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1.
Nat Commun ; 11(1): 583, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996670

RESUMO

Medulloblastoma (MB) is the most common malignant brain tumor in children and among the subtypes, Group 3 MB has the worst outcome. Here, we perform an in vivo, patient-specific screen leading to the identification of Otx2 and c-MYC as strong Group 3 MB inducers. We validated our findings in human cerebellar organoids where Otx2/c-MYC give rise to MB-like organoids harboring a DNA methylation signature that clusters with human Group 3 tumors. Furthermore, we show that SMARCA4 is able to reduce Otx2/c-MYC tumorigenic activity in vivo and in human cerebellar organoids while SMARCA4 T910M, a mutant form found in human MB patients, inhibits the wild-type protein function. Finally, treatment with Tazemetostat, a EZH2-specific inhibitor, reduces Otx2/c-MYC tumorigenesis in ex vivo culture and human cerebellar organoids. In conclusion, human cerebellar organoids can be efficiently used to understand the role of genes found altered in cancer patients and represent a reliable tool for developing personalized therapies.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Organoides/metabolismo , Organoides/patologia , Benzamidas/antagonistas & inibidores , Carcinogênese , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Humanos , Meduloblastoma/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridonas/antagonistas & inibidores , Células-Tronco , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Cancer Sci ; 111(1): 137-147, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31724799

RESUMO

As a member of the epidermal growth factor receptor (EGFR) family, ERBB3 plays an essential role in development and disease independent of inherently inactive kinase domain. Recently, ERBB3 has been found to bind to ATP and has catalytic activity in vitro. However, the biological function of ERBB3 kinase activity remains elusive in vivo. Here we have identified the physiological function of inactivated ERBB3 kinase activity by creating Erbb3-K740M knockin mice in which ATP cannot bind to ERBB3. Unlike Erbb3 knockout mice, kinase-inactive Erbb3K740M homozygous mice were born in Mendelian ratios and showed normal development. After dextran sulfate sodium-induced colitis, the kinase-inactive Erbb3 mutant mice showed normal recovery. However, the outgrowth of ileal organoids by neuregulin-1 treatment was more attenuated in Erbb3 mutant mice than in WT mice. Moreover, in combination with the ApcMin mouse, the proportion of polyps less than 1 mm in diameter in mutant mice was higher than in control mice and an increase in the number of apoptotic cells was observed in polyps from mutant mice compared with polyps from control mice. Taken together, the ERBB3 kinase activity contributes to the outgrowth of ileal organoids and intestinal tumorigenesis, and the development of ERBB3 kinase inhibitors, including epidermal growth factor receptor family members, can be a potential way to target colorectal cancer.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Intestinos/patologia , Organoides/metabolismo , Organoides/patologia , Receptor ErbB-3/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Carcinogênese/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Intestinos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Organoides/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Pólipos/tratamento farmacológico , Pólipos/patologia , Inibidores de Proteínas Quinases/farmacologia
3.
Nat Cell Biol ; 21(11): 1321-1333, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31685987

RESUMO

Following severe or chronic liver injury, adult ductal cells (cholangiocytes) contribute to regeneration by restoring both hepatocytes and cholangiocytes. We recently showed that ductal cells clonally expand as self-renewing liver organoids that retain their differentiation capacity into both hepatocytes and ductal cells. However, the molecular mechanisms by which adult ductal-committed cells acquire cellular plasticity, initiate organoids and regenerate the damaged tissue remain largely unknown. Here, we describe that ductal cells undergo a transient, genome-wide, remodelling of their transcriptome and epigenome during organoid initiation and in vivo following tissue damage. TET1-mediated hydroxymethylation licences differentiated ductal cells to initiate organoids and activate the regenerative programme through the transcriptional regulation of stem-cell genes and regenerative pathways including the YAP-Hippo signalling. Our results argue in favour of the remodelling of genomic methylome/hydroxymethylome landscapes as a general mechanism by which differentiated cells exit a committed state in response to tissue damage.


