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Amyotrophic lateral sclerosis (ALS) causes progressive degeneration of the motor neurons. In this study, we delivered the genetic construct including the whole locus of human mutant superoxide dismutase 1 (SOD1) with the promoter region of human SOD1 into porcine zygotes using intracytoplasmic sperm injection-mediated gene transfer, and we thereby generated a pig model of human mutant SOD1-mediated familial ALS. The established ALS pig model exhibited an initial abnormality of motor neurons with accumulated misfolded SOD1. The ALS pig model, with a body size similar to that of human beings, will provide opportunities for cell and gene therapy platforms in preclinical translational research.
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Esclerose Lateral Amiotrófica , Superóxido Dismutase-1 , Animais , Humanos , Masculino , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Modelos Animais de Doenças , Neurônios Motores/patologia , Mutação , Sêmen , Superóxido Dismutase-1/genética , SuínosRESUMO
In amyotrophic lateral sclerosis (ALS), early diagnosis is essential for both current and potential treatments. To find a supportive approach for the diagnosis, we constructed an artificial intelligence-based prediction model of ALS using induced pluripotent stem cells (iPSCs). Images of spinal motor neurons derived from healthy control subject and ALS patient iPSCs were analyzed by a convolutional neural network, and the algorithm achieved an area under the curve of 0.97 for classifying healthy control and ALS. This prediction model by deep learning algorithm with iPSC technology could support the diagnosis and may provide proactive treatment of ALS through future prospective research. ANN NEUROL 2021;89:1226-1233.
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Esclerose Lateral Amiotrófica/diagnóstico , Aprendizado Profundo , Diagnóstico Precoce , Células-Tronco Pluripotentes Induzidas , Neurônios Motores , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Human pluripotent stem cells have the potential to differentiate into various cell types including skeletal muscles (SkM), and they are applied to regenerative medicine or in vitro modelling for intractable diseases. A simple differentiation method is required for SkM cells to accelerate neuromuscular disease studies. Here, we established a simple method to convert human pluripotent stem cells into SkM cells by using temperature-sensitive Sendai virus (SeV) vector encoding myoblast determination protein 1 (SeV-Myod1), a myogenic master transcription factor. SeV-Myod1 treatment converted human embryonic stem cells (ESCs) into SkM cells, which expressed SkM markers including myosin heavy chain (MHC). We then removed the SeV vector by temporal treatment at a high temperature of 38â, which also accelerated mesodermal differentiation, and found that SkM cells exhibited fibre-like morphology. Finally, after removal of the residual human ESCs by pluripotent stem cell-targeting delivery of cytotoxic compound, we generated SkM cells with 80% MHC positivity and responsiveness to electrical stimulation. This simple method for myogenic differentiation was applicable to human-induced pluripotent stem cells and will be beneficial for investigations of disease mechanisms and drug discovery in the future.
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Diferenciação Celular , Vetores Genéticos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Vírus Sendai , Cálcio/metabolismo , Sinalização do Cálcio , Diferenciação Celular/genética , Células Cultivadas , Reprogramação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Imunofluorescência , Expressão Gênica , Vetores Genéticos/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Desenvolvimento Muscular/genética , Vírus Sendai/genética , Temperatura , TransgenesRESUMO
Amyloid ß-protein (Aß) molecules tend to aggregate and subsequently form low MW (LMW) oligomers, high MW (HMW) aggregates such as protofibrils, and ultimately fibrils. These Aß species can generally form amyloid plaques implicated in the neurodegeneration of Alzheimer disease (AD), but therapies designed to reduce plaque load have not demonstrated clinical efficacy. Recent evidence implicates amyloid oligomers in AD neuropathology, but the precise mechanisms are uncertain. We examined the mechanisms of neuronal dysfunction from HMW-Aß1-42 exposure by measuring membrane integrity, reactive oxygen species (ROS) generation, membrane lipid peroxidation, membrane fluidity, intracellular calcium regulation, passive membrane electrophysiological properties, and long-term potentiation (LTP). HMW-Aß1-42 disturbed membrane integrity by inducing ROS generation and lipid peroxidation, resulting in decreased membrane fluidity, intracellular calcium dysregulation, depolarization, and impaired LTP. The damaging effects of HMW-Aß1-42 were significantly greater than those of LMW-Aß1-42. Therapeutic reduction of HMW-Aß1-42 may prevent AD progression by ameliorating direct neuronal membrane damage.-Yasumoto, T., Takamura, Y., Tsuji, M., Watanabe-Nakayama, T., Imamura, K., Inoue, H., Nakamura, S., Inoue, T., Kimura, A., Yano, S., Nishijo, H., Kiuchi, Y., Teplow, D. B., Ono, K. High molecular weight amyloid ß1-42 oligomers induce neurotoxicity via plasma membrane damage.
