviral therapies during pregnancy. To elucidate the potential mechanisms underlying the DTG-induced NTDs, we sought to assess the potential neurotoxicity of DTG in stem cell-derived brain organoids. The gene expression of brain organoids developed in the presence of DTG was analyzed by RNA sequencing, Optical Coherence Tomography (OCT), Optical Coherence Elastography (OCE), and Brillouin microscopy. The sequencing data shows that DTG induces the expression of the folate receptor (FOLR1) and modifies the expression of genes required for neurogenesis. The Brillouin frequency shift observed at the surface of DTG-exposed brain organoids indicates an increase in superficial tissue stiffness. In contrast, reverberant OCE measurements indicate decreased organoid volumes and internal stiffness.

Stem cell-derived brain organoids for controlled studies of transcranial neuromodulation

Transcranial neuromodulation methods have the potential to diagnose and treat brain disorders at their neural source in a personalized manner. However, it has been difficult to investigate the direct effects of transcranial neuromodulation on neurons in human brain tissue. Here, we show that human brain organoids provide a detailed and artifact-free window into neuromodulation-evoked electrophysiological effects. We derived human cortical organoids from induced pluripotent stem cells and implanted 32-channel electrode arrays. Each organoid was positioned in the center of the human skull and subjected to low-intensity transcranial focused ultrasound. We found that ultrasonic stimuli modulated network activity in the gamma and delta ranges of the frequency spectrum. The effects on the neural networks were a function of the ultrasound stimulation frequency. High gamma activity remained elevated for at least 20 minutes following stimulation offset. This approach is expected to provide controlled studies of the effects of ultrasound and other transcranial neuromodulation modalities on human brain tissue.

BIOL-09. PROTEOMIC ANALYSES REVEAL THAT CO-CULTURE OF DIFFUSE INTRINSIC PONTINE GLIOME (DIPG) WITH CORTICAL ORGANOIDS ALTERS CELL ADHESION, DNA SYNTHESIS AND REPLICATION, AND DENDRITIC GROWTH SIGNALLING

Abstract Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly brain cancers in children for which there is currently no effective treatment. In part, this can be attributed to preclinical models that lack essential elements of the in vivo tissue environment, resulting in treatments that appear promising preclinically, but fail to result in effective cures. Recently developed co-culture models combining stem cell-derived brain organoids with brain cancer cells provide tissue dimensionality and a human-relevant tissue-like microenvironment. As these models are technically challenging and time consuming it is imperative to establish whether interaction with the organoid influences DIPG biology and thus warrants their use. To address this question, we cultured DIPG cells with GFP-expressing cortical organoids. We created “mosaic” co-cultures enriched for tumour cell-neuronal cell interactions, where disaggregated spheroids and organoids were mixed and allowed to reform, versus “assembloid” co-cultures enriched for tumour cell-tumour cell interactions, where preformed tumour spheroids and organoids were combined. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to analyse the proteomes of DIPG fractions isolated by flow-assisted cell sorting. Control proteomes from DIPG spheroids were compared with DIPG cells isolated from mosaic and assembloid co-cultures. This revealed that tumour cell adhesion was reduced, and DNA synthesis and replication were increased, in DIPG cells under either co-culture condition. By contrast, the mosaic co-culture was alone associated with pathways implicated in dendrite growth. We propose that co-culture with brain organoids is a valuable tool to parse the contribution of the brain microenvironment to DIPG tumour biology.

Generation of iPSC-derived human forebrain organoids assembling bilateral eye primordia.

Induced pluripotent stem cell-derived brain organoids enable the developmental complexities of the human brain to be deconstructed. During embryogenesis, optic vesicles (OVs), the eye primordium attached to the forebrain, develop from diencephalon. However, most 3D culturing methods generate either brain or retinal organoids individually. Here we describe a protocol to generate organoids with both forebrain entities, which we call OV-containing brain organoids (OVB organoids). In this protocol, we first induce neural differentiation (days 0-5) and collect neurospheres, which we culture in a neurosphere medium to initiate their patterning and further self-assembly (days 5-10). Then, upon transfer to spinner flasks containing OVB medium (days 10-30), neurospheres develop into forebrain organoids with one or two pigmented dots restricted to one pole, displaying forebrain entities of ventral and dorsal cortical progenitors and preoptic areas. Further long-term culture results in photosensitive OVB organoids constituting complementary cell types of OVs, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections and electrically active neuronal networks. OVB organoids provide a system to help dissect interorgan interactions between the OVs as sensory organs and the brain as a processing unit, and can help model early eye patterning defects, including congenital retinal dystrophy. To conduct the protocol, experience in sterile cell culture and maintenance of human induced pluripotent stem cells is essential; theoretical knowledge of brain development is advantageous. Furthermore, specialized expertise in 3D organoid culture and imaging for the analysis is needed.