Assuntos
Proteínas de Ligação a DNA/genética , Epigênese Genética , Regeneração Hepática/genética , Fígado/metabolismo , Organoides/metabolismo , Proteínas Proto-Oncogênicas/genética , Transcriptoma , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ductos Biliares/citologia , Ductos Biliares/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Fígado/citologia , Masculino , Camundongos Transgênicos , Organoides/citologia , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais
4.
Nat Cell Biol ; 21(11): 1425-1435, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31685994

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) shows great cellular heterogeneity, with pronounced epithelial and mesenchymal cancer cell populations. However, the cellular hierarchy underlying PDAC cell diversity is unknown. Here we identify the tetraspanin CD9 as a marker of PDAC tumour-initiating cells. CD9high cells had increased organoid formation capability, and generated tumour grafts in vivo at limiting dilutions. Tumours initiated from CD9high cells recapitulated the cellular heterogeneity of primary PDAC, whereas CD9low cells produced only duct-like epithelial progeny. CD9 knockdown decreased the growth of PDAC organoids, and heterozygous CD9 deletion in Pdx1-Cre; LSL-KRasG12D; p53F/F mice prolonged overall survival. Mechanistically, CD9 promoted the plasma membrane localization of the glutamine transporter ASCT2, enhancing glutamine uptake in PDAC cells. Thus, our study identifies a PDAC subpopulation capable of initiating PDAC and giving rise to PDAC heterogeneity, suggesting that the cellular diversity of PDAC is generated by PDAC stem cell differentiation.


Assuntos
Sistema ASC de Transporte de Aminoácidos/genética , Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Glutamina/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/genética , Tetraspanina 29/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor/metabolismo , Células-Tronco Neoplásicas/patologia , Organoides/metabolismo , Organoides/patologia , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Análise de Sobrevida , Tetraspanina 29/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
BMC Cancer ; 19(1): 970, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31638925

RESUMO

BACKGROUND: Neuroblastoma (NB) is a paediatric tumour of the sympathetic nervous system. Half of all cases are defined high-risk with an overall survival less than 40% at 5 years from diagnosis. The lack of in vitro models able to recapitulate the intrinsic heterogeneity of primary NB tumours has hindered progress in understanding disease pathogenesis and therapy response. METHODS: Here we describe the establishment of 6 patient-derived organoids (PDOs) from cells of NB tumour biopsies capable of self-organising in a structure resembling the tissue of origin. RESULTS: PDOs recapitulate the histological architecture typical of the NB tumour. Moreover, PDOs expressed NB specific markers such as neural cell adhesion molecules, NB84 antigen, synaptophysin (SYP), chromogranin A (CHGA) and neural cell adhesion molecule NCAM (CD56). Analyses of whole genome genotyping array revealed that PDOs maintained patient-specific chromosomal aberrations such as MYCN amplification, deletion of 1p and gain of chromosome 17q. Furthermore, the PDOs showed stemness features and retained cellular heterogeneity reflecting the high heterogeneity of NB tumours. CONCLUSIONS: We were able to create a novel preclinical model for NB exhibiting self-renewal property and allowing to obtain a reservoir of NB patients' biological material useful for the study of NB molecular pathogenesis and to test drugs for personalised treatments.


Assuntos
Doenças do Sistema Nervoso Autônomo/genética , Doenças do Sistema Nervoso Autônomo/patologia , Modelos Biológicos , Neuroblastoma/genética , Neuroblastoma/patologia , Organoides/patologia , Doenças do Sistema Nervoso Autônomo/metabolismo , Biomarcadores Tumorais/metabolismo , Biópsia , Criança , Pré-Escolar , Cromogranina A/metabolismo , Aberrações Cromossômicas , Amplificação de Genes/genética , Humanos , Lactente , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/metabolismo , Organoides/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Sinaptofisina/metabolismo
6.
PLoS Pathog ; 15(10): e1008003, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31581229

RESUMO

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>103 cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a+/stx2c+ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium.


