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Modeling of T cell-mediated autoimmune pituitary disease using human induced pluripotent stem cell-originated organoid.
Anti-pituitary-specific transcription factor (PIT)-1 hypophysitis is an autoimmune disease characterized by hormone secretion impairment from PIT-1-expressing pituitary cells, accompanied by malignancies with ectopic PIT-1 expression. Cytotoxic T cells (CTL) targeting PIT-1-positive cells have been implicated in disease development, yet direct evidence is lacking. As human leukocyte antigen (HLA)-matching is required for modeling T cell-mediated autoimmune diseases, we employ induced pluripotent stem cells (iPSC) to generate pituitary organoids harboring the patients' HLA haplotype and coculture the organoids with PIT-1-reactive CTLs isolated from the patients' peripheral blood mononuclear cells. The coculture demonstrates specific CTL-mediated cytotoxicity against PIT-1-positive cells exclusively in autologous conditions, with this cytotoxicity inhibited by immunosuppressive agents such as dexamethasone and cyclosporin A. Multiple combinations of epitopes, CTLs, and HLA molecules are responsible for pathogenesis. These data demonstrate CTL-mediated autoimmunity in anti-PIT-1 hypophysitis and highlight the potential application of this strategy for other T cell-mediated autoimmune diseases.
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Modular automated microfluidic cell culture platform reduces glycolytic stress in cerebral cortex organoids.
Organ-on-a-chip systems combine microfluidics, cell biology, and tissue engineering to culture 3D organ-specific in vitro models that recapitulate the biology and physiology of their in vivo counterparts. Here, we have developed a multiplex platform that automates the culture of individual organoids in isolated microenvironments at user-defined media flow rates. Programmable workflows allow the use of multiple reagent reservoirs that may be applied to direct differentiation, study temporal variables, and grow cultures long term. Novel techniques in polydimethylsiloxane (PDMS) chip fabrication are described here that enable features on the upper and lower planes of a single PDMS substrate. RNA sequencing (RNA-seq) analysis of automated cerebral cortex organoid cultures shows benefits in reducing glycolytic and endoplasmic reticulum stress compared to conventional in vitro cell cultures.
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Tropism of SARS-CoV-2 for human cortical astrocytes.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.
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Autism genes converge on asynchronous development of shared neuron classes.
Genetic risk for autism spectrum disorder (ASD) is associated with hundreds of genes spanning a wide range of biological functions1-6. The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals7,8. Here we used organoid models of the human cerebral cortex to identify cell-type-specific developmental abnormalities that result from haploinsufficiency in three ASD risk genes-SUV420H1 (also known as KMT5B), ARID1B and CHD8-in multiple cell lines from different donors, using single-cell RNA-sequencing (scRNA-seq) analysis of more than 745,000 cells and proteomic analysis of individual organoids, to identify phenotypic convergence. Each of the three mutations confers asynchronous development of two main cortical neuronal lineages-γ-aminobutyric-acid-releasing (GABAergic) neurons and deep-layer excitatory projection neurons-but acts through largely distinct molecular pathways. Although these phenotypes are consistent across cell lines, their expressivity is influenced by the individual genomic context, in a manner that is dependent on both the risk gene and the developmental defect. Calcium imaging in intact organoids shows that these early-stage developmental changes are followed by abnormal circuit activity. This research uncovers cell-type-specific neurodevelopmental abnormalities that are shared across ASD risk genes and are finely modulated by human genomic context, finding convergence in the neurobiological basis of how different risk genes contribute to ASD pathology.
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[Relation between lipofuscin content and organelle density in pyramidal cells of laminae III and V of area 10 (Brodmann) of the cerebral frontal lobe of humans of different ages].
The areal portion of the lipofuscin granules and mitochondria contained in ultrathin sections, the length of rough endoplasmic reticulum (RER) and the numerical density of ribosomes were determined in the pyramidal cells of layers III and V of area 10 (Brodmann) of the frontal brains of six newly died persons of different ages. This revealed considerable differences in lipofuscin contents and numerical density of organelles between the pyramidal cells and greater values, on an average, for the pyramidal cells of layer III. The comparison of averages for the single brains did not show the continuity nor the age dependence of changes but rather the individual variability of these processes. The expected decrease of the density of organelles with increasing lipofuscin contents was found more frequently only at extremely high values of lipofuscin contents. It becomes evident that even under the conditions of lipofuscin accumulation the permanently necessary physiological renewal of cell structures involved in metabolism is guaranteed over a longer period of time and that their full performance is almost completely unimpaired during this phase.
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Disruption of lysosomal proteolysis in astrocytes facilitates midbrain organoid proteostasis failure in an early-onset Parkinson's disease model.
Accumulation of advanced glycation end products (AGEs) on biopolymers accompanies cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early onset Parkinson's Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, α-synuclein (α-syn) phosphorylation to increase, and proteins to aggregate. We demonstrated these processes are at least partly driven by astrocytes, as DJ1 loss reduces their capacity to provide metabolic support and triggers acquisition of a pro-inflammatory phenotype. Consistently, in co-cultures, we find that DJ1-expressing astrocytes are able to reverse the proteolysis deficits of DJ1 knockout midbrain neurons. In conclusion, astrocytes' capacity to clear toxic damaged proteins is critical to preserve neuronal function and their dysfunction contributes to the neurodegeneration observed in a DJ1 loss-of-function PD model.
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Transcriptome and epigenome landscape of human cortical development modeled in organoids.
Genes implicated in neuropsychiatric disorders are active in human fetal brain, yet difficult to study in a longitudinal fashion. We demonstrate that organoids from human pluripotent cells model cerebral cortical development on the molecular level before 16 weeks postconception. A multiomics analysis revealed differentially active genes and enhancers, with the greatest changes occurring at the transition from stem cells to progenitors. Networks of converging gene and enhancer modules were assembled into six and four global patterns of expression and activity across time. A pattern with progressive down-regulation was enriched with human-gained enhancers, suggesting their importance in early human brain development. A few convergent gene and enhancer modules were enriched in autism-associated genes and genomic variants in autistic children. The organoid model helps identify functional elements that may drive disease onset.
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Non-genetic neuromodulation with graphene optoelectronic actuators for disease models, stem cell maturation, and biohybrid robotics.
Light can serve as a tunable trigger for neurobioengineering technologies, enabling probing, control, and enhancement of brain function with unmatched spatiotemporal precision. Yet, these technologies often require genetic or structural alterations of neurons, disrupting their natural activity. Here, we introduce the Graphene-Mediated Optical Stimulation (GraMOS) platform, which leverages graphene's optoelectronic properties and its ability to efficiently convert light into electricity. Using GraMOS in longitudinal studies, we found that repeated optical stimulation enhances the maturation of hiPSC-derived neurons and brain organoids, underscoring GraMOS's potential for regenerative medicine and neurodevelopmental studies. To explore its potential for disease modeling, we applied short-term GraMOS to Alzheimer's stem cell models, uncovering disease-associated alterations in neuronal activity. Finally, we demonstrated a proof-of-concept for neuroengineering applications by directing robotic movements with GraMOS-triggered signals from graphene-interfaced brain organoids. By enabling precise, non-invasive neural control across timescales from milliseconds to months, GraMOS opens new avenues in neurodevelopment, disease treatment, and robotics.
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Towards neuronal organoids: a method for long-term culturing of high-density hippocampal neurons.
One of the goals in neuroscience is to obtain tractable laboratory cultures that closely recapitulate in vivo systems while still providing ease of use in the lab. Because neurons can exist in the body over a lifetime, long-term culture systems are necessary so as to closely mimic the physiological conditions under laboratory culture conditions. Ideally, such a neuronal organoid culture would contain multiple cell types, be highly differentiated, and have a high density of interconnected cells. However, before these types of cultures can be created, certain problems associated with long-term neuronal culturing must be addressed. We sought to develop a new protocol which may further prolong the duration and integrity of E18 rat hippocampal cultures. We have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes-particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons.
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Epilepsy-associated SCN2A-L1342P mutation drives network hyperexcitability and widespread transcriptomic changes in human cortical organoids.
OBJECTIVE: SCN2A pathogenic mutations, such as the recurrent heterozygous Nav1.2-L1342P, are monogenic causes of epilepsy. In this human-induced pluripotent stem cell-derived model system, we aim to investigate the molecular and cellular mechanisms underlying SCN2A-L1342P-associated pathology. METHODS: Using a human male induced pluripotent stem cell (iPSC) reference line (KOLF) carrying the Nav1.2-L1342P mutation, we generated three-dimensional (3D) cortical organoids for functional studies. Patch-clamp, multi-electrode array (MEA) recordings, immunocytochemistry, and RNA sequencing were used to characterize the disease phenotypes. RESULTS: Nav1.2-L1342P organoid neurons displayed increased intrinsic excitability and amplified excitatory post-synaptic currents, which are consistent with an increase in excitatory synapse formation revealed by SYN1/PSD95 immunostaining. Moreover, elevated network firing activity, as demonstrated by MEA, indicates a pronounced network hyperexcitability. Transcriptomic profiling of organoids carrying the Nav1.2-L1342P mutation further revealed significant alterations in synaptic, glutamatergic, developmental, and senescence/apoptotic pathways. SIGNIFICANCE: Our findings demonstrate that the Nav1.2-L1342P mutation drives a multifaceted disease phenotype, including network hyperexcitability and disruption of pathways related to neuronal and synaptic functions. These results advance our understanding of SCN2A-related developmental and epileptic encephalopathy (DEE), laying a foundation for personalized interventions.