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1.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800815

RESUMO

In the last decade, different research groups in the academic setting have developed induced pluripotent stem cell-based protocols to generate three-dimensional, multicellular, neural organoids. Their use to model brain biology, early neural development, and human diseases has provided new insights into the pathophysiology of neuropsychiatric and neurological disorders, including microcephaly, autism, Parkinson's disease, and Alzheimer's disease. However, the adoption of organoid technology for large-scale drug screening in the industry has been hampered by challenges with reproducibility, scalability, and translatability to human disease. Potential technical solutions to expand their use in drug discovery pipelines include Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to create isogenic models, single-cell RNA sequencing to characterize the model at a cellular level, and machine learning to analyze complex data sets. In addition, high-content imaging, automated liquid handling, and standardized assays represent other valuable tools toward this goal. Though several open issues still hamper the full implementation of the organoid technology outside academia, rapid progress in this field will help to prompt its translation toward large-scale drug screening for neurological disorders.


Assuntos
Descoberta de Drogas/métodos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Doenças do Sistema Nervoso/tratamento farmacológico , Organoides/efeitos dos fármacos , Animais , Automação , Encéfalo/citologia , Sistemas CRISPR-Cas , Técnicas de Cultura de Células , Colágeno , Combinação de Medicamentos , Avaliação Pré-Clínica de Medicamentos/métodos , Indústria Farmacêutica/organização & administração , Previsões , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Laminina , Aprendizado de Máquina , Microscopia/métodos , Doenças do Sistema Nervoso/patologia , Proteoglicanas , RNA-Seq , Reprodutibilidade dos Testes , Análise de Célula Única
2.
J Vis Exp ; (169)2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33749682

RESUMO

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel). We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina. The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.


Assuntos
Descoberta de Drogas , Organoides/fisiologia , Retina/fisiologia , Testes de Toxicidade , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/fisiologia , Fenofibrato/toxicidade , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Organoides/efeitos dos fármacos , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/toxicidade
3.
Nat Commun ; 12(1): 1836, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758175

RESUMO

To prevent damage to the host or its commensal microbiota, epithelial tissues must match the intensity of the immune response to the severity of a biological threat. Toll-like receptors allow epithelial cells to identify microbe associated molecular patterns. However, the mechanisms that mitigate biological noise in single cells to ensure quantitatively appropriate responses remain unclear. Here we address this question using single cell and single molecule approaches in mammary epithelial cells and primary organoids. We find that epithelial tissues respond to bacterial microbe associated molecular patterns by activating a subset of cells in an all-or-nothing (i.e. digital) manner. The maximum fraction of responsive cells is regulated by a bimodal epigenetic switch that licenses the TLR2 promoter for transcription across multiple generations. This mechanism confers a flexible memory of inflammatory events as well as unique spatio-temporal control of epithelial tissue-level immune responses. We propose that epigenetic licensing in individual cells allows for long-term, quantitative fine-tuning of population-level responses.


Assuntos
Bactérias/imunologia , Células Epiteliais/imunologia , Imunidade Inata , Lipopeptídeos/imunologia , NF-kappa B/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Bactérias/metabolismo , Linhagem Celular , Citocinas/metabolismo , Citocinas/farmacologia , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Flagelina/farmacologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Processamento de Imagem Assistida por Computador , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Hibridização in Situ Fluorescente , Glândulas Mamárias Animais , Camundongos , Organoides/efeitos dos fármacos , Organoides/imunologia , Organoides/metabolismo , Regiões Promotoras Genéticas , RNA-Seq , Transdução de Sinais/imunologia , Análise de Célula Única , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/genética , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo
4.
Life Sci ; 271: 119195, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33581125

RESUMO

AIMS: Ulcerative colitis and Crohn's disease, collectively known as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestine for which key elements in disease initiation and perpetuation are defects in epithelial barrier integrity. Achieving mucosal healing is essential to ameliorate disease outcome and so new therapies leading to epithelial homeostasis and repair are under investigation. This study was designed to determine the mechanisms by which IL-22 regulates intestinal epithelial cell function. MAIN METHODS: Human intestinal organoids and resections, as well as mice were used to evaluate the effect of IL-22 on stem cell expansion, proliferation and expression of mucus components. IL-22 effect on barrier function was assessed in polarized T-84 cell monolayers. Butyrate co-treatments and organoid co-cultures with immune cells were performed to monitor the impact of microbial-derived metabolites and inflammatory environments on IL-22 responses. KEY FINDINGS: IL-22 led to epithelial stem cell expansion, proliferation, barrier dysfunction and anti-microbial peptide production in human and mouse models evaluated. IL-22 also altered the mucus layer by inducing an increase in membrane mucus but a decrease in secreted mucus and goblet cell content. IL-22 had the same effect on anti-microbial peptides and membrane mucus in both healthy and IBD human samples. In contrast, this IL-22-associated epithelial phenotype was different when treatments were performed in presence of butyrate and organoids co-cultured with immune cells. SIGNIFICANCE: Our data indicate that IL-22 promotes epithelial regeneration, innate defense and membrane mucus production, strongly supporting the potential clinical utility of IL-22 as a mucosal healing therapy in IBD.


