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Evaluation of UBE3A antibodies in mice and human cerebral organoids.
UBE3A is an E3 ubiquitin ligase encoded by the neurally imprinted UBE3A gene. The abundance and subcellular distribution of UBE3A has been the topic of many previous studies as its dosage and localization has been linked to neurodevelopmental disorders including Autism, Dup15q syndrome, and Angelman syndrome. While commercially available antibodies have been widely employed to determine UBE3A localization, an extensive analysis and comparison of the performance of different UBE3A antibodies has not been conducted. Here we evaluated the specificities of seven commercial UBE3A antibodies in two of the major experimental models used in UBE3A research, mouse and human pluripotent stem cell-derived neural cells and tissues. We tested these antibodies in their two most common assays, immunofluorescence and western blot. In addition, we also assessed the ability of these antibodies to capture dynamic spatiotemporal changes of UBE3A by utilizing human cerebral organoid models. Our results reveal that among the seven antibodies tested, three antibodies demonstrated substantial nonspecific immunoreactivity. While four antibodies show specific localization patterns in both mouse brain sections and human cerebral organoids, these antibodies varied significantly in background signals and staining patterns in undifferentiated human pluripotent stem cells.
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Human oligodendrocyte progenitor cells mediate synapse elimination through TAM receptor activation.
Oligodendrocyte progenitor cells (OPCs) have been implicated in synaptic remodelling in animal models, but the underlying mechanisms and their relevance to human brain development remain unclear. Here, we generate a human multi-lineage forebrain organoid model in which OPCs, together with microglia, form close contacts with synapses and spontaneously internalize synaptic material. Single-nucleus transcriptomic profiling with unbiased cell-cell communication analysis identifies the growth arrest-specific gene 6 (GAS6)-TYRO3, AXL, and MERTK (TAM) receptor axis as a key signalling pathway, with neurons and microglia expressing GAS6 and a subset of OPCs expressing AXL. Further, dose-dependent pharmacological inhibition of TAM receptors demonstrates the importance of AXL, and targeted reduction of AXL expression in OPCs impairs synaptic uptake. These findings reveal a role for GAS6-AXL signalling in driving synaptic internalisation by AXL+ OPCs during early human brain development.
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Patient-derived iPSC-cerebral organoid modeling of the 17q11.2 microdeletion syndrome establishes CRLF3 as a critical regulator of neurogenesis.
Neurodevelopmental disorders are often caused by chromosomal microdeletions comprising numerous contiguous genes. A subset of neurofibromatosis type 1 (NF1) patients with severe developmental delays and intellectual disability harbors such a microdeletion event on chromosome 17q11.2, involving the NF1 gene and flanking regions (NF1 total gene deletion [NF1-TGD]). Using patient-derived human induced pluripotent stem cell (hiPSC)-forebrain cerebral organoids (hCOs), we identify both neural stem cell (NSC) proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NSC proliferation results from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects are caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrate a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T>C, p.Leu389Pro). Collectively, these findings identify a causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1.
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Loss of NARS1 impairs progenitor proliferation in cortical brain organoids and leads to microcephaly.
Asparaginyl-tRNA synthetase1 (NARS1) is a member of the ubiquitously expressed cytoplasmic Class IIa family of tRNA synthetases required for protein translation. Here, we identify biallelic missense and frameshift mutations in NARS1 in seven patients from three unrelated families with microcephaly and neurodevelopmental delay. Patient cells show reduced NARS1 protein, impaired NARS1 activity and impaired global protein synthesis. Cortical brain organoid modeling shows reduced proliferation of radial glial cells (RGCs), leading to smaller organoids characteristic of microcephaly. Single-cell analysis reveals altered constituents of both astrocytic and RGC lineages, suggesting a requirement for NARS1 in RGC proliferation. Our findings demonstrate that NARS1 is required to meet protein synthetic needs and to support RGC proliferation in human brain development.
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Factors Affecting Stem Cell-Based Regenerative Approaches in Retinal Degeneration.
Inherited and age-associated vision loss is often associated with degeneration of the cells of the retina, the light-sensitive layer at the back of the eye. The mammalian retina, being a postmitotic neural tissue, does not have the capacity to repair itself through endogenous regeneration. There has been considerable excitement for the development of cell replacement approaches since the isolation and development of culture methods for human pluripotent stem cells, as well as the generation of induced pluripotent stem cells. This has now been combined with novel three-dimensional organoid culture systems that closely mimic human retinal development in vitro. In this review, we cover the current state of the field, with emphasis on the cell delivery challenges, role of the recipient immunological microenvironment, and challenges related to connectivity between transplanted cells and host circuitry both locally and centrally to the different areas of the brain.
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SARS-CoV-2 Infects the Brain Choroid Plexus and Disrupts the Blood-CSF Barrier in Human Brain Organoids.
Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, leads to respiratory symptoms that can be fatal. However, neurological symptoms have also been observed in some patients. The cause of these complications is currently unknown. Here, we use human-pluripotent-stem-cell-derived brain organoids to examine SARS-CoV-2 neurotropism. We find expression of viral receptor ACE2 in mature choroid plexus cells expressing abundant lipoproteins, but not in neurons or other cell types. We challenge organoids with SARS-CoV-2 spike pseudovirus and live virus to demonstrate viral tropism for choroid plexus epithelial cells but little to no infection of neurons or glia. We find that infected cells are apolipoprotein- and ACE2-expressing cells of the choroid plexus epithelial barrier. Finally, we show that infection with SARS-CoV-2 damages the choroid plexus epithelium, leading to leakage across this important barrier that normally prevents entry of pathogens, immune cells, and cytokines into cerebrospinal fluid and the brain.
