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Page 54 of 545-
Next-Generation Human Cerebral Organoids as Powerful Tools To Advance NeuroHIV Research.
Long-term effective use of antiretroviral therapy (ART) among people with HIV (PWH) has significantly reduced the burden of disease, yet a cure for HIV has not been universally achieved, likely due to the persistence of an HIV reservoir. The central nervous system (CNS) is an understudied HIV sanctuary. Importantly, due to viral persistence in the brain, cognitive disturbances persist to various degrees at high rates in PWH despite suppressive ART. Given the complexity and accessibility of the CNS compartment and that it is a physiologically and anatomically unique immune site, human studies to reveal molecular mechanisms of viral entry, reservoir establishment, and the cellular and structural interactions leading to viral persistence and brain injury to advance a cure and either prevent or limit cognitive impairments in PWH remain challenging. Recent advances in human brain organoids show that they can mimic the intercellular dynamics of the human brain and may recapitulate many of the events involved in HIV infection of the brain (neuroHIV). Human brain organoids can be produced, spontaneously or with addition of growth factors and at immature or mature states, and have become stronger models to study neurovirulent viral infections of the CNS. While organoids provide opportunities to study neuroHIV, obstacles such as the need to incorporate microglia need to be overcome to fully utilize this model. Here, we review the current achievements in brain organoid biology and their relevance to neuroHIV research efforts.
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Engineering Human Brain Assembloids by Microfluidics.
Brain assembloids offer a highly promising strategy to model human brain development and disease, and advance potential studies in regenerative medicine, therapeutic screening, and drug discovery, while it is challenging to produce uniform brain organoids and assemble them flexibly by conventional methods. Here, a multidisciplinary engineered strategy to generate human brain assembloids with desired patterning based on microfluidic technology is presented. By encapsulating human induced pluripotent stem cells in microcapsules via microfluidic electrospray, brain region-specific organoids are efficiently formed, which are then introduced into a microfluidic chip consisting of a bottom layer with a micropillar array and a movable upper layer with a complementary microhole array. These brain organoids can settle into microholes and fuse into brain assembloids. As varied organoid microcapsules with designed 1D sequences or 2D arrays can be assembled into the vertical microholes, large coding amounts of fused brain assembloids with desired patterning can be produced. It is found that brain assembloids composed of cortical, hippocampal, and thalamic organoids can grow and function well, characterized with active neural migration and interaction. These features indicate that the suggested flexible, scalable, and controlled microfluidic systems are remarkably potential in wide applications of brain assembloids in neurological and biomedical fields.
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Modeling HIV-1 infection and NeuroHIV in hiPSCs-derived cerebral organoid cultures.
The human immunodeficiency virus (HIV) epidemic is an ongoing global health problem affecting 38 million people worldwide with nearly 1.6 million new infections every year. Despite the advent of combined antiretroviral therapy (cART), a large percentage of people with HIV (PWH) still develop neurological deficits, grouped into the term of HIV-associated neurocognitive disorders (HAND). Investigating the neuropathology of HIV is important for understanding mechanisms associated with cognitive impairment seen in PWH. The major obstacle for studying neuroHIV is the lack of suitable in vitro human culture models that could shed light into the HIV-CNS interactions. Recent advances in induced pluripotent stem cell (iPSC) culture and 3D brain organoid systems have allowed the generation of 2D and 3D culture methods that possess a potential to serve as a model of neurotropic viral diseases, including HIV. In this study, we first generated and characterized several hiPSC lines from healthy human donor skin fibroblast cells. hiPSCs were then used for the generation of microglia-containing human cerebral organoids (hCOs). Once fully characterized, hCOs were infected with HIV-1 in the presence and absence of cART regimens and viral infection was studied by cellular, molecular/biochemical, and virological assays. Our results revealed that hCOs were productively infected with HIV-1 as evident by viral p24-ELISA in culture media, RT-qPCR and RNAscope analysis of viral RNA, as well as ddPCR analysis of proviral HIV-1 in genomic DNA samples. More interestingly, replication and gene expression of HIV-1 were also greatly suppressed by cART in hCOs as early as 7 days post-infections. Our results suggest that hCOs derived from hiPSCs support HIV-1 replication and gene expression and may serve as a unique platform to better understand neuropathology of HIV infection in the brain.
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Delayed forebrain excitatory and inhibitory neurogenesis in STRADA-related megalencephaly via mTOR hyperactivity.
Biallelic pathogenic variants in STRADA (STE20-related adaptor alpha), an upstream regulator of the mechanistic target of rapamycin (mTOR) pathway, result in megalencephaly, drug-resistant epilepsy, and severe intellectual disability. This study explores how mTOR pathway hyperactivity alters cell fate specification in dorsal and ventral forebrain development using STRADA knockout human stem cell-derived brain organoids. In both dorsal and ventral forebrain STRADA knockout organoids, neurogenesis is delayed, with a predilection for progenitor renewal, increased proliferation and an expanded outer radial glia population. Ventrally, interneuron subtypes shift to an increase in neuropeptide Y-expressing cells. Inhibition of the mTOR pathway with rapamycin rescues most phenotypes. When mTOR pathway variants are present in all cells of the developing brain, overproduction of interneurons and altered interneuron cell fate may underlie mechanisms of megalencephaly, epilepsy, and cognitive impairment. Our findings suggest that mTOR inhibition during fetal brain development could be a potential therapeutic strategy in STRADA deficiency.