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Peptídeos beta-Amiloides/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Cálcio/metabolismo , Linhagem Celular Tumoral , Eletrofisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Fluidez de Membrana , Microscopia de Força Atômica , Peso Molecular , Técnicas de Patch-Clamp , Espécies Reativas de Oxigênio/metabolismoRESUMO
Recent reports, including ours, have indicated that microRNA (miR)-33 located within the intron of sterol regulatory element binding protein (SREBP) 2 controls cholesterol homeostasis and can be a potential therapeutic target for the treatment of atherosclerosis. Here, we show that SPAST, which encodes a microtubule-severing protein called SPASTIN, was a novel target gene of miR-33 in human. Actually, the miR-33 binding site in the SPAST 3'-UTR is conserved not in mice but in mid to large mammals, and it is impossible to clarify the role of miR-33 on SPAST in mice. We demonstrated that inhibition of miR-33a, a major form of miR-33 in human neurons, via locked nucleic acid (LNA)-anti-miR ameliorated the pathological phenotype in hereditary spastic paraplegia (HSP)-SPG4 patient induced pluripotent stem cell (iPSC)-derived cortical neurons. Thus, miR-33a can be a potential therapeutic target for the treatment of HSP-SPG4.
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Terapia Genética/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/genética , Células-Tronco Neurais/metabolismo , Neuritos/metabolismo , Oligonucleotídeos/genética , Paraplegia Espástica Hereditária/terapia , Espastina/genética , Regiões 3' não Traduzidas , Sítios de Ligação , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Células-Tronco Neurais/patologia , Neuritos/patologia , Neurogênese , Oligonucleotídeos/metabolismo , Fenótipo , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Paraplegia Espástica Hereditária/patologia , Espastina/metabolismoRESUMO
Dopamine (DA) is a neurotransmitter in the brain, playing a central role in several disease conditions, including tetrahydrobiopterin (BH4) metabolism disorders and Parkinson's disease (PD). BH4 metabolism disorders present a variety of clinical manifestations including motor disturbance via altered DA metabolism, since BH4 is a cofactor for tyrosine hydroxylase (TH), a rate-limiting enzyme for DA synthesis. Genetically, BH4 metabolism disorders are, in an autosomal recessive pattern, caused by a variant in genes encoding enzymes for BH4 synthesis or recycling, including 6-pyruvoyltetrahydropterin synthase (PTPS) or dihydropteridine reductase (DHPR), respectively. Although BH4 metabolism disorders and its metabolisms have been studied, it is unclear how gene variants cause aberrant DA synthesis in patient neurons. Here, we generated induced pluripotent stem cells (iPSCs) from BH4 metabolism disorder patients with PTPS or DHPR variants, corrected the gene variant in the iPSCs using the CRISPR/Cas9 system, and differentiated the BH4 metabolism disorder patient- and isogenic control iPSCs into midbrain DA neurons. We found that by the gene correction, the BH4 amount, TH protein level and extracellular DA level were restored in DA neuronal culture using PTPS deficiency iPSCs. Furthermore, the pharmacological correction by BH4 precursor sepiapterin treatment also improved the phenotypes of PTPS deficiency. These results suggest that patient iPSCs with BH4 metabolism disorders provide an opportunity for screening substances for treating aberrant DA synthesis-related disorders.
Assuntos
Biopterinas/análogos & derivados , Dopamina/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Doenças Metabólicas/genética , Doença de Parkinson/genética , Biopterinas/metabolismo , Diferenciação Celular/genética , Dopamina/biossíntese , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Genótipo , Humanos , Cariótipo , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Pterinas/metabolismo , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
Chromosome 15q11.2-13.1 duplication (Dup15q) syndrome is one of the most common autism spectrum disorders (ASDs) associated with copy number variants (CNVs). For the analysis of CNV-relevant pathological cellular phenotypes, a CNV-corrected isogenic cell line is useful for excluding the influence of genetic background. Here, we devised a strategy to remove the isodicentric chromosome 15 by inserting a puro-ΔTK selection cassette into the extra chromosome using the CRISPR-Cas9 system, followed by a subsequent two-step drug selection. A series of assays, including qPCR-based copy number analysis and karyotype analysis, confirmed the elimination of the extra chromosome. Furthermore, cerebral organoids were generated from the parental Dup15q iPSCs and their isogenic iPSCs. scRNA-seq analysis revealed the alteration of expression levels in ion-channel-related genes and synapse-related genes in glutamatergic and GABAergic neurons in Dup15q organoids, respectively. The established isogenic cell line is a valuable resource for unraveling cellular and molecular alterations associated with Dup15q syndrome.
Assuntos
Cromossomos Humanos Par 15 , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 15/metabolismo , Duplicação Cromossômica , Variações do Número de Cópias de DNA , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Organoides/metabolismo , Aberrações Cromossômicas , Deficiência IntelectualRESUMO
Neural organoids consist of three-dimensional tissue derived from pluripotent stem cells that could recapitulate key features of the human brain. During the past decade, organoid technology has evolved in the field of human brain science by increasing the quality and applicability of its products. Among them, a novel approach involving the design of neural organoids engineered by mechanical forces has emerged. This review describes previous approaches for the generation of neural organoids, the engineering of neural organoids by mechanical forces, and future challenges for the application of mechanical forces in the design of neural organoids.
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Synucleinopathies refer to a group of disorders characterized by SNCA/α-synuclein (α-Syn)-containing cytoplasmic inclusions and neuronal cell loss in the nervous system including the cortex, a common feature being cognitive impairment. Still, the molecular pathogenesis of cognitive decline remains poorly understood, hampering the development of effective treatments. Here, we generated induced pluripotent stem cells (iPSCs) derived from familial Parkinson's disease (PD) patients carrying SNCA A53T mutation, differentiating them into cortical neurons by a direct conversion method. Patient iPSCs-derived cortical neurons harboring mutant α-Syn exhibited increased α-Syn-positive aggregates, shorter neurites, and time-dependent vulnerability. Furthermore, RNA-sequencing analysis, followed by biochemical validation, identified the activation of the ERK1/2 and JNK cascades in cortical neurons with SNCA A53T mutation. This result was consistent with a reverted phenotype of neuronal death in cortical neurons when treated with ERK1/2 and JNK inhibitors, respectively. Our findings emphasize the role of ERK1/2 and JNK cascades in the vulnerability of cortical neurons in synucleinopathies, and they could pave the way toward therapeutic advancements for synucleinopathies.
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Sinucleinopatias , alfa-Sinucleína , Humanos , Sistema de Sinalização das MAP Quinases , Neurônios , NeuritosRESUMO
Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein-coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.
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Células-Tronco Pluripotentes Induzidas , Organoides , Distrofias Retinianas , Peixe-Zebra , Animais , Humanos , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Luz/efeitos adversos , Mutação , Organoides/metabolismo , Retina/metabolismo , Retina/patologia , Distrofias Retinianas/terapia , Distrofias Retinianas/genética , Distrofias Retinianas/metabolismoRESUMO
Induced pluripotent stem cells (iPSC) are derived from somatic cells. These somatic cells have had their gene expression experimentally reprogrammed to an embryonic stem cell-like pluripotent state, gaining the capacity to differentiate various cell types in the three embryonic germ layers. Thus, iPSC technology makes it possible to obtain neuronal cells from any human cells. iPSC can be generated from various kinds of somatic cells and from patients with neurodegenerative diseases. Disease modelling using iPSC technology would elucidate the pathogenesis of such diseases and contribute to related drug discoveries. In this review, we discuss the recent advances in iPSC technology as well as its potential applications.
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Células-Tronco Pluripotentes Induzidas/fisiologia , Doenças Neurodegenerativas/fisiopatologia , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Proteínas de Transporte/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Análise Mutacional de DNA , Modelos Animais de Doenças , Descoberta de Drogas , Disautonomia Familiar/tratamento farmacológico , Disautonomia Familiar/genética , Disautonomia Familiar/fisiopatologia , Éxons/genética , Humanos , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Neurônios/fisiologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , RNA Mensageiro/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Fatores de Elongação da TranscriçãoRESUMO
In this study, we compared the disaster relief practices of nurses who worked in welfare shelters in Iwate and Miyagi Prefectures, areas in which only natural disasters occurred, and nurses who worked in Fukushima Prefecture, an area in which both nuclear and natural disasters occurred during the Great East Japan Earthquake in 2011, in order to identify commonalities and differences between them. We conducted semi-structured interviews with two nurses from each prefecture. The results revealed that "nursing practice with minimal available materials and personnel" and "nursing practice based on knowledge and experience as a nurse" were common themes in the content of nursing practices, whereas "securing human resources during disasters and considering ideal welfare evacuation centers" and "recording the difficulties in dealing with nuclear disasters" were uncommon themes. The findings confirmed that even in Fukushima Prefecture, in which the nuclear disaster occurred, participants did not talk about their concerns regarding radiation exposure while working at welfare evacuation shelters where people with special requirements were evacuated, and that they were expected to respond in the same way as they would in natural disasters. However, participants reported several difficulties relating to nuclear disasters that should be considered in future disaster support.
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Desastres , Terremotos , Acidente Nuclear de Fukushima , Exposição à Radiação , Humanos , Abrigo de Emergência , JapãoRESUMO
Mutations within Superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS), accounting for approximately 20% of familial cases. The pathological feature is a loss of motor neurons with enhanced formation of intracellular misfolded SOD1. Homozygous SOD1-D90A in familial ALS has been reported to show slow disease progression. Here, we reported a rare case of a slowly progressive ALS patient harboring a novel SOD1 homozygous mutation D92G (homD92G). The neuronal cell line overexpressing SOD1-D92G showed a lower ratio of the insoluble/soluble fraction of SOD1 with fine aggregates of the misfolded SOD1 and lower cellular toxicity than those overexpressing SOD1-G93A, a mutation that generally causes rapid disease progression. Next, we analyzed spinal motor neurons derived from induced pluripotent stem cells (iPSC) of a healthy control subject and ALS patients carrying SOD1-homD92G or heterozygous SOD1-L144FVX mutation. Lower levels of misfolded SOD1 and cell loss were observed in the motor neurons differentiated from patient-derived iPSCs carrying SOD1-homD92G than in those carrying SOD1-L144FVX. Taken together, SOD1-homD92G has a lower propensity to aggregate and induce cellular toxicity than SOD1-G93A or SOD1-L144FVX, and these cellular phenotypes could be associated with the clinical course of slowly progressive ALS.