Assuntos
Doenças dos Bovinos/transmissão , Células Epiteliais/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli O157/efeitos dos fármacos , Íleo/microbiologia , Organoides/microbiologia , Toxina Shiga II/farmacologia , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/microbiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/isolamento & purificação , Íleo/citologia , Íleo/metabolismo , Masculino , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Virulência
7.
Nat Commun ; 10(1): 4491, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31582751

RESUMO

Maintaining long-term euglycemia after intraportal islet transplantation is hampered by the considerable islet loss in the peri-transplant period attributed to inflammation, ischemia and poor angiogenesis. Here, we show that viable and functional islet organoids can be successfully generated from dissociated islet cells (ICs) and human amniotic epithelial cells (hAECs). Incorporation of hAECs into islet organoids markedly enhances engraftment, viability and graft function in a mouse type 1 diabetes model. Our results demonstrate that the integration of hAECs into islet cell organoids has great potential in the development of cell-based therapies for type 1 diabetes. Engineering of functional mini-organs using this strategy will allow the exploration of more favorable implantation sites, and can be expanded to unlimited (stem-cell-derived or xenogeneic) sources of insulin-producing cells.


Assuntos
Diabetes Mellitus Tipo 1/terapia , Células Epiteliais/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Organoides/transplante , Engenharia Tecidual/métodos , Âmnio/citologia , Animais , Sobrevivência Celular , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/induzido quimicamente , Células Epiteliais/transplante , Sobrevivência de Enxerto , Xenoenxertos/irrigação sanguínea , Xenoenxertos/metabolismo , Xenoenxertos/transplante , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos SCID , Organoides/irrigação sanguínea , Organoides/metabolismo , Ratos , Ratos Sprague-Dawley , Medicina Regenerativa/métodos , Esferoides Celulares , Estreptozocina , Técnicas de Cultura de Tecidos/métodos , Transplante Heterólogo/métodos
8.
Pediatr Surg Int ; 35(12): 1363-1368, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31576466

RESUMO

AIM OF THE STUDY: Human breast milk reduces the risk and severity of necrotizing enterocolitis (NEC). Exosomes are extracellular vesicles (EVs) found in high concentrations in milk, and they mediate intercellular communication and immune responses. The aim of this study is to compare the protective effects of exosomes that are derived from different time periods of breast milk production against intestinal injury using an ex vivo intestinal organoid model. METHODS: Colostrum, transitional and mature breast milk samples from healthy lactating mothers were collected. Exosomes were isolated using serial ultracentrifugation and filtration. Exosomes' presence was confirmed using transmission electron microscopy (TEM) and western blot. To form the intestinal organoids, terminal ileum was harvested from neonatal mice pups at postnatal day 9, crypts were isolated and organoids were cultured in matrigel. Organoids were either cultured with exposure to lipopolysaccharide (LPS), or in treatment groups where both LPS and exosomes were added in the culturing medium. Inflammatory markers and organoids viability were evaluated. MAIN RESULTS: Human milk-derived exosomes were successfully isolated and characterized. LPS administration reduced the size of intestinal organoids, induced inflammation through increasing TNFα and TLR4 expression, and stimulated intestinal regeneration. Colostrum, transitional and mature human milk-derived exosome treatment all prevented inflammatory injury, while exosomes derived from colostrum were most effective at reducing inflammatory cytokine. CONCLUSIONS: Human breast milk-derived exosomes were able to protect intestine organoids against epithelial injury induced by LPS. Colostrum exosomes offer the best protective effect among the breast-milk derived exosomes. Human milk exosomes can be protective against the development of intestinal injury such as that seen in NEC.