Assuntos
Células Epiteliais/fisiologia , Homeostase/fisiologia , Interleucinas/fisiologia , Interleucinas/uso terapêutico , Mucosa Intestinal/fisiologia , Animais , Linhagem Celular , Técnicas de Cocultura , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Células Epiteliais/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Interleucinas/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Organoides/efeitos dos fármacos , Organoides/fisiologia
5.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530458

RESUMO

A high-throughput drug screen identifies potentially promising therapeutics for clinical trials. However, limitations that persist in current disease modeling with limited physiological relevancy of human patients skew drug responses, hamper translation of clinical efficacy, and contribute to high clinical attritions. The emergence of induced pluripotent stem cell (iPSC) technology revolutionizes the paradigm of drug discovery. In particular, iPSC-based three-dimensional (3D) tissue engineering that appears as a promising vehicle of in vitro disease modeling provides more sophisticated tissue architectures and micro-environmental cues than a traditional two-dimensional (2D) culture. Here we discuss 3D based organoids/spheroids that construct the advanced modeling with evolved structural complexity, which propels drug discovery by exhibiting more human specific and diverse pathologies that are not perceived in 2D or animal models. We will then focus on various central nerve system (CNS) disease modeling using human iPSCs, leading to uncovering disease pathogenesis that guides the development of therapeutic strategies. Finally, we will address new opportunities of iPSC-assisted drug discovery with multi-disciplinary approaches from bioengineering to Omics technology. Despite technological challenges, iPSC-derived cytoarchitectures through interactions of diverse cell types mimic patients' CNS and serve as a platform for therapeutic development and personalized precision medicine.


Assuntos
Doenças do Sistema Nervoso Central/tratamento farmacológico , Descoberta de Drogas/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Engenharia Tecidual/métodos , Animais , /patologia , Doenças do Sistema Nervoso Central/patologia , Descoberta de Drogas/instrumentação , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Dispositivos Lab-On-A-Chip , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/patologia , Engenharia Tecidual/instrumentação , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/patologia
6.
Mol Pharmacol ; 99(4): 256-265, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33547249

RESUMO

The high failure rate of drugs in the clinical pipeline is likely in part the result of inadequate preclinical models, particularly those for neurologic disorders and neurodegenerative disease. Such preclinical animal models often suffer from fundamental species differences and rarely recapitulate all facets of neurologic conditions, whereas conventional two-dimensional (2D) in vitro models fail to capture the three-dimensional spatial organization and cell-to-cell interactions of brain tissue that are presumed to be critical to the function of the central nervous system. Recent studies have suggested that stem cell-derived neuronal organoids are more physiologically relevant than 2D neuronal cultures because of their cytoarchitecture, electrophysiological properties, human origin, and gene expression. Hence there is interest in incorporating such physiologically relevant models into compound screening and lead optimization efforts within drug discovery. However, despite their perceived relevance, compared with previously used preclinical models, little is known regarding their predictive value. In fact, some have been wary to broadly adopt organoid technology for drug discovery because of the low-throughput and tedious generation protocols, inherent variability, and lack of compatible moderate-to-high-throughput screening assays. Consequently, microfluidic platforms, specialized bioreactors, and automated assays have been and are being developed to address these deficits. This mini review provides an overview of the gaps to broader implementation of neuronal organoids in a drug discovery setting as well as emerging technologies that may better enable their utilization. SIGNIFICANCE STATEMENT: Neuronal organoid models offer the potential for a more physiological system in which to study neurological diseases, and efforts are being made to employ them not only in mechanistic studies but also in profiling/screening purposes within drug discovery. In addition to exploring the utility of neuronal organoid models within this context, efforts in the field aim to standardize such models for consistency and adaptation to screening platforms for throughput evaluation. This review covers potential impact of and hurdles to implementation.