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Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/metabolismo , Animais , Modelos Animais de Doenças , Progressão da Doença , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Mutação , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
Retinal dystrophies (RDs) lead to irreversible vision impairment with no radical treatment. Although photoreceptor cells (PRCs) differentiated from human induced pluripotent stem cells (iPSCs) are essential for the study of RDs as a scalable source, current differentiation methods for PRCs require multiple steps. To address these issues, we developed a method to generate PRCs from human iPSCs by introducing the transcription factors, CRX and NEUROD1. This approach enabled us to generate induced photoreceptor-like cells (iPRCs) expressing PRC markers. Single-cell RNA sequencing revealed the transcriptome of iPRCs in which the genes associated with phototransduction were expressed. Generated iPRCs exhibited their functional properties in calcium imaging. Furthermore, light-induced damage on iPRCs was inhibited by an antioxidant compound. This simple approach would facilitate the availability of materials for PRC-related research and provide a useful application for disease modeling and drug discovery.
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Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by the loss of motor neurons, and development of effective medicines is urgently required. Induced pluripotent stem cell (iPSC)-based drug repurposing identified the Src/c-Abl inhibitor bosutinib, which is approved for the treatment of chronic myelogenous leukemia (CML), as a candidate for the molecular targeted therapy of ALS. Methods: An open-label, multicentre, dose-escalation phase 1 study using a 3 + 3 design was conducted in 4 hospitals in Japan to evaluate the safety and tolerability of bosutinib in patients with ALS. Furthermore, the exploratory efficacy was evaluated using Revised ALS Functional Rating Scale (ALSFRS-R), predictive biomarkers including plasma neurofilament light chain (NFL) were explored, and single-cell RNA sequencing of iPSC-derived motor neurons was conducted. Patients, whose total ALSFRS-R scores decreased by 1-3 points during the 12-week, received escalating doses starting from 100 mg quaque die (QD) up to 400 mg QD based on dose-limiting toxicity (DLT) occurrence, and all participants who received one dose of the study drug were included in the primary analysis. This trial is registered with ClinicalTrials.gov, NCT04744532, as Induced pluripotent stem cell-based Drug Repurposing for Amyotrophic Lateral Sclerosis Medicine (iDReAM) study. Findings: Between March 29, 2019 and May 7, 2021, 20 patients were enrolled, 13 of whom received bosutinib treatment and 12 were included in the safety and efficacy analyses. No DLTs were observed up to 300 mg QD, but DLTs were observed in 3/3 patients of the 400 mg QD cohort. In all patients receiving 100 mg-400 mg, the prevalent adverse events (AEs) were gastrointestinal AEs in 12 patients (92.3%), liver function related AEs in 7 patients (53.8%), and rash in 3 patients (23.1%). The safety profile was consistent with that known for CML treatment, and ALS-specific AEs were not observed. A subset of patients (5/9 patients) was found to respond well to bosutinib treatment over the 12-week treatment period. It was found that the treatment-responsive patients could be distinguished by their lower levels of plasma NFL. Furthermore, single-cell RNA sequencing of iPSC-derived motor neurons revealed the pathogenesis related molecular signature in patients with ALS showing responsiveness to bosutinib. Interpretation: This is the first trial of a Src/c-Abl inhibitor, bosutinib, for patients with ALS. The safety and tolerability of bosutinib up to 300 mg, not 400 mg, in ALS were described, and responsiveness of patients on motor function was observed. Since this was an open-label trial within a short period with a limited number of patients, further clinical trials will be required. Funding: AMED and iPS Cell Research Fund.
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Human motor neurons are important materials for the research of the pathogenesis and drug discovery of motor neuron diseases. Various methods to generate motor neurons (MNs) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) by the addition of signaling molecules have been reported. However, they require multiple steps and complicated processes. Here we describe an approach for generating human MNs from ESCs/iPSCs using a single Sendai virus vector encoding three transcription factors-Lhx3, Ngn2, and Isl1. This approach enabled us to generate MNs in one step, adding Sendai virus vector in culture medium. This simple method significantly reduces the efforts to generate MNs, and it provides a useful tool for motor neuron disease research.
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Diferenciação Celular , Células-Tronco Embrionárias/citologia , Vetores Genéticos , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios Motores/citologia , Vírus Sendai , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Vírus Sendai/genética , Fatores de Transcrição/genética , TransgenesRESUMO
Idiopathic basal ganglia calcification (IBGC) is a rare neurodegenerative disease, characterized by abnormal calcium deposits in basal ganglia of the brain. The affected individuals exhibit movement disorders, and progressive deterioration of cognitive and psychiatric ability. The genetic cause of the disease is mutation in one of several different genes, SLC20A2, PDGFB, PDGFRB, XPR1 or MYORG, which inheritably or sporadically occurs. Here we generated an induced pluripotent stem cell (iPSC) line from an IBGC patient, which is likely be a powerful tool for revealing the pathomechanisms and exploring potential therapeutic candidates of IBGC.
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Doenças dos Gânglios da Base , Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Gânglios da Base/metabolismo , Doenças dos Gânglios da Base/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Receptor do Retrovírus Politrópico e XenotrópicoRESUMO
Schizophrenia (SCZ) is one of the major psychiatric disorders. The genetic factor is certainly influential in the onset of the disease but is not decisive. There is no identified molecular/cellular marker of the disease, and the pathomechanism is still unknown. In this study, we generated human induced pluripotent stem cells (iPSCs) derived from SCZ-discordant fraternal twins, and they could contribute to elucidation of the pathomechanism of SCZ.
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Células-Tronco Pluripotentes Induzidas , Esquizofrenia , Humanos , Esquizofrenia/genética , Gêmeos DizigóticosRESUMO
Organoid technology provides an opportunity to generate brain-like structures by recapitulating developmental steps in the manner of self-organization. Here we examined the vertical-mixing effect on brain organoid structures using bioreactors and established inverted brain organoids. The organoids generated by vertical mixing showed neurons that migrated from the outer periphery to the inner core of organoids, in contrast to orbital mixing. Computational analysis of flow dynamics clarified that, by comparison with orbital mixing, vertical mixing maintained the high turbulent energy around organoids, and continuously kept inter-organoid distances by dispersing and adding uniform rheological force on organoids. To uncover the mechanisms of the inverted structure, we investigated the direction of primary cilia, a cellular mechanosensor. Primary cilia of neural progenitors by vertical mixing were aligned in a multidirectional manner, and those by orbital mixing in a bidirectional manner. Single-cell RNA sequencing revealed that neurons of inverted brain organoids presented a GABAergic character of the ventral forebrain. These results suggest that controlling fluid dynamics by biomechanical engineering can direct stem cell differentiation of brain organoids, and that inverted brain organoids will be applicable for studying human brain development and disorders in the future.
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Reatores Biológicos , Encéfalo/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Organoides/citologia , HumanosRESUMO
Human pathogenic RNA viruses are threats to public health because they are prone to escaping the human immune system through mutations of genomic RNA, thereby causing local outbreaks and global pandemics of emerging or re-emerging viral diseases. While specific therapeutics and vaccines are being developed, a broad-spectrum therapeutic agent for RNA viruses would be beneficial for targeting newly emerging and mutated RNA viruses. In this study, we conducted a screen of repurposed drugs using Sendai virus (an RNA virus of the family Paramyxoviridae), with human-induced pluripotent stem cells (iPSCs) to explore existing drugs that may present anti-RNA viral activity. Selected hit compounds were evaluated for their efficacy against two important human pathogens: Ebola virus (EBOV) using Huh7 cells and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using Vero E6 cells. Selective estrogen receptor modulators (SERMs), including raloxifene, exhibited antiviral activities against EBOV and SARS-CoV-2. Pioglitazone, a PPARγ agonist, also exhibited antiviral activities against SARS-CoV-2, and both raloxifene and pioglitazone presented a synergistic antiviral effect. Finally, we demonstrated that SERMs blocked entry steps of SARS-CoV-2 into host cells. These findings suggest that the identified FDA-approved drugs can modulate host cell susceptibility against RNA viruses.