Assuntos
Colostro/metabolismo , Enterocolite Necrosante/prevenção & controle , Exossomos/metabolismo , Mucosa Intestinal/metabolismo , Leite Humano/metabolismo , Organoides/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Lactação , Camundongos , Camundongos Endogâmicos C57BL
9.
PLoS Biol ; 17(10): e3000498, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613879

RESUMO

During gastrulation, the pluripotent epiblast self-organizes into the 3 germ layers-endoderm, mesoderm and ectoderm, which eventually form the entire embryo. Decades of research in the mouse embryo have revealed that a signaling cascade involving the Bone Morphogenic Protein (BMP), WNT, and NODAL pathways is necessary for gastrulation. In vivo, WNT and NODAL ligands are expressed near the site of gastrulation in the posterior of the embryo, and knockout of these ligands leads to a failure to gastrulate. These data have led to the prevailing view that a signaling gradient in WNT and NODAL underlies patterning during gastrulation; however, the activities of these pathways in space and time have never been directly observed. In this study, we quantify BMP, WNT, and NODAL signaling dynamics in an in vitro model of human gastrulation. Our data suggest that BMP signaling initiates waves of WNT and NODAL signaling activity that move toward the colony center at a constant rate. Using a simple mathematical model, we show that this wave-like behavior is inconsistent with a reaction-diffusion-based Turing system, indicating that there is no stable signaling gradient of WNT/NODAL. Instead, the final signaling state is homogeneous, and spatial differences arise only from boundary effects. We further show that the durations of WNT and NODAL signaling control mesoderm differentiation, while the duration of BMP signaling controls differentiation of CDX2-positive extra-embryonic cells. The identity of these extra-embryonic cells has been controversial, and we use RNA sequencing (RNA-seq) to obtain their transcriptomes and show that they closely resemble human trophoblast cells in vivo. The domain of BMP signaling is identical to the domain of differentiation of these trophoblast-like cells; however, neither WNT nor NODAL forms a spatial pattern that maps directly to the mesodermal region, suggesting that mesoderm differentiation is controlled dynamically by the combinatorial effect of multiple signals. We synthesize our data into a mathematical model that accurately recapitulates signaling dynamics and predicts cell fate patterning upon chemical and physical perturbations. Taken together, our study shows that the dynamics of signaling events in the BMP, WNT, and NODAL cascade in the absence of a stable signaling gradient control fate patterning of human gastruloids.


Assuntos
Proteína Morfogenética Óssea 4/genética , Gastrulação/genética , Mesoderma/metabolismo , Proteína Nodal/genética , Transdução de Sinais , Proteínas Wnt/genética , Benzotiazóis/farmacologia , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Gástrula/citologia , Gástrula/efeitos dos fármacos , Gástrula/metabolismo , Gastrulação/efeitos dos fármacos , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Modelos Biológicos , Modelos Estatísticos , Proteína Nodal/deficiência , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Proteínas Wnt/metabolismo
10.
Nat Commun ; 10(1): 4407, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562298

RESUMO

Understanding urothelial stem cell biology and differentiation has been limited by the lack of methods for their unlimited propagation. Here, we establish mouse urothelial organoids that can be maintained uninterruptedly for >1 year. Organoid growth is dependent on EGF and Wnt activators. High CD49f/ITGA6 expression features a subpopulation of organoid-forming cells expressing basal markers. Upon differentiation, multilayered organoids undergo reduced proliferation, decreased cell layer number, urothelial program activation, and acquisition of barrier function. Pharmacological modulation of PPARγ and EGFR promotes differentiation. RNA sequencing highlighted genesets enriched in proliferative organoids (i.e. ribosome) and transcriptional networks involved in differentiation, including expression of Wnt ligands and Notch components. Single-cell RNA sequencing (scRNA-Seq) analysis of the organoids revealed five clusters with distinct gene expression profiles. Together, with the use of γ-secretase inhibitors and scRNA-Seq, confirms that Notch signaling is required for differentiation. Urothelial organoids provide a powerful tool to study cell regeneration and differentiation.