Assuntos
Descoberta de Drogas/métodos , Doenças Neurodegenerativas/tratamento farmacológico , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Organoides/efeitos dos fármacos , Organoides/fisiologia , Animais , Descoberta de Drogas/tendências , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Doenças Neurodegenerativas/fisiopatologia
7.
Cell Stem Cell ; 28(2): 331-342.e5, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33450186

RESUMO

ApoE4, a strong genetic risk factor for Alzheimer disease, has been associated with increased risk for severe COVID-19. However, it is unclear whether ApoE4 alters COVID-19 susceptibility or severity, and the role of direct viral infection in brain cells remains obscure. We tested the neurotropism of SARS-CoV2 in human-induced pluripotent stem cell (hiPSC) models and observed low-grade infection of neurons and astrocytes that is boosted in neuron-astrocyte co-cultures and organoids. We then generated isogenic ApoE3/3 and ApoE4/4 hiPSCs and found an increased rate of SARS-CoV-2 infection in ApoE4/4 neurons and astrocytes. ApoE4 astrocytes exhibited enlarged size and elevated nuclear fragmentation upon SARS-CoV-2 infection. Finally, we show that remdesivir treatment inhibits SARS-CoV2 infection of hiPSC neurons and astrocytes. These findings suggest that ApoE4 may play a causal role in COVID-19 severity. Understanding how risk factors impact COVID-19 susceptibility and severity will help us understand the potential long-term effects in different patient populations.


Assuntos
Apolipoproteínas E/metabolismo , Encéfalo/patologia , Encéfalo/virologia , Células-Tronco Pluripotentes Induzidas/virologia , Tropismo/fisiologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Antivirais/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Astrócitos/virologia , Diferenciação Celular , Chlorocebus aethiops , Humanos , Degeneração Neural/patologia , Neuritos/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/virologia , Organoides/efeitos dos fármacos , Organoides/patologia , Organoides/virologia , Isoformas de Proteínas/metabolismo , Sinapses/patologia , Células Vero
8.
Int J Mol Sci ; 22(2)2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440617

RESUMO

For obvious reasons, such as, e.g., ethical concerns or sample accessibility, model systems are of highest importance to study the underlying molecular mechanisms of human maladies with the aim to develop innovative and effective therapeutic strategies. Since many years, animal models and highly proliferative transformed cell lines are successfully used for disease modelling, drug discovery, target validation, and preclinical testing. Still, species-specific differences regarding genetics and physiology and the limited suitability of immortalized cell lines to draw conclusions on normal human cells or specific cell types, are undeniable shortcomings. The progress in human pluripotent stem cell research now allows the growth of a virtually limitless supply of normal and DNA-edited human cells, which can be differentiated into various specific cell types. However, cells in the human body never fulfill their functions in mono-lineage isolation and diseases always develop in complex multicellular ecosystems. The recent advances in stem cell-based 3D organoid technologies allow a more accurate in vitro recapitulation of human pathologies. Embryoids are a specific type of such multicellular structures that do not only mimic a single organ or tissue, but the entire human conceptus or at least relevant components of it. Here we briefly describe the currently existing in vitro human embryo models and discuss their putative future relevance for disease modelling and drug discovery.


Assuntos
Descoberta de Drogas , Embrião de Mamíferos/efeitos dos fármacos , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Descoberta de Drogas/métodos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Modelos Animais , Organoides/citologia , Organoides/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
9.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33430111

RESUMO

Articular cartilage experiences mechanical constraints leading to chondral defects that inevitably evolve into osteoarthritis (OA), because cartilage has poor intrinsic repair capacity. Although OA is an incurable degenerative disease, several dietary supplements may help improve OA outcomes. In this study, we investigated the effects of Dielen® hydrolyzed fish collagens from skin (Promerim®30 and Promerim®60) and cartilage (Promerim®40) to analyze the phenotype and metabolism of equine articular chondrocytes (eACs) cultured as organoids. Here, our findings demonstrated the absence of cytotoxicity and the beneficial effect of Promerim® hydrolysates on eAC metabolic activity under physioxia; further, Promerim®30 also delayed eAC senescence. To assess the effect of Promerim® in a cartilage-like tissue, eACs were cultured as organoids under hypoxia with or without BMP-2 and/or IL-1ß. In some instances, alone or in the presence of IL-1ß, Promerim®30 and Promerim®40 increased protein synthesis of collagen types I and II, while decreasing transcript levels of proteases involved in OA pathogenesis, namely Htra1, and the metalloproteinases Mmp1-3, Adamts5, and Cox2. Both Promerim® hydrolysates also decreased Htra1 protein amounts, particularly in inflammatory conditions. The effect of Promerim® was enhanced under inflammatory conditions, possibly due to a decrease in the synthesis of inflammation-associated molecules. Finally, Promerim® favored in vitro repair in a scratch wound assay through an increase in cell proliferation or migration. Altogether, these data show that Promerim®30 and 40 hold promise as dietary supplements to relieve OA symptoms in patients and to delay OA progression.


Assuntos
Cartilagem Articular/efeitos dos fármacos , Colágeno/biossíntese , Organoides/efeitos dos fármacos , Osteoartrite/tratamento farmacológico , Animais , Cartilagem Articular/crescimento & desenvolvimento , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Cavalos , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/patologia , Organoides/crescimento & desenvolvimento , Pele/química
10.
Cancer Sci ; 112(3): 1196-1208, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33423358

RESUMO

5-Fluorouracil (5-FU) is one of the most frequently used pharmacological agents in the treatment of colorectal cancer (CRC). Resistance to chemotherapy is a major cause of treatment failure of CRC, and it is a well known fact that cancer stem cells play a significant role in the acquisition of drug resistance. In this study, we focused on the KHDRBS3 gene that encodes KH RNA Binding Domain Containing, Signal Transduction Associated 3. We first clarified the relationship between KHDRBS3 and 5-FU resistance. We then observed higher expression levels of KHDRBS3 in KRAS-mutant organoids and cell lines in comparison with KRAS wild-type organoids and cell lines. Immunohistochemical analysis using CRC cases revealed that the prognosis of KHDRBS3-positive patients was significantly worse compared with that of KHDRBS3-negative patients. Univariate and multivariate Cox proportional hazards analyses showed that KHDRBS3 was an independent prognostic factor in patients with CRC. We determined that KHDRBS3 might play a crucial role in the acquisition of stem cell properties, such as drug resistance and spheroid/organoid formation, by regulating CD44 variant expression and the Wnt signaling pathway. In an immunodeficient mouse model, KHDRBS3-positive cells showed efficient tumor formation and formed metastatic lesions in the lungs. These results indicated that KHDRBS3 plays a crucial role in drug resistance and anchorage-independent growth by maintaining stem cell-like features in CRC cells. KHDRBS3 could be a promising candidate marker for predicting chemotherapeutic effect and prognosis in CRC patients.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais/terapia , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Ligação a RNA/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Colectomia , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Receptores de Hialuronatos/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Células-Tronco Neoplásicas/patologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas de Ligação a RNA/genética , Análise de Sobrevida , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Nat Biomed Eng ; 5(1): 11-25, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33318650

RESUMO

Engineered human mini-brains, made possible by knowledge from the convergence of precision microengineering and cell biology, permit systematic studies of complex neurological processes and of pathogenesis beyond what can be done with animal models. By culturing human brain cells with physiological microenvironmental cues, human mini-brain models reconstitute the arrangement of structural tissues and some of the complex biological functions of the human brain. In this Review, we highlight the most significant developments that have led to microphysiological human mini-brain models. We introduce the history of mini-brain development, review methods for creating mini-brain models in static conditions, and discuss relevant state-of-the-art dynamic cell-culture systems. We also review human mini-brain models that reconstruct aspects of major neurological disorders under static or dynamic conditions. Engineered human mini-brains will contribute to advancing the study of the physiology and aetiology of neurological disorders, and to the development of personalized medicines for them.


Assuntos
Encéfalo , Modelos Biológicos , Organoides , Técnicas de Cultura de Tecidos , Doença de Alzheimer , Encéfalo/anatomia & histologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Lesões Encefálicas Traumáticas , Neoplasias Encefálicas , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Miniaturização , Organoides/anatomia & histologia , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/fisiologia
12.
Int J Mol Sci ; 22(1)2020 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-33375140

RESUMO

Zika virus (ZIKV) acquired a special relevance due to the pandemic that occurred in the Americas in 2015, when an important number of fetal microcephaly cases occurred. Since then, numerous studies have tried to elucidate the pathogenic mechanisms and the potential therapeutic approaches to combat the virus. Cellular and animal models have proved to be a basic resource for this research, with the more recent addition of organoids as a more realistic and physiological 3D culture for the study of ZIKV. Nanotechnology can also offer a promising therapeutic tool, as the nanoparticles developed by this field can penetrate cells and deliver a wide array of drugs in a very specific and controlled way inside the cells. These two state-of-the-art scientific tools clearly provide a very relevant resource for the study of ZIKV, and will help researchers find an effective treatment or vaccine against the virus.


Assuntos
Antivirais/farmacologia , Nanotecnologia/métodos , Técnicas de Cultura de Órgãos/métodos , Organoides/efeitos dos fármacos , Infecção por Zika virus/tratamento farmacológico , Zika virus/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Humanos , Nanomedicina/métodos , Organoides/virologia , Zika virus/fisiologia , Infecção por Zika virus/virologia
13.
Cell Death Dis ; 11(12): 1059, 2020 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-33311479

RESUMO

Recent studies on developing three-dimensional (3D) brain organoids from stem cells have allowed the generation of in vitro models of neural disease and have enabled the screening of drugs because these organoids mimic the complexity of neural tissue. Niemann-Pick disease, type C (NPC) is a neurodegenerative lysosomal storage disorder caused by mutations in the NPC1 or NPC2. The pathological features underlying NPC are characterized by the abnormal accumulation of cholesterol in acidic compartments, including late endosomes and lysosomes. Due to the inaccessibility of brain tissues from human NPC patients, we developed NPC brain organoids with induced neural stem cells from NPC patient-derived fibroblasts. NPC organoids exhibit significantly reduced size and proliferative ability, which are accompanied by accumulation of cholesterol, impairment in neuronal differentiation, and autophagic flux and dysfunction of lysosomes; therefore, NPC organoids can recapitulate the main phenotypes of NPC patients. Furthermore, these pathological phenotypes observed in NPC organoids were reversed by treatment with valproic acid and HPBCD, which are known to be an effective treatment for several neurodegenerative diseases. Our data present patient-specific phenotypes in 3D organoid-based models of NPC and highlight the application of this model to drug screening in vitro.


Assuntos
Encéfalo/patologia , Modelos Biológicos , Células-Tronco Neurais/patologia , Doença de Niemann-Pick Tipo C/patologia , Organoides/patologia , Autofagia/efeitos dos fármacos , Biomarcadores/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Niemann-Pick Tipo C/genética , Organoides/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácido Valproico/farmacologia
14.
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
15.
Nat Commun ; 11(1): 5271, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077832

RESUMO

Three-dimensional (3D) cell culture technologies, such as organoids, are physiologically relevant models for basic and clinical applications. Automated microfluidics offers advantages in high-throughput and precision analysis of cells but is not yet compatible with organoids. Here, we present an automated, high-throughput, microfluidic 3D organoid culture and analysis system to facilitate preclinical research and personalized therapies. Our system provides combinatorial and dynamic drug treatments to hundreds of cultures and enables real-time analysis of organoids. We validate our system by performing individual, combinatorial, and sequential drug screens on human-derived pancreatic tumor organoids. We observe significant differences in the response of individual patient-based organoids to drug treatments and find that temporally-modified drug treatments can be more effective than constant-dose monotherapy or combination therapy in vitro. This integrated platform advances organoids models to screen and mirror real patient treatment courses with potential to facilitate treatment decisions for personalized therapy.


Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Microfluídica/métodos , Organoides/efeitos dos fármacos , Automação , Técnicas de Cultura de Células , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Humanos , Microfluídica/instrumentação , Neoplasias Pancreáticas/tratamento farmacológico
16.
Nat Commun ; 11(1): 5485, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127883

RESUMO

Cancer patient classification using predictive biomarkers for anti-cancer drug responses is essential for improving therapeutic outcomes. However, current machine-learning-based predictions of drug response often fail to identify robust translational biomarkers from preclinical models. Here, we present a machine-learning framework to identify robust drug biomarkers by taking advantage of network-based analyses using pharmacogenomic data derived from three-dimensional organoid culture models. The biomarkers identified by our approach accurately predict the drug responses of 114 colorectal cancer patients treated with 5-fluorouracil and 77 bladder cancer patients treated with cisplatin. We further confirm our biomarkers using external transcriptomic datasets of drug-sensitive and -resistant isogenic cancer cell lines. Finally, concordance analysis between the transcriptomic biomarkers and independent somatic mutation-based biomarkers further validate our method. This work presents a method to predict cancer patient drug responses using pharmacogenomic data derived from organoid models by combining the application of gene modules and network-based approaches.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Aprendizado de Máquina , Organoides/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Bexiga Urinária/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Desenvolvimento de Medicamentos/métodos , Fluoruracila/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Organoides/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Transcriptoma , Bexiga Urinária/efeitos dos fármacos , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo
17.
Nat Commun ; 11(1): 5321, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087710

RESUMO

5-Fluorouracil (5-FU) remains the first-line treatment for colorectal cancer (CRC). Although 5-FU initially de-bulks the tumor mass, recurrence after chemotherapy is the barrier to effective clinical outcomes for CRC patients. Here, we demonstrate that p53 promotes WNT3 transcription, leading to activation of the WNT/ß-catenin pathway in ApcMin/+/Lgr5EGFP mice, CRC patient-derived tumor organoids (PDTOs) and patient-derived tumor cells (PDCs). Through this regulation, 5-FU induces activation and enrichment of cancer stem cells (CSCs) in the residual tumors, contributing to recurrence after treatment. Combinatorial treatment of a WNT inhibitor and 5-FU effectively suppresses the CSCs and reduces tumor regrowth after discontinuation of treatment. These findings indicate p53 as a critical mediator of 5-FU-induced CSC activation via the WNT/ß-catenin signaling pathway and highlight the significance of combinatorial treatment of WNT inhibitor and 5-FU as a compelling therapeutic strategy to improve the poor outcomes of current 5-FU-based therapies for CRC patients.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Fluoruracila/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Camundongos Transgênicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Pirazinas/administração & dosagem , Piridinas/administração & dosagem , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Proteína Wnt3/genética , Proteína Wnt3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
18.
PLoS One ; 15(9): e0238862, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32898185

RESUMO

A model that recapitulates development of acquired therapeutic resistance is needed to improve oncology drug development and patient outcomes. To achieve this end, we established methods for the preparation and growth of spheroids from primary human lung adenocarcinomas, including methods to culture, passage, monitor growth, and evaluate changes in mutational profile over time. Primary lung tumor spheroids were cultured in Matrigel® with varying concentrations of erlotinib, a small molecule kinase inhibitor of epidermal growth factor receptor (EGFR) that is ineffective against KRAS mutant cells. Subtle changes in spheroid size and number were observed within the first two weeks of culture. Spheroids were cultured for up to 24 weeks, during which time interactions between different cell types, movement, and assembly into heterogeneous organoid structures were documented. Allele-specific competitive blocker PCR (ACB-PCR) was used to quantify low frequency BRAF V600E, KRAS G12D, KRAS G12V, and PIK3CA H1047R mutant subpopulations in tumor tissue residue (TR) samples and cultured spheroids. Mutant subpopulations, including multiple mutant subpopulations, were quite prevalent. Twelve examples of mutant enrichment were found in eight of the 14 tumors analyzed, based on the criteria that a statistically-significant increase in mutant fraction was observed relative to both the TR and the no-erlotinib control. Of the mutants quantified in erlotinib-treated cultures, PIK3CA H1047 mutant subpopulations increased most often (5/14 tumors), which is consistent with clinical observations. Thus, this ex vivo lung tumor spheroid model replicates the cellular and mutational tumor heterogeneity of human lung adenocarcinomas and can be used to assess the outgrowth of mutant subpopulations. Spheroid cultures with characterized mutant subpopulations could be used to investigate the efficacy of lung cancer combination therapies.


Assuntos
Biomarcadores Tumorais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Cloridrato de Erlotinib/farmacologia , Neoplasias Pulmonares/patologia , Mutação , Organoides/patologia , Esferoides Celulares/patologia , Idoso , Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Organoides/efeitos dos fármacos , Organoides/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas
19.
PLoS One ; 15(8): e0235319, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810173

RESUMO

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Organoides/efeitos dos fármacos , Tanquirases/antagonistas & inibidores , Adulto , Animais , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/patologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Organoides/patologia
20.
Nat Commun ; 11(1): 4178, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826895

RESUMO

Friedreich's ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.


Assuntos
Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Gânglios Espinais/metabolismo , Edição de Genes/métodos , Proteínas de Ligação ao Ferro/genética , Organoides/metabolismo , Células Receptoras Sensoriais/metabolismo , Antioxidantes/farmacologia , Sistemas CRISPR-Cas , Diferenciação Celular , Cromatina/metabolismo , Ataxia de Friedreich/tratamento farmacológico , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Predisposição Genética para Doença/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons , Mitocôndrias/metabolismo , Organoides/efeitos dos fármacos , Organoides/patologia , Células Receptoras Sensoriais/patologia , Análise de Sequência de RNA , Transcriptoma
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