Organoid intelligence (OI): the new frontier in biocomputing and intelligence-in-a-dish

Recent advances in human stem cell-derived brain organoids promise to replicate critical molecular and cellular aspects of learning and memory and possibly aspects of cognition in vitro. Coining the term “organoid intelligence” (OI) to encompass these developments, we present a collaborative program to implement the vision of a multidisciplinary field of OI. This aims to establish OI as a form of genuine biological computing that harnesses brain organoids using scientific and bioengineering advances in an ethically responsible manner. Standardized, 3D, myelinated brain organoids can now be produced with high cell density and enriched levels of glial cells and gene expression critical for learning. Integrated microfluidic perfusion systems can support scalable and durable culturing, and spatiotemporal chemical signaling. Novel 3D microelectrode arrays permit high-resolution spatiotemporal electrophysiological signaling and recording to explore the capacity of brain organoids to recapitulate the molecular mechanisms of learning and memory formation and, ultimately, their computational potential. Technologies that could enable novel biocomputing models via stimulus-response training and organoid-computer interfaces are in development. We envisage complex, networked interfaces whereby brain organoids are connected with real-world sensors and output devices, and ultimately with each other and with sensory organ organoids (e.g. retinal organoids), and are trained using biofeedback, big-data warehousing, and machine learning methods. In parallel, we emphasize an embedded ethics approach to analyze the ethical aspects raised by OI research in an iterative, collaborative manner involving all relevant stakeholders. The many possible applications of this research urge the strategic development of OI as a scientific discipline. We anticipate OI-based biocomputing systems to allow faster decision-making, continuous learning during tasks, and greater energy and data efficiency. Furthermore, the development of “intelligence-in-a-dish” could help elucidate the pathophysiology of devastating developmental and degenerative diseases (such as dementia), potentially aiding the identification of novel therapeutic approaches to address major global unmet needs.

Mechanoinhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis.

Fas (CD95/APO-1) is a transmembrane death receptor that transduces apoptotic signals upon binding to its ligand and assembling into a death-inducing signaling complex (DISC). Intracellular trafficking of Fas receptors, including recycling from endosomes to the plasma membrane, plays a vital role in ligand-induced assembly of DISC. Although Fas is highly expressed in tumor cells, insufficient expression of these receptors on the cell surface makes cancer cells insensitive to the Fas-induced apoptosis. Here we show that inhibition of endocytosis increases the formation of Fas microaggregates on the plasma membrane and sensitizes cancer cells to Fas-induced apoptosis. We have identified a clinically used vasodilator, Fasudil, that slows down endocytosis by increasing plasma membrane tension. Fasudil enhanced apoptosis in cancerous cells when combined with exogenous soluble Fas ligand (FasL), whereas the synergistic effect was substantially weaker in nonmalignant cells. Additionally, the FasL and Fasudil combination prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft U87 tumor model in nude mice. Our results demonstrate that FasL treatment has strong potential as an apoptosis-directed cancer therapy when the formation of Fas microaggregates is augmented by slowing down endocytosis dynamics.

Modeling Autism Spectrum Disorders with Induced Pluripotent Stem Cell-Derived Brain Organoids.

Autism spectrum disorders (ASD) are a group of complex neurodevelopmental disorders that affect communication and social interactions and present with restricted interests and repetitive behavior patterns. The susceptibility to ASD is strongly influenced by genetic/heritable factors; however, there is still a large gap in understanding the cellular and molecular mechanisms underlying the neurobiology of ASD. Significant progress has been made in identifying ASD risk genes and the possible convergent pathways regulated by these gene networks during development. The breakthrough of cellular reprogramming technology has allowed the generation of induced pluripotent stem cells (iPSCs) from individuals with syndromic and idiopathic ASD, providing patient-specific cell models for mechanistic studies. In the past decade, protocols for developing brain organoids from these cells have been established, leading to significant advances in the in vitro reproducibility of the early steps of human brain development. Here, we reviewed the most relevant literature regarding the application of brain organoids to the study of ASD, providing the current state of the art, and discussing the impact of such models on the field, limitations, and opportunities for future development.

Developmental exposure to thyroid disruptors: misprogramming of the brain's stem cells in later life?

Developmental exposure to thyroid disruptors: misprogramming of the brain's stem cells in later life?

Induced pluripotent stem cell-derived brain organoids as potential human model system for chemotherapy induced CNS toxicity.

Neurotoxic phenomena are among the most common side effects of cytotoxic agents. The development of chemotherapy-induced polyneuropathy (CIPN) is a well-recognized adverse reaction in the peripheral nervous system, while changes of cognitive functions (post-chemotherapy cognitive impairment (PCCI)) are more diffuse and have only recently drawn scientific interest. PCCI in patients most often displays as short-term memory loss, reduced multitasking ability or deficits in language. Not least, due to a lack of preclinical human model systems, the underlying molecular mechanisms are poorly understood, and treatments are missing. We thus investigated whether induced pluripotent stem cell (iPSC)-derived brain organoids can serve as a human model system for the study of chemotherapy induced central nervous system toxicity. We robustly generated mature brain organoids from iPSC-derived neuronal precursor cells (NPC), which showed a typical composition with 1) dividing NPCs forming ventricle like structures 2) matured neurons and 3) supporting glial cells closer to the surface. Furthermore, upon stimulation the brain organoids showed functional signaling. When exposed to increasing concentrations of paclitaxel, a frequently used chemotherapy drug, we observed time dependent neurotoxicity with an EC50 of 153 nM, comparable to a published murine model system. Histological analysis after paclitaxel exposure demonstrated dose dependent apoptosis induction and reduced proliferation in the organoids with further Western blot analyses indicating the degradation of neuronal calcium sensor one protein (NCS-1) and activation of Caspase-3. We could also provide evidence that paclitaxel treatment negatively affects the pool of neuronal and astrocyte precursor cells as well as mature neurons. In summary our data suggests that human iPSC derived brain organoids are a promising preclinical model system to investigate molecular mechanisms underlying PCCI and to develop novel prevention and treatment strategies.

EPEN-10. Elucidating the biological difference between pediatric supratentorial ependymomas using genetically engineered brain tumor organoids.

Abstract Ependymomas (EPN) are neuroepithelial tumors of the central nervous system occurring both in adults and children. DNA methylation profiling has identified ten distinct molecular groups. In the supratentorial (ST) compartment, ZFTA-fusion positive and YAP1-fusion positive ST-EPN represent the two main groups in pediatric patients that are histologically very similar but have a completely different clinical behavior. While most ST-EPN-YAP1 patients survive, only 50% of ZFTA positive patients survive longer than 5 years. Improving survival of ZFTA positive patients requires better understanding of the tumor biology. Despite having different oncogenic fusions, it is unclear whether the cellular origin and tumor biology is related or not and why they respond differently to treatment. The development of human stem cell-derived brain organoids provides a novel opportunity to model these ST-EPN in vitro. These organoids mimic embryonal brain development, during which the cells-of-origin of ST-EPN as well as their micro-environment are present. To induce the development of ST-EPN in vitro, we genetically manipulated cerebral organoids to overexpress the group-specific oncogenic fusion proteins. Electroporating constructs expressing the different fusion proteins into organoids early in brain development (day 11), induced ectopic growth after approximately 30 days. Based on immunohistochemistry, we observed a different phenotype using either the ZFTA or YAP1 fusion constructs, as well as (over)expression of group-specific markers in the organoids. Preliminary transcriptomic analyses suggest that these genetically modified brain organoids represent models with features of human ST-EPN tumors. Ongoing single-cell RNA-sequencing and epigenomic analyses during normal organoid and tumor development will further reveal how well these models represent human primary tumors, how they develop in time, and possibly whether they originate from similar cells or different cells within these organoids. Hence, these models will uniquely contribute to the molecular and biological understanding of ependymomas and can be used to identify new targeted therapies.

Modeling PCDH19 Related Epilepsy in Human Pluripotent Stem Cell Derived Brain Organoids

Modeling PCDH19 Related Epilepsy in Human Pluripotent Stem Cell Derived Brain Organoids

Stretchable Mesh Nanoelectronics for 3D Single-Cell Chronic Electrophysiology from Developing Brain Organoids.

Human induced pluripotent stem cell derived brain organoids have shown great potential for studies of human brain development and neurological disorders. However, quantifying the evolution of the electrical properties of brain organoids during development is currently limited by the measurement techniques, which cannot provide long-term stable 3D bioelectrical interfaces with developing brain organoids. Here, a cyborg brain organoid platform is reported, in which "tissue-like" stretchable mesh nanoelectronics are designed to match the mechanical properties of brain organoids and to be folded by the organogenetic process of progenitor or stem cells, distributing stretchable electrode arrays across the 3D organoids. The tissue-wide integrated stretchable electrode arrays show no interruption to brain organoid development, adapt to the volume and morphological changes during brain organoid organogenesis, and provide long-term stable electrical contacts with neurons within brain organoids during development. The seamless and noninvasive coupling of electrodes to neurons enables long-term stable, continuous recording and captures the emergence of single-cell action potentials from early-stage brain organoid development.

Recapitulating influenza virus infection and facilitating antiviral and neuroprotective screening in tractable brain organoids.

Human pluripotent stem cell derived brain organoids offer an unprecedented opportunity for various applications as in vitro model. Currently, human brain organoids as models have been used to understand virus-induced neurotoxicity.Methods: The brain organoids were separately challenged by multiple viruses including influenza viruses (H1N1-WSN and H3N2-HKT68), Enteroviruses (EV68 and EV71) and Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) to investigate the impaired effect of these viruses on human brain development.Results: The brain organoids challenged by influenza viruses had decreased overall organoid size, while enteroviruses infected brain organoids displayed the opposite result. Then, we found WSN preferentially infected MAP2+ neurons compared to SOX2+ neural stem cells (NSCs) and GFAP+ astrocytes in brain organoids, and induced apoptosis of NSCs and neurons, and released inflammatory factors (TNF-α, INF-γ, and IL-6), facilitating brain damage. Furthermore, transcriptional profiling revealed several co-upregulated genes (CSAG3 and OAS2) and co-downregulated genes (CDC20B, KCNJ13, OTX2-AS1) after WSN infection for 24 hpi and 96 hpi, implicating target for antiviral drugs development. Finally, we explored compound PYC-12 could significantly suppress virus infection, apoptosis, and inflammatory responses.Conclusions: Collectively, we established a tractable experimental model to investigate the impact and mechanism of virus infection on human brain development.

N-methyl-d-aspartate Receptor-mediated Preconditioning Mitigates Excitotoxicity in Human Induced Pluripotent Stem Cell-derived Brain Organoids

Studies in rodent models of acute and chronic neurodegenerative disorders have uncovered that glutamate-induced excitotoxic cell death is mediated primarily by extrasynaptic N-methyl-d-aspartate receptors (NMDARs). Rodent neurons can also build up in an activity-dependent manner a protective shield against excitotoxicity. This form of acquired neuroprotection is induced by preconditioning with low doses of NMDA or by activation of synaptic NMDARs triggered by bursts of action potentials. Whether NMDARs in human neurons have similar dichotomous actions in cell death and survival is unknown. To investigate this, we established an induced pluripotent stem cell (iPSC)-derived forebrain organoid model for excitotoxic cell death and explored conditions of NMDAR activation that promote neuronal survival when applied prior to a toxic insult. We found that glutamate-induced excitotoxicity in human iPSC-derived neurons is mediated by NMDARs. Treatment of organoids with high concentrations of glutamate or NMDA caused the typical excitotoxicity pathology, comprising structural disintegration, neurite blebbing, shut-off of the transcription factor CRE binding protein (CREB), and cell death. In contrast, bath-applied low doses of NMDA elicited synaptic activity, a robust and sustained increase in CREB phosphorylation as well as function, and upregulation of immediate-early genes, including neuroprotective genes. Moreover, we found that conditions of enhanced synaptic activity increased survival of human iPSC-derived neurons if applied as pre-treatment before toxic NMDA application. These results revealed that both toxic and protective actions of NMDARs are preserved in human neurons. The experimental platform described in this study may prove useful for the validation of neuroprotective gene products and drugs in human neurons.

Molecular Features of the Measles Virus Viral Fusion Complex That Favor Infection and Spread in the Brain.

ABSTRACTMeasles virus (MeV) bearing a single amino acid change in the fusion protein (F)—L454W—was isolated from two patients who died of MeV central nervous system (CNS) infection. This mutation in F confers an advantage over wild-type virus in the CNS, contributing to disease in these patients. Using murine ex vivo organotypic brain cultures and human induced pluripotent stem cell-derived brain organoids, we show that CNS adaptive mutations in F enhance the spread of virus ex vivo. The spread of virus in human brain organoids is blocked by an inhibitory peptide that targets F, confirming that dissemination in the brain tissue is attributable to F. A single mutation in MeV F thus alters the fusion complex to render MeV more neuropathogenic.

SARS-CoV-2 targets glial cells in human cortical organoids.

SummaryCoronavirus disease 2019 (COVID-19) patients have manifested a variety of neurological complications, and there is still much to reveal regarding the neurotropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human stem cell-derived brain organoids offer a valuable in vitro approach to study the cellular effects of SARS-CoV-2 on the brain. Here we used human embryonic stem cell-derived cortical organoids to investigate whether SARS-CoV-2 could infect brain tissue in vitro and found that cortical organoids could be infected at low viral titers and within 6 h. Importantly, we show that glial cells and cells of the choroid plexus were preferentially targeted in our model, but not neurons. Interestingly, we also found expression of angiotensin-converting enzyme 2 in SARS-CoV-2 infected cells; however, viral replication and cell death involving DNA fragmentation does not occur. We believe that our model is a tractable platform to study the cellular effects of SARS-CoV-2 infection in brain tissue.

Artificially Induced Pluripotent Stem Cell-Derived Whole-Brain Organoid for Modelling the Pathophysiology of Metachromatic Leukodystrophy and Drug Repurposing.

Metachromatic leukodystrophy (MLD) is a rare neurodegenerative disease that results from a deficiency of the lysosomal enzyme arylsulfatase A (ARSA). Worldwide, there are between one in 40,000 and one in 160,000 people living with the disease. While there are currently no effective treatments for MLD, induced pluripotent stem cell-derived brain organoids have the potential to provide a better understanding of MLD pathogenesis. However, developing brain organoid models is expensive, time consuming and may not accurately reflect disease progression. Using accurate and inexpensive computer simulations of human brain organoids could overcome the current limitations. Artificially induced whole-brain organoids (aiWBO) have the potential to greatly expand our ability to model MLD and guide future wet lab research. In this study, we have upgraded and validated our artificially induced whole-brain organoid platform (NEUBOrg) using our previously validated machine learning platform, DeepNEU (v6.2). Using this upgraded NEUBorg, we have generated aiWBO simulations of MLD and provided a novel approach to evaluate factors associated with MLD pathogenesis, disease progression and new potential therapeutic options.

Rapid and Efficient Generation of Myelinating Human Oligodendrocytes in Organoids.

Human stem cell derived brain organoids are increasingly gaining attention as an ideal model system for investigating neurological diseases, particularly those that involve myelination defects. However, current protocols for generating brain organoids with sufficiently mature oligodendrocytes that deposit myelin on endogenously produced neurons are lengthy and complicated. Taking advantage of a human pluripotent stem cell line that reports on SOX10 expression, we developed a protocol that involves a 42 day exposure of neuroectoderm-derived organoids to a cocktail of growth factors and small molecules that collectively foster oligodendrocyte specification and survival. Importantly, the resulting day 42 brain organoids contain both myelinating oligodendrocytes, cortical neuronal cells and astrocytes. These oligodendrocyte brain organoids therefore constitute a valuable and tractable platform for functional neurogenomics and drug screening for white matter diseases.

NEUBOrg: Artificially Induced Pluripotent Stem Cell-Derived Brain Organoid to Model and Study Genetics of Alzheimer's Disease Progression.

Alzheimer’s disease (AD) is the most common type of neurodegenerative diseases. There are over 44 million people living with the disease worldwide. While there are currently no effective treatments for AD, induced pluripotent stem cell-derived brain organoids have the potential to provide a better understanding of Alzheimer’s pathogenesis. Nevertheless, developing brain organoid models is expensive, time consuming and often does not reflect disease progression. Using accurate and inexpensive computer simulations of human brain organoids can overcome the current limitations. Induced whole brain organoids (aiWBO) will greatly expand our ability to model AD and can guide wet lab research. In this study, we have successfully developed and validated artificially induced a whole brain organoid platform (NEUBOrg) using our previously validated machine learning platform, DeepNEU (v6.1). Using NEUBorg platform, we have generated aiWBO simulations of AD and provided a novel approach to test genetic risk factors associated with AD progression and pathogenesis.

Three-dimensional model of glioblastoma by co-culturing tumor stem cells with human brain organoids.

ABSTRACTEmerging three-dimensional (3D) cultures of glioblastoma are becoming powerful models to study glioblastoma stem cell behavior and the impact of cell–cell and cell–microenvironment interactions on tumor growth and invasion. Here we describe a method for culturing human glioblastoma stem cells (GSCs) in 3D by co-culturing them with pluripotent stem cell-derived brain organoids. This requires multiple coordinated steps, including the generation of cerebral organoids, and the growth and fluorescence tagging of GSCs. We highlight how to recognize optimal organoid generation and how to efficiently mark GSCs, before describing optimized co-culture conditions. We show that GSCs can efficiently integrate into brain organoids and maintain a significant degree of cell fate heterogeneity, paving the way for the analysis of GSC fate behavior and lineage progression. These results establish the 3D culture system as a viable and versatile GBM model for investigating tumor cell biology and GSC heterogeneity.This article has an associated First Person interview with the first author of the paper.