Assuntos
Diferenciação Celular/genética , Integrina alfa6/genética , Organoides/metabolismo , Receptores Notch/metabolismo , Células-Tronco/metabolismo , Urotélio/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Integrina alfa6/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Organoides/citologia , Organoides/efeitos dos fármacos , Receptores Notch/genética , Análise de Célula Única/métodos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Urotélio/citologia
11.
Nat Commun ; 10(1): 4430, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562326

RESUMO

Zika virus (ZIKV) invades and persists in the central nervous system (CNS), causing severe neurological diseases. However the virus journey, from the bloodstream to tissues through a mature endothelium, remains unclear. Here, we show that ZIKV-infected monocytes represent suitable carriers for viral dissemination to the CNS using human primary monocytes, cerebral organoids derived from embryonic stem cells, organotypic mouse cerebellar slices, a xenotypic human-zebrafish model, and human fetus brain samples. We find that ZIKV-exposed monocytes exhibit higher expression of adhesion molecules, and higher abilities to attach onto the vessel wall and transmigrate across endothelia. This phenotype is associated to enhanced monocyte-mediated ZIKV dissemination to neural cells. Together, our data show that ZIKV manipulates the monocyte adhesive properties and enhances monocyte transmigration and viral dissemination to neural cells. Monocyte transmigration may represent an important mechanism required for viral tissue invasion and persistence that could be specifically targeted for therapeutic intervention.


Assuntos
Moléculas de Adesão Celular/metabolismo , Monócitos/metabolismo , Monócitos/virologia , Neurônios/metabolismo , Migração Transendotelial e Transepitelial/fisiologia , Infecção por Zika virus/metabolismo , Zika virus/fisiologia , Zika virus/patogenicidade , Animais , Adesão Celular/fisiologia , Sobrevivência Celular , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Cerebelo/patologia , Cerebelo/virologia , Modelos Animais de Doenças , Células-Tronco Embrionárias , Endotélio/virologia , Feminino , Humanos , Monócitos/patologia , Neurônios/patologia , Neurônios/virologia , Organoides/metabolismo , Organoides/patologia , Peixe-Zebra , Infecção por Zika virus/patologia , Infecção por Zika virus/virologia
12.
Nat Cell Biol ; 21(8): 1041-1051, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31371824

RESUMO

Endometrial disorders represent a major gynaecological burden. Current research models fail to recapitulate the nature and heterogeneity of these diseases, thereby hampering scientific and clinical progress. Here we developed long-term expandable organoids from a broad spectrum of endometrial pathologies. Organoids from endometriosis show disease-associated traits and cancer-linked mutations. Endometrial cancer-derived organoids accurately capture cancer subtypes, replicate the mutational landscape of the tumours and display patient-specific drug responses. Organoids were also established from precancerous pathologies encompassing endometrial hyperplasia and Lynch syndrome, and inherited gene mutations were maintained. Endometrial disease organoids reproduced the original lesion when transplanted in vivo. In summary, we developed multiple organoid models that capture endometrial disease diversity and will provide powerful research models and drug screening and discovery tools.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Neoplasias do Endométrio/patologia , Organoides/patologia , Doenças Uterinas/patologia , Técnicas de Cultura de Células/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/metabolismo , Endométrio/patologia , Feminino , Humanos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Doenças Uterinas/tratamento farmacológico , Doenças Uterinas/metabolismo
13.
Nat Rev Nephrol ; 15(10): 613-624, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31383997

RESUMO

Kidney organoids are regarded as important tools with which to study the development of the normal and diseased human kidney. Since the first reports of human pluripotent stem cell-derived kidney organoids 5 years ago, kidney organoids have been successfully used to model glomerular and tubular diseases. In parallel, advances in single-cell RNA sequencing have led to identification of a variety of cell types in the organoids, and have shown these to be similar to, but more immature than, human kidney cells in vivo. Protocols for the in vitro expansion of stem cell-derived nephron progenitor cells (NPCs), as well as those for the selective induction of specific lineages, especially glomerular podocytes, have also been reported. Although most current organoids are based on the induction of NPCs, an induction protocol for ureteric buds (collecting duct precursors) has also been developed, and approaches to generate more complex kidney structures may soon be possible. Maturation of organoids is a major challenge, and more detailed analysis of the developing kidney at a single cell level is needed. Eventually, organotypic kidney structures equipped with nephrons, collecting ducts, ureters, stroma and vascular flow are required to generate transplantable kidneys; such attempts are in progress.


Assuntos
Rim/citologia , Organoides/patologia , Animais , Humanos , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Organoides/metabolismo , Organoides/fisiopatologia
14.
Nat Methods ; 16(9): 879-886, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31384046

RESUMO

Although messenger RNAs are key molecules for understanding life, until now, no method has existed to determine the full-length sequence of endogenous mRNAs including their poly(A) tails. Moreover, although non-A nucleotides can be incorporated in poly(A) tails, there also exists no method to accurately sequence them. Here, we present full-length poly(A) and mRNA sequencing (FLAM-seq), a rapid and simple method for high-quality sequencing of entire mRNAs. We report a complementary DNA library preparation method coupled to single-molecule sequencing to perform FLAM-seq. Using human cell lines, brain organoids and Caenorhabditis elegans we show that FLAM-seq delivers high-quality full-length mRNA sequences for thousands of different genes per sample. We find that 3' untranslated region length is correlated with poly(A) tail length, that alternative polyadenylation sites and alternative promoters for the same gene are linked to different tail lengths, and that tails contain a substantial number of cytosines.


Assuntos
Encéfalo/metabolismo , Organoides/metabolismo , Poli A/química , Poli A/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Animais , Caenorhabditis elegans , Regulação da Expressão Gênica , Células HeLa , Humanos , Poli A/genética , Poliadenilação , Regiões Promotoras Genéticas , RNA Mensageiro/genética
15.
Regul Toxicol Pharmacol ; 108: 104449, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31449916

RESUMO

Multidrug resistance associated protein 2 (MRP2) is an important efflux transporter involved in clinical drug disposition and drug-drug interactions. The study of MRP2-mediated drug transport has become an integral part of drug discovery and development. In particular, screening of specific MRP2 inhibitors will help overcome the multidrug resistance in cancer. In this report, a new method for rapid and sensitive detection of Mrp2 function was established via using mouse small intestinal organoids. Firstly, small intestinal crypts isolated from mouse intestine were induced by Noggin, R-spondin1 and EGF to develop three-dimensional (3D) organoids. Secondly, the 3D organoids were characterized by the physical and physiological structure of Mrp2-mediated drug transport. Finally, Mrp2 fluorescent substrate 5(6)-carboxyl- 2', 7'-dichlorofluorescein (CDF) and its inhibitor MK-571 and probenecid were used to demonstrate Mrp2-mediated CDF transport in 3D organoids. The results showed that the small intestinal organoids have a physiological structure for Mrp2-mediated compound transport. Moreover, MK-571 and probenecid, inhibitors of MRP2, significantly decreased the accumulation of CDF in 3D organoids. In summary, a novel intestinal organoid model has been successfully established for the rapid and effective study of Mrp2-mediated drug transport.


Assuntos
Intestino Delgado/metabolismo , Modelos Biológicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Organoides/metabolismo , Animais , Fluoresceínas/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Probenecid/farmacologia , Propionatos/farmacologia , Quinolinas/farmacologia
16.
PLoS One ; 14(7): e0213114, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31295264

RESUMO

BACKGROUND: 2-Cl-C.OXT-A (COA-Cl) is a novel synthesized adenosine analog that activates Sphingosine-1-phosphate 1 receptor (S1P1R) and combines with the adenosine A1 receptor (A1R) in G proteins and was shown to enhance angiogenesis and improve the brain function in rat stroke models. However, the role of COA-Cl in hearts remains unclear. COA-Cl, which has a similar structure to xanthine derivatives, has the potential to suppress phosphodiesterase (PDE), which is an important factor involved in the beating of heart muscle. METHODS AND RESULTS: Cardiac organoids with fibroblasts, human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs), and hiPSC-derived endothelial cells (hiPSC-ECs) were cultured until they started beating. The beating and contraction of organoids were observed before and after the application of COA-Cl. COA-Cl significantly increased the beating rate and fractional area change in organoids. To elucidate the mechanism underlying these effects of COA-Cl on cardiac myocytes, pure hiPSC-CM spheroids were evaluated in the presence/absence of Suramin (antagonist of A1R). The effects of COA-Cl, SEW2871 (direct stimulator of S1P1R), two positive inotropes (Isoproterenol [ISO] and Forskolin [FSK]), and negative inotrope (Propranolol [PRP]) on spheroids were assessed based on the beating rates and cAMP levels. COA-Cl stimulated the beating rates about 1.5-fold compared with ISO and FSK, while PRP suppressed the beating rate. However, no marked changes were observed with SEW2871. COA-Cl, ISO, and FSK increased the cAMP level. In contrast, the level of cAMP did not change with PRP or SEW2871 treatment. The results were the same in the presence of Suramin as absence. Furthermore, an enzyme analysis showed that COA-Cl suppressed the PDE activity by half. CONCLUSIONS: COA-Cl, which has neovascularization effects, suppressed PDE and increased the contraction of cardiac organoids, independent of S1P1R and A1R. These findings suggest that COA-Cl may be useful as an inotropic agent for promoting angiogenesis in the future.


Assuntos
Adenosina/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Adenosina/análogos & derivados , Linhagem Celular , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/metabolismo
17.
Endocrinology ; 160(10): 2282-2297, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31290979

RESUMO

The human endometrium is the inner lining of the uterus consisting of stromal and epithelial (secretory and ciliated) cells. It undergoes a hormonally regulated monthly cycle of growth, differentiation, and desquamation. However, how these cyclic changes control the balance between secretory and ciliated cells remains unclear. Here, we established endometrial organoids to investigate the estrogen (E2)-driven control of cell fate decisions in human endometrial epithelium. We demonstrate that they preserve the structure, expression patterns, secretory properties, and E2 responsiveness of their tissue of origin. Next, we show that the induction of ciliated cells is orchestrated by the coordinated action of E2 and NOTCH signaling. Although E2 is the primary driver, inhibition of NOTCH signaling provides a permissive environment. However, inhibition of NOTCH alone is not sufficient to trigger ciliogenesis. Overall, we provide insights into endometrial biology and propose endometrial organoids as a robust and powerful model for studying ciliogenesis in vitro.


Assuntos
Cílios/fisiologia , Endométrio/fisiologia , Estrogênios/metabolismo , Organoides/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Técnicas de Cultura de Tecidos
18.
Nature ; 572(7768): 199-204, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31292543

RESUMO

The human liver is an essential multifunctional organ. The incidence of liver diseases is rising and there are limited treatment options. However, the cellular composition of the liver remains poorly understood. Here we performed single-cell RNA sequencing of about 10,000 cells from normal liver tissue from nine human donors to construct a human liver cell atlas. Our analysis identified previously unknown subtypes of endothelial cells, Kupffer cells, and hepatocytes, with transcriptome-wide zonation of some of these populations. We show that the EPCAM+ population is heterogeneous, comprising hepatocyte-biased and cholangiocyte populations as well as a TROP2int progenitor population with strong potential to form bipotent liver organoids. As a proof-of-principle, we used our atlas to unravel the phenotypic changes that occur in hepatocellular carcinoma cells and in human hepatocytes and liver endothelial cells engrafted into a mouse liver. Our human liver cell atlas provides a powerful resource to enable the discovery of previously unknown cell types in normal and diseased livers.


Assuntos
Células Epiteliais/citologia , Hepatócitos/citologia , Fígado/citologia , Células-Tronco/citologia , Adulto , Animais , Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular/metabolismo , Quimera/imunologia , Quimera/metabolismo , Células Endoteliais/citologia , Células Endoteliais/imunologia , Células Epiteliais/imunologia , Feminino , Regulação da Expressão Gênica , Hepatócitos/imunologia , Hepatócitos/metabolismo , Humanