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2.
Elife ; 102021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33393462

RESUMO

Coronavirus entry is mediated by the spike protein that binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines, and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells and, more frequently, formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy.


Assuntos
Organoides/virologia , Sistema Respiratório/virologia , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus , Motivos de Aminoácidos , Animais , Fusão Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Vírus da SARS/química , Vírus da SARS/fisiologia , Serina Endopeptidases , Células Vero
3.
J Exp Med ; 218(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33433624

RESUMO

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus on the consequences of CNS infections. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in infected and neighboring neurons. However, no evidence for type I interferon responses was detected. We demonstrate that neuronal infection can be prevented by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate SARS-CoV-2 neuroinvasion in vivo. Finally, in autopsies from patients who died of COVID-19, we detect SARS-CoV-2 in cortical neurons and note pathological features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV-2 and an unexpected consequence of direct infection of neurons by SARS-CoV-2.


Assuntos
Anticorpos Bloqueadores/química , Córtex Cerebral , Neurônios , /metabolismo , /antagonistas & inibidores , Animais , /patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/virologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , Organoides/metabolismo , Organoides/patologia , Organoides/virologia
4.
Nat Commun ; 12(1): 109, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397937

RESUMO

Zn2+ plays important roles in metabolism and signaling regulation. Subcellular Zn2+ compartmentalization is essential for organelle functions and cell biology, but there is currently no method to determine Zn2+ signaling relationships among more than two different organelles with one probe. Here, we report simultaneous Zn2+ tracking in multiple organelles (Zn-STIMO), a method that uses structured illumination microscopy (SIM) and a single Zn2+ fluorescent probe, allowing super-resolution morphology-correlated organelle identification in living cells. To guarantee SIM imaging quality for organelle identification, we develop a new turn-on Zn2+ fluorescent probe, NapBu-BPEA, by regulating the lipophilicity of naphthalimide-derived Zn2+ probes to make it accumulate in multiple organelles except the nucleus. Zn-STIMO with this probe shows that CCCP-induced mitophagy in HeLa cells is associated with labile Zn2+ enhancement. Therefore, direct organelle identification supported by SIM imaging makes Zn-STIMO a reliable method to determine labile Zn2+ dynamics in various organelles with one probe. Finally, SIM imaging of pluripotent stem cell-derived organoids with NapBu-BPEA demonstrates the potential of super-resolution morphology-correlated organelle identification to track biospecies and events in specific organelles within organoids.


Assuntos
Rastreamento de Células , Organelas/metabolismo , Zinco/metabolismo , Autofagossomos/metabolismo , Autofagia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Sobrevivência Celular , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Espaço Intracelular/metabolismo , Lisossomos/metabolismo , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Naftalimidas/metabolismo , Organoides/metabolismo , Espectrometria de Fluorescência
5.
Life Sci ; 264: 118682, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33127519

RESUMO

AIMS: Menthacarin is a herbal combination that is clinically used for the treatment of functional gastrointestinal disorders (FGIDs). In several clinical studies, Menthacarin reduced visceral hypersensitivity-related symptoms. Pathogenesis of visceral hypersensitivity is multifactorial. This involves several cell types and different transient receptor potential ion channels (TRPs); these ion channels are highly conductive for calcium ions. Since transient changes in cytosolic calcium levels are crucial for many functions of living cells, we investigated if Menthacarin can induce calcium influx in sensory, largely nociceptive, neurons from dorsal root ganglia (DRG), peritoneal macrophages (PMs) and colonic organoids. MAIN METHODS: We employed the calcium imaging technique on sensory neurons from DRG, PMs and colonic organoids isolated from mice. All cells were superfused by Menthacarin at several concentrations (600, 1200, 1800 µg/ml) during the experiments, followed by calcium ionophor ionomycin (Iono., 1 µM) as a positive control. KEY FINDINGS: Menthacarin induced concentration-dependent calcium ion influx in all investigated cell types. Furthermore, repeated applications of Menthacarin induced tachyphylaxis (desensitisation) of calcium responses in sensory neurons and colonic organoids. SIGNIFICANCE: Menthacarin-induced calcium influx into sensory neurons, macrophages and colonic organoids is probably related to its clinical desensitising effects in patients with FGIDs.


Assuntos
Canais de Cálcio/metabolismo , Colo/metabolismo , Macrófagos/metabolismo , Organoides/metabolismo , Preparações de Plantas/farmacologia , Células Receptoras Sensoriais/metabolismo , Animais , Colo/citologia , Colo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Óleos Voláteis/química , Óleos Voláteis/farmacologia , Técnicas de Cultura de Órgãos , Organoides/efeitos dos fármacos , Preparações de Plantas/química , Células Receptoras Sensoriais/efeitos dos fármacos
6.
Oral Dis ; 27(1): 52-63, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32531849

RESUMO

OBJECTIVE: Hyposalivation-related xerostomia is an irreversible, untreatable, and frequent condition after radiotherapy for head and neck cancer. Stem cell therapy is an attractive option of treatment, but demands knowledge of stem cell functioning. Therefore, we aimed to develop a murine parotid gland organoid model to explore radiation response of stem cells in vitro. MATERIALS AND METHODS: Single cells derived from murine parotid gland organoids were passaged in Matrigel with defined medium to assess self-renewal and differentiation potential. Single cells were irradiated and plated in a 3D clonogenic stem cell survival assay to assess submandibular and parotid gland radiation response. RESULTS: Single cells derived from parotid gland organoids were able to extensively self-renew and differentiate into all major tissue cell types, indicating the presence of potential stem cells. FACS selection for known salivary gland stem cell markers CD24/CD29 did not further enrich for stem cells. The parotid gland organoid-derived stem cells displayed radiation dose-response curves similar to the submandibular gland. CONCLUSIONS: Murine parotid gland organoids harbor stem cells with long-term expansion and differentiation potential. This model is useful for mechanistic studies of stem cell radiation response and suggests similar radiosensitivity for the parotid and submandibular gland organoids.


Assuntos
Neoplasias de Cabeça e Pescoço , Radiação , Xerostomia , Animais , Camundongos , Organoides , Glândula Parótida , Glândulas Salivares , Glândula Submandibular
10.
Pest Manag Sci ; 77(1): 55-63, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32865304

RESUMO

Mammalian intestinal organoids are multicellular structures that closely resemble the structure of the intestinal epithelium and can be generated in vitro from intestinal stem cells under appropriate culture conditions. This technology has transformed pharmaceutical research and drug development in human medicine. For the insect gut, no biotechnological platform equivalent to organoid cultures has been described yet. Comparison of the regulation of intestinal homeostasis and growth between insects and mammals has revealed significant similarities but also important differences. In contrast to mammals, the differentiation potential of available insect cell lines is limited and can not be exploited for in vitro permeability assays to measure the uptake of insecticides. The successful development of in vitro models could be a result of the emergence of molecular mechanisms of self-organization and signaling in the intestine that are unique to mammals. It is nevertheless considered that the technology gap is a consequence of vast differences in knowledge, particularly with respect to culture conditions that maintain the differentation potential of insect midgut cells. From the viewpoint of pest control, advanced in vitro models of the insect midgut would be very desirable because of its key barrier function for orally ingested insecticides with hemolymphatic target and its role in insecticide resistance. © 2020 Society of Chemical Industry.


Assuntos
Microbioma Gastrointestinal , Organoides , Animais , Humanos , Insetos , Mamíferos , Tecnologia
11.
Sci Rep ; 10(1): 20987, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33268815

RESUMO

Homeostatic control of neuronal excitability by modulation of synaptic inhibition (I) and excitation (E) of the principal neurons is important during brain maturation. The fundamental features of in-utero brain development, including local synaptic E-I ratio and bioenergetics, can be modeled by cerebral organoids (CO) that have exhibited highly regular nested oscillatory network events. Therefore, we evaluated a 'Phase Zero' clinical study platform combining broadband Vis/near-infrared(NIR) spectroscopy and electrophysiology with studying E-I ratio based on the spectral exponent of local field potentials and bioenergetics based on the activity of mitochondrial Cytochrome-C Oxidase (CCO). We found a significant effect of the age of the healthy controls iPSC CO from 23 days to 3 months on the CCO activity (chi-square (2, N = 10) = 20, p = 4.5400e-05), and spectral exponent between 30-50 Hz (chi-square (2, N = 16) = 13.88, p = 0.001). Also, a significant effect of drugs, choline (CHO), idebenone (IDB), R-alpha-lipoic acid plus acetyl-L-carnitine (LCLA), was found on the CCO activity (chi-square (3, N = 10) = 25.44, p = 1.2492e-05), spectral exponent between 1 and 20 Hz (chi-square (3, N = 16) = 43.5, p = 1.9273e-09) and 30-50 Hz (chi-square (3, N = 16) = 23.47, p = 3.2148e-05) in 34 days old CO from schizophrenia (SCZ) patients iPSC. We present the feasibility of a multimodal approach, combining electrophysiology and broadband Vis-NIR spectroscopy, to monitor neurodevelopment in brain organoid models that can complement traditional drug design approaches to test clinically meaningful hypotheses.