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Enterovirus D68 Infection in Human Primary Airway and Brain Organoids: No Additional Role for Heparan Sulfate Binding for Neurotropism.
Enterovirus D68 (EV-D68) is an RNA virus that can cause outbreaks of acute flaccid paralysis (AFP), a polio-like disease. Before 2010, EV-D68 was a rare pathogen associated with mild respiratory symptoms, but the recent EV-D68 related increase in severe respiratory illness and outbreaks of AFP is not yet understood. An explanation for the rise in severe disease is that it may be due to changes in the viral genome resulting in neurotropism. In this regard, in addition to sialic acid, binding to heparan sulfate proteoglycans (HSPGs) has been identified as a feature for viral entry of some EV-D68 strains in cell lines. Studies in human primary organotypic cultures that recapitulate human physiology will address the relevance of these HSPG-binding mutations for EV-D68 infection in vivo. Therefore, in this work, we studied the replication and neurotropism of previously determined sialic acid-dependent and HSPG-dependent strains using primary human airway epithelial (HAE) cultures and induced human pluripotent stem cell (iPSC)-derived brain organoids. All three strains (B2/2042, B2/947, and A1/1348) used in this study infected HAE cultures and human brain organoids (shown for the first time). Receptor-blocking experiments in both cultures confirm that B2/2042 infection is solely dependent on sialic acid, while B2/947 and A1/1348 (HSPG to a lesser extent) binds to sialic acid and HSPG for cell entry. Our data suggest that HSPG-binding can be used by EV-D68 for entry in human physiological models but offers no advantage for EV-D68 infection of brain cells. IMPORTANCE Recent outbreaks of enterovirus D68, a nonpolio enterovirus, is associated with a serious neurological condition in young children, acute flaccid myelitis (AFM). As there is no antiviral treatment or vaccine available for EV-D68 it is important to better understand how EV-D68 causes AFM and why only recent outbreaks are associated with AFM. We investigated if a change in receptor usage of EV-D68 increases the virulence of EV-D68 in the airway or the central nervous system and thus could explain the increase in AFM cases. We studied this using physiologically relevant human airway epithelium and cerebral organoid cultures that are physiologically relevant human models. Our data suggest that heparan sulfate proteoglycans can be used by EV-D68 as an additional entry receptor in human physiological models but offers no advantage for EV-D68 infection of brain cells, and our data show the potential of these 46 innovative models for virology.
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[Ultrastructural changes in the brains of rats after sustaining acute emotional stress].
The ultrastructure of the neurones and synapses of the parietal area of the cortex and reticular formation of the brain of rats was studied after emotional stress induced by 2-hour immobilization, as well as 1 and 24 hours after discontinuance of immobilization. Two types of ultrastructural changes were revealed. Some of the changes were reversible and functional in nature and were largely pertinent to the alterations on the cytoplasma part, with these alterations being appreciably reduced 24 hours after the discontinuance of immobilization. The other ones (vacuolization of neuronal nuclei and marked changes in the synapses) were more pronounced and resistant and were seen 24 hours after the immobilization was discontinued. It is suggested that stable changes that do not disappear 24 hours after the immobilization stress might form the pathomorphological basis for the clinical syndromes of cerebrovisceral disorders that develop during long-term periods after the stress.
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Profiling human brain vascular cells using single-cell transcriptomics and organoids.
Angiogenesis and neurogenesis are functionally interconnected during brain development. However, the study of the vasculature has trailed other brain cell types because they are delicate and of low abundance. Here we describe a protocol extension to purify prenatal human brain endothelial and mural cells with FACS and utilize them in downstream applications, including transcriptomics, culture and organoid transplantation. This approach is simple, efficient and generates high yields from small amounts of tissue. When the experiment is completed within a 24 h postmortem interval, these healthy cells produce high-quality data in single-cell transcriptomics experiments. These vascular cells can be cultured, passaged and expanded for many in vitro assays, including Matrigel vascular tube formation, microfluidic chambers and metabolic measurements. Under these culture conditions, primary vascular cells maintain expression of cell-type markers for at least 3 weeks. Finally, we describe how to use primary vascular cells for transplantation into cortical organoids, which captures key features of neurovascular interactions in prenatal human brain development. In terms of timing, tissue processing and staining requires ~3 h, followed by an additional 3 h of FACS. The transplant procedure of primary, FACS-purified vascular cells into cortical organoids requires an additional 2 h. The time required for different transcriptomic and epigenomic protocols can vary based on the specific application, and we offer strategies to mitigate batch effects and optimize data quality. In sum, this vasculo-centric approach offers an integrated platform to interrogate neurovascular interactions and human brain vascular development.
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Protocol for minimizing necrotic core formation in iPSC-derived cortical organoids.
Cortical organoids (COs) derived from human induced pluripotent stem cells (hiPSCs) provide a unique opportunity to model human neurodevelopment in vitro. Here, we present a protocol to prevent necrotic core formation without requiring organoid slicing. We describe steps for hiPSC culturing, preparation of single-cell suspension, seeding, and embryoid body (EB) and neuroectoderm formation. We then detail procedures for CO formation and maintenance without necrotic core formation. This protocol sustains long-term viability and preserves structural integrity by enhancing oxygen and nutrient diffusion.