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1.
Nat Commun ; 12(1): 1368, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649334

RESUMO

The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/ß-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic ß-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of ß-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with ß-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune ß-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/ß-catenin output requires selective modulation of gene expression by transcriptional co-factors.


Assuntos
Mucosa Intestinal/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , beta Catenina/química , beta Catenina/metabolismo , Algoritmos , Animais , Sequência de Bases , Diferenciação Celular , Proliferação de Células , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Homeostase , Hiperplasia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Organoides/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais
3.
EMBO J ; 40(5): e107651, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33576058

RESUMO

Defining the pulmonary cell types infected by SARS-CoV-2 and finding ways to prevent subsequent tissue damage are key goals for controlling COVID-19. Recent work establishing a human lung organoid-derived air-liquid interface model permissive to SARS-CoV-2 infection identifies alveolar type II cells as the primary cell type infected, reports an infection-induced interferon response and demonstrates the effectiveness of interferon lambda 1 treatment in dampening lung infection.


Assuntos
Células Epiteliais Alveolares/metabolismo , Modelos Biológicos , Organoides/metabolismo , Replicação Viral , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , /patologia , Humanos , Organoides/patologia , Organoides/virologia
4.
Stem Cell Reports ; 16(3): 437-445, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33631122

RESUMO

COVID-19 is a transmissible respiratory disease caused by a novel coronavirus, SARS-CoV-2, and has become a global health emergency. There is an urgent need for robust and practical in vitro model systems to investigate viral pathogenesis. Here, we generated human induced pluripotent stem cell (iPSC)-derived lung organoids (LORGs), cerebral organoids (CORGs), neural progenitor cells (NPCs), neurons, and astrocytes. LORGs containing epithelial cells, alveolar types 1 and 2, highly express ACE2 and TMPRSS2 and are permissive to SARS-CoV-2 infection. SARS-CoV-2 infection induces interferons, cytokines, and chemokines and activates critical inflammasome pathway genes. Spike protein inhibitor, EK1 peptide, and TMPRSS2 inhibitors (camostat/nafamostat) block viral entry in LORGs. Conversely, CORGs, NPCs, astrocytes, and neurons express low levels of ACE2 and TMPRSS2 and correspondingly are not highly permissive to SARS-CoV-2 infection. Infection in neuronal cells activates TLR3/7, OAS2, complement system, and apoptotic genes. These findings will aid in understanding COVID-19 pathogenesis and facilitate drug discovery.


Assuntos
Encéfalo/virologia , Células-Tronco Pluripotentes Induzidas/virologia , Pulmão/virologia , Células-Tronco Neurais/virologia , Organoides/virologia , /patogenicidade , Apoptose/fisiologia , Encéfalo/metabolismo , Células Cultivadas , Proteínas do Sistema Complemento/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/metabolismo , Inflamação/virologia , Pulmão/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Neurônios/virologia , Organoides/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Células-Tronco/virologia
5.
Int J Mol Sci ; 22(3)2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525682

RESUMO

Angiotensin-converting enzyme 2 (ACE2) was identified as the main host cell receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its subsequent infection. In some coronavirus disease 2019 (COVID-19) patients, it has been reported that the nervous tissues and the eyes were also affected. However, evidence supporting that the retina is a target tissue for SARS-CoV-2 infection is still lacking. This present study aimed to investigate whether ACE2 expression plays a role in human retinal neurons during SARS-CoV-2 infection. Human induced pluripotent stem cell (hiPSC)-derived retinal organoids and monolayer cultures derived from dissociated retinal organoids were generated. To validate the potential entry of SARS-CoV-2 infection in the retina, we showed that hiPSC-derived retinal organoids and monolayer cultures endogenously express ACE2 and transmembrane serine protease 2 (TMPRSS2) on the mRNA level. Immunofluorescence staining confirmed the protein expression of ACE2 and TMPRSS2 in retinal organoids and monolayer cultures. Furthermore, using the SARS-CoV-2 pseudovirus spike protein with GFP expression system, we found that retinal organoids and monolayer cultures can potentially be infected by the SARS-CoV-2 pseudovirus. Collectively, our findings highlighted the potential of iPSC-derived retinal organoids as the models for ACE2 receptor-based SARS-CoV-2 infection.


Assuntos
/genética , Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Retina/citologia , /fisiologia , /metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/citologia , Organoides/metabolismo , Retina/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Internalização do Vírus
7.
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
8.
Nat Protoc ; 16(2): 919-936, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33432231

RESUMO

Human organoids are emerging as a valuable resource to investigate human organ development and disease. The applicability of human organoids has been limited, partly due to the oversimplified architecture of the current technology, which generates single-tissue organoids that lack inter-organ structural connections. Thus, engineering organoid systems that incorporate connectivity between neighboring organs is a critical unmet challenge in an evolving organoid field. Here, we describe a protocol for the continuous patterning of hepatic, biliary and pancreatic (HBP) structures from a 3D culture of human pluripotent stem cells (PSCs). After differentiating PSCs into anterior and posterior gut spheroids, the two spheroids are fused together in one well. Subsequently, self-patterning of multi-organ (i.e., HBP) domains occurs within the boundary region of the two spheroids, even in the absence of any extrinsic factors. Long-term culture of HBP structures induces differentiation of the domains into segregated organs complete with developmentally relevant invagination and epithelial branching. This in-a-dish model of human hepato-biliary-pancreatic organogenesis provides a unique platform for studying human development, congenital disorders, drug development and therapeutic transplantation. More broadly, our approach could potentially be used to establish inter-organ connectivity models for other organ systems derived from stem cell cultures.


Assuntos
Técnicas de Cultura de Células/métodos , Organoides/citologia , Engenharia Tecidual/métodos , Ductos Biliares/citologia , Diferenciação Celular/fisiologia , Humanos , Fígado/citologia , Organogênese/fisiologia , Organoides/metabolismo , Pâncreas/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
9.
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
10.
Nat Commun ; 12(1): 270, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431892

RESUMO

Intestinal stem cells (ISCs) residing in the crypts are critical for the continual self-renewal and rapid recovery of the intestinal epithelium. The regulatory mechanism of ISCs is not fully understood. Here we report that CREPT, a recently identified tumor-promoting protein, is required for the maintenance of murine ISCs. CREPT is preferably expressed in the crypts but not in the villi. Deletion of CREPT in the intestinal epithelium of mice (Vil-CREPTKO) results in lower body weight and slow migration of epithelial cells in the intestine. Vil-CREPTKO intestine fails to regenerate after X-ray irradiation and dextran sulfate sodium (DSS) treatment. Accordingly, the deletion of CREPT decreases the expression of genes related to the proliferation and differentiation of ISCs and reduces Lgr5+ cell numbers at homeostasis. We identify that CREPT deficiency downregulates Wnt signaling by impairing ß-catenin accumulation in the nucleus of the crypt cells during regeneration. Our study provides a previously undefined regulator of ISCs.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Intestinos/fisiologia , Proteínas de Neoplasias/metabolismo , Regeneração/fisiologia , Células-Tronco/metabolismo , Animais , Contagem de Células , Proteínas de Ciclo Celular/deficiência , Diferenciação Celular , Proliferação de Células , Epitélio/metabolismo , Deleção de Genes , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Proteínas de Neoplasias/deficiência , Organoides/metabolismo , Células-Tronco/citologia , Via de Sinalização Wnt , Raios X , beta Catenina/metabolismo
11.
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
13.
Nat Protoc ; 16(2): 579-602, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33328611

RESUMO

Cerebral organoids, or brain organoids, can be generated from a wide array of emerging technologies for modeling brain development and disease. The fact that they are cultured in vitro makes them easily accessible both genetically and for live assays such as fluorescence imaging. In this Protocol Extension, we describe a modified version of our original protocol (published in 2014) that can be used to reliably generate cerebral organoids of a telencephalic identity and maintain long-term viability for later stages of neural development, including axon outgrowth and neuronal maturation. The method builds upon earlier cerebral organoid methodology, with modifications of embryoid body size and shape to increase surface area and slice culture to maintain nutrient and oxygen access to the interior regions of the organoid, enabling long-term culture. We also describe approaches for introducing exogenous plasmid constructs and for sparse cell labeling to image neuronal axon outgrowth and maturation over time. Together, these methods allow for modeling of later events in cortical development, which are important for neurodevelopmental disease modeling. The protocols described can be easily performed by an experimenter with stem cell culture experience and take 2-3 months to complete, with long-term maturation occurring over several months.


Assuntos
Técnicas de Cultura de Células/métodos , Neurônios/citologia , Organoides/citologia , Encéfalo/citologia , Corpos Embrioides/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Neurogênese/fisiologia , Organoides/metabolismo
14.
Methods Mol Biol ; 2258: 259-272, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340366

RESUMO

As the field of organoid development matures, the need to transplant organoids to evaluate and characterize their functionality grows. Decades of research developing islet organoid transplantation for the treatment of type 1 diabetes can contribute substantially to accelerating diverse tissue organoid transplantation. Biomaterials-based organoid delivery methods offer the potential to maximize organoid survival and engraftment. In this protocol, we describe a vasculogenic degradable hydrogel vehicle and a method to deliver organoids to intraperitoneal tissue. Further, we describe a method to fluorescently label and image functional vasculature within the graft as a tool to investigate organoid engraftment.


Assuntos
Ilhotas Pancreáticas/irrigação sanguínea , Microscopia Confocal , Neovascularização Fisiológica , Organoides/irrigação sanguínea , Organoides/transplante , Engenharia Tecidual , Indutores da Angiogênese/farmacologia , Animais , Técnicas de Cultura de Células , Células Cultivadas , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Hidrogéis , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas , Maleimidas/química , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Organoides/metabolismo , Polietilenoglicóis/química , Ratos , Fixação de Tecidos , Fator A de Crescimento do Endotélio Vascular/farmacologia
15.
Proc Natl Acad Sci U S A ; 117(52): 33597-33607, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33318207

RESUMO

Axon injury is a hallmark of many neurodegenerative diseases, often resulting in neuronal cell death and functional impairment. Dual leucine zipper kinase (DLK) has emerged as a key mediator of this process. However, while DLK inhibition is robustly protective in a wide range of neurodegenerative disease models, it also inhibits axonal regeneration. Indeed, there are no genetic perturbations that are known to both improve long-term survival and promote regeneration. To identify such a neuroprotective target, we conducted a set of complementary high-throughput screens using a protein kinase inhibitor library in human stem cell-derived retinal ganglion cells (hRGCs). Overlapping compounds that promoted both neuroprotection and neurite outgrowth were bioinformatically deconvoluted to identify specific kinases that regulated neuronal death and axon regeneration. This work identified the role of germinal cell kinase four (GCK-IV) kinases in cell death and additionally revealed their unexpected activity in suppressing axon regeneration. Using an adeno-associated virus (AAV) approach, coupled with genome editing, we validated that GCK-IV kinase knockout improves neuronal survival, comparable to that of DLK knockout, while simultaneously promoting axon regeneration. Finally, we also found that GCK-IV kinase inhibition also prevented the attrition of RGCs in developing retinal organoid cultures without compromising axon outgrowth, addressing a major issue in the field of stem cell-derived retinas. Together, these results demonstrate a role for the GCK-IV kinases in dissociating the cell death and axonal outgrowth in neurons and their druggability provides for therapeutic options for neurodegenerative diseases.


Assuntos
Axônios/enzimologia , Axônios/patologia , Sistema Nervoso Central/patologia , Quinases do Centro Germinativo/metabolismo , Regeneração Nervosa , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dependovirus/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Organoides/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Int J Mol Sci ; 21(24)2020 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-33327663

RESUMO

Three-dimensional (3-D) cell culture models, such as spheroids, organoids, and organotypic cultures, are more physiologically representative of the human tumor microenvironment (TME) than traditional two-dimensional (2-D) cell culture models. They have been used as in vitro models to investigate various aspects of oral cancer but, to date, have not be widely used in investigations of the process of oral carcinogenesis. The aim of this scoping review was to evaluate the use of 3-D cell cultures in oral squamous cell carcinoma (OSCC) research, with a particular emphasis on oral carcinogenesis studies. Databases (PubMed, Scopus, and Web of Science) were systematically searched to identify research applying 3-D cell culture techniques to cells from normal, dysplastic, and malignant oral mucosae. A total of 119 studies were included for qualitative analysis including 53 studies utilizing spheroids, 62 utilizing organotypic cultures, and 4 using organoids. We found that 3-D oral carcinogenesis studies had been limited to just two organotypic culture models and that to date, spheroids and organoids had not been utilized for this purpose. Spheroid culture was most frequently used as a tumorosphere forming assay and the organoids cultured from human OSCCs most often used in drug sensitivity testing. These results indicate that there are significant opportunities to utilize 3-D cell culture to explore the development of oral cancer, particularly as the physiological relevance of these models continues to improve.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas/metabolismo , Técnicas de Cultura de Células , Humanos , Neoplasias Bucais/metabolismo , Organoides/citologia , Organoides/metabolismo , Esferoides Celulares
17.
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
18.
Nat Commun ; 11(1): 5352, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097693

RESUMO

Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding ß-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.


Assuntos
Edição de Genes/métodos , Organoides/metabolismo , beta Catenina/genética , Sistemas CRISPR-Cas , Linhagem Celular , Proliferação de Células , ATPases Transportadoras de Cobre/genética , Desoxirribonuclease I/metabolismo , Diacilglicerol O-Aciltransferase/genética , Células HEK293 , Degeneração Hepatolenticular/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Reparo de DNA por Recombinação , Células-Tronco , Reparo Gênico Alvo-Dirigido/métodos
19.
Med Sci (Paris) ; 36(10): 879-885, 2020 Oct.
Artigo em Francês | MEDLINE | ID: mdl-33026330

RESUMO

Pancreatic islet transplantation is a valid cure for selected type-1 diabetic patients. It offers a minimally invasive ß-cell replacement approach and has proven its capacity to significantly enhance patients quality of life. However, these insulin-secreting mini-organs suffer from the loss of intrinsic vascularization and extra-cellular matrix occurring during isolation, resulting in hypoxic stress and necrosis. In addition, they have to face inflammatory and immune destruction once transplanted in the liver. Organoid generation represents a strategy to overcome these obstacles by allowing size and shape control as well as composition. It does offer the possibility to add supporting cells such as endothelial cells, in order to facilitate revascularization or cells releasing anti-inflammatory and/or immunomodulatory factors. This review describes the limitations of pancreatic islet transplantation and details the benefits offered by organoids as a cornerstone toward the generation of a bioartificial pancreas.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Organoides/metabolismo , Pâncreas Artificial , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/terapia , Humanos , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Organoides/citologia , Pâncreas Artificial/provisão & distribução , Técnicas de Cultura de Tecidos/métodos
20.
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
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