Assuntos
Encéfalo/crescimento & desenvolvimento , Organoides/crescimento & desenvolvimento , Acetilcarnitina/farmacologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Estudos de Casos e Controles , Linhagem Celular , Colina/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Eletrofisiologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Mitocôndrias/metabolismo , Organoides/efeitos dos fármacos , Organoides/fisiologia , Estudo de Prova de Conceito , Esquizofrenia/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho , Ácido Tióctico/farmacologia , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia
12.
Cell Stem Cell ; 27(6): 876-889.e12, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33232663

RESUMO

SARS-CoV-2 infection has led to a global health crisis, and yet our understanding of the disease and potential treatment options remains limited. The infection occurs through binding of the virus with angiotensin converting enzyme 2 (ACE2) on the cell membrane. Here, we established a screening strategy to identify drugs that reduce ACE2 levels in human embryonic stem cell (hESC)-derived cardiac cells and lung organoids. Target analysis of hit compounds revealed androgen signaling as a key modulator of ACE2 levels. Treatment with antiandrogenic drugs reduced ACE2 expression and protected hESC-derived lung organoids against SARS-CoV-2 infection. Finally, clinical data on COVID-19 patients demonstrated that prostate diseases, which are linked to elevated androgen, are significant risk factors and that genetic variants that increase androgen levels are associated with higher disease severity. These findings offer insights on the mechanism of disproportionate disease susceptibility in men and identify antiandrogenic drugs as candidate therapeutics for COVID-19.


Assuntos
Androgênios/metabolismo , /metabolismo , Gravidade do Paciente , Transdução de Sinais , Adulto , Antagonistas de Androgênios , Androgênios/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Antivirais/uso terapêutico , /tratamento farmacológico , Células Cultivadas , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Organoides/efeitos dos fármacos , Organoides/virologia , Fatores de Risco , Fatores Sexuais , Células Vero
13.
Nature ; 588(7839): 670-675, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33238290

RESUMO

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.


Assuntos
/virologia , Pulmão/citologia , Modelos Biológicos , Organoides/citologia , Organoides/virologia , Técnicas de Cultura de Tecidos , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , /patologia , Diferenciação Celular , Divisão Celular , Células Clonais/citologia , Células Clonais/metabolismo , Células Clonais/virologia , Humanos , Técnicas In Vitro , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/fisiologia , Integrina alfa6/análise , Integrina beta4/análise , Queratina-5/análise , Organoides/metabolismo , Pneumonia Viral/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Análise de Célula Única , Receptor de TWEAK/análise
14.
Toxicology ; 446: 152614, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33199268

RESUMO

Many in vitro gastrointestinal models have been developed with the hope that they will continue to improve in their similarity to the organs from which they were isolated. Intestinal organoids isolated from various species are now being used to investigate physiology and pathophysiology. In this study, intestinal stem cells were isolated from adult rat duodenum and culture conditions were optimized to promote the growth, differentiation and development of 3D organoids. We optimized and characterized rat duodenal organoids with light and electron microscopy, immunofluorescence and notably, global mRNA expression. The metabolic capacity of these cultures was investigated using probe substrates for multiple phase I and phase II drug metabolizing enzymes and found to be in line with previous results from intestinal primary cultures and a significant improvement over immortalized cell lines. Over the course of differentiation, the gene expression profiles of the rat duodenal organoids were consistent with expected trends in differentiation to various cell lineages reflecting the duodenum in vivo. Further, incubations of these cultures with naproxen and celecoxib resulted in cytotoxicity consistent with the direct cytotoxic effects of these drugs to duodenum in vivo. Based on these characteristics, the rat duodenal organoids described herein will provide a novel platform for investigating a wide variety of mechanistic questions.


Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Diferenciação Celular/efeitos dos fármacos , Duodeno/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Organoides/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Duodeno/citologia , Duodeno/metabolismo , Feminino , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Organoides/metabolismo , Ratos , Ratos Sprague-Dawley , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
15.
Nat Commun ; 11(1): 5540, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139712

RESUMO

APOE4 is the strongest genetic risk factor associated with late-onset Alzheimer's disease (AD). To address the underlying mechanism, we develop cerebral organoid models using induced pluripotent stem cells (iPSCs) with APOE ε3/ε3 or ε4/ε4 genotype from individuals with either normal cognition or AD dementia. Cerebral organoids from AD patients carrying APOE ε4/ε4 show greater apoptosis and decreased synaptic integrity. While AD patient-derived cerebral organoids have increased levels of Aß and phosphorylated tau compared to healthy subject-derived cerebral organoids, APOE4 exacerbates tau pathology in both healthy subject-derived and AD patient-derived organoids. Transcriptomics analysis by RNA-sequencing reveals that cerebral organoids from AD patients are associated with an enhancement of stress granules and disrupted RNA metabolism. Importantly, isogenic conversion of APOE4 to APOE3 attenuates the APOE4-related phenotypes in cerebral organoids from AD patients. Together, our study using human iPSC-organoids recapitulates APOE4-related phenotypes and suggests APOE4-related degenerative pathways contributing to AD pathogenesis.


Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/metabolismo , Sinapses/metabolismo , Doença de Alzheimer/genética , Apolipoproteína E4/genética , Regulação da Expressão Gênica , Genótipo , Humanos , Organoides/patologia , RNA/metabolismo , Transcriptoma
17.
Artigo em Inglês | MEDLINE | ID: mdl-33202948

RESUMO

Growing evidence links prenatal exposure to particulate matter (PM2.5) with reduced lung function and incidence of pulmonary diseases in infancy and childhood. However, the underlying biological mechanisms of how prenatal PM2.5 exposure affects the lungs are incompletely understood, which explains the lack of an ideal in vitro lung development model. Human pluripotent stem cells (hPSCs) have been successfully employed for in vitro developmental toxicity evaluations due to their unique ability to differentiate into any type of cell in the body. In this study, we investigated the developmental toxicity of diesel fine PM (dPM2.5) exposure during hPSC-derived alveolar epithelial cell (AEC) differentiation and three-dimensional (3D) multicellular alveolar organoid (AO) development. We found that dPM2.5 (50 and 100 µg/mL) treatment disturbed the AEC differentiation, accompanied by upregulation of nicotinamide adenine dinucleotide phosphate oxidases and inflammation. Exposure to dPM2.5 also promoted epithelial-to-mesenchymal transition during AEC and AO development via activation of extracellular signal-regulated kinase signaling, while dPM2.5 had no effect on surfactant protein C expression in hPSC-derived AECs. Notably, we provided evidence, for the first time, that angiotensin-converting enzyme 2, a receptor to mediate the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) entry into target cells, and the cofactor transmembrane protease serine 2 were significantly upregulated in both hPSC-AECs and AOs treated with dPM2.5. In conclusion, we demonstrated the potential alveolar development toxicity and the increase of SARS-Cov-2 susceptibility of PM2.5. Our findings suggest that an hPSC-based 2D and 3D alveolar induction system could be a useful in vitro platform for evaluating the adverse effects of environmental toxins and for virus research.


Assuntos
Infecções por Coronavirus , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Pandemias , Material Particulado/toxicidade , Peptidil Dipeptidase A/genética , Células-Tronco Pluripotentes/efeitos dos fármacos , Pneumonia Viral , Betacoronavirus , Células Epiteliais/efeitos dos fármacos , Humanos , Organoides/efeitos dos fármacos , Regulação para Cima , Emissões de Veículos/toxicidade
20.
Cell Stem Cell ; 27(6): 951-961.e5, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33113348

RESUMO

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.


Assuntos
Barreira Hematoencefálica/virologia , Plexo Corióideo/virologia , /fisiologia , /metabolismo , Animais , Chlorocebus aethiops , Células HEK293 , Humanos , Modelos Biológicos , Organoides/virologia , Células Vero , Tropismo Viral , Internalização do Vírus
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