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Three-Dimensional Microfluidic Platform with Neural Organoids: Model System for Unraveling Synapses.
Unraveling the large number of various signals in the brain under the influence of physical and chemical cues that govern the formation of individual neurons, axons, dendrites, and their functional synapses during the development of neural network is a challenging task. To understand this task, microfluidic devices equipped with microchannels for reconstitution of cell/tissue-culture environments have been studied. Microfluidic devices are emerging as powerful tools in neurobiology, since they are capable of controlling and manipulating the microenvironment of the brain in a precise manner. They can enhance the physiological relevance of three-dimensional (3D) cell culture by allowing spatial control over fluids in micrometer-sized channels. Recent technological advancement in designing microfluidic platforms for studying neural communication, disease progression, and detection of neurotransmitters enhance our fundamental knowledge and understanding. However, more such advanced and innovative interventions are required. This Viewpoint focuses on highlighting a few of them with future scope of further advancement in this field.
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Electron microscopy studies on Banzi virus particle and its development in the suckling mice brains.
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Rotenone-driven DNA hypermethylation of the miR-6991-3p promoter induces death of mouse brain organoids.
Rotenone has potential chemical toxicity in the nervous system of both insects and mammals, but its deep molecular biological mechanisms have not been clarified. Here, the epigenetic regulatory mechanism underlying the toxicity of rotenone was studied using murine brain organoids (mBOs). Transmission electron microscopy indicated that rotenone destroyed mBOs'mitochondrial structure. RRBS-Seq showed that some promoter regions from the DLK1-DIO3 imprinted microRNA clusters were hypomethylated. But, rotenone stimulated hypermethylation significantly on the promoter DNA of miR-6991-3p. MiR-6991-3p in the rotenone-treated mBOs had the greatest decreased miRNA expression compared with the control. Meanwhile, luciferase report assay indicated that miR-6991-3p induced a decrease in luciferase activity via binding to specific sites on the 3'UTR of DEDD2 gene. To overexpression of miR-6991-3p attenuated mBO proliferated inhibition and cell death, accumulation for lipid peroxidation products significantly by rotenone inducing. Subsequently, results of cell staining and molecular biology experiment revealed that overexpression for miR-6991-3p significantly weakened expression levels of death-related genes (DEDD2, caspase-8, caspase-3, and caspase-1), but significantly elevated expression levels of cell proliferation-related genes (Ki67 and BCL2) in rotenone treated mBOs group. Here, we reveal a novel epigenetic mechanism of rotenone-induced neuronal death, in which rotenone induced promoter DNA hypermethylation of miR-6991-3p in the DLK1-DIO3 imprinted cluster. This caused miR-6991-3p transcriptional activity to be downregulated, which subsequently significantly increased the expression of its target gene, DEDD2, ultimately leading to neural organoid cell death.
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Antioxidants Ameliorates Ionizing Radiation-Induced Microcephaly in Cerebral Organoid Derived from Human-induced Pluripotent Stem Cells.
Ionizing radiation exposure induces DNA damage and chromosome aberrations through both direct and indirect effect. The indirect effects are primarily mediated by the generation of hydroxyl radicals, a process attributed to radiation. Dimethyl sulfoxide (DMSO) and ascorbic acid (AA) are known as radical scavengers and have radioprotective effects. Radiation therapy is widely employed in the treatment of malignant tumors such as glioblastoma; however, its side effects, including cognitive impairments resulting from damage to healthy neurons, pose significant challenges. To ameliorate these effects, radioprotective reagents have been sought. In this study, we used cerebral organoids derived from human-induced pluripotent stem cells to address the radioprotective effect of radical scavengers, DMSO and AA in brain exposure. Although exposure to radiation for 20-day-old cerebral organoids results in DNA double-strand breaks and apoptosis leading to microcephaly phenotype, treatment with DMSO or AA not only before but also after radiation alleviated DNA damage, cell death, and the microcephaly phenotype. Our results suggest that DMSO and AA are candidates for the radioprotective reagents for brain tumor therapy.
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Minibrain Storm : Cerebral Organoids Aren't Real Brains?But They Provide a Powerful Platform for Modeling Brain Diseases Like Zika Infection, Alzheimer's, and Even Autism.
Floating in a Petri dish, they look like tiny tapioca pearls in peach broth, a couple dozen in number and none much larger than the tip of a ballpoint pen. But under a microscope, dense, lumpy bodies come into focus, outlined by wispy coronas.
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Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons.
Recent advances in 3D culture systems have led to the generation of brain organoids that resemble different human brain regions; however, a 3D organoid model of the midbrain containing functional midbrain dopaminergic (mDA) neurons has not been reported. We developed a method to differentiate human pluripotent stem cells into a large multicellular organoid-like structure that contains distinct layers of neuronal cells expressing characteristic markers of human midbrain. Importantly, we detected electrically active and functionally mature mDA neurons and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using 2D methods or MLOs generated from mouse embryonic stem cells, our human MLOs produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a tractable in vitro system to study the human midbrain and its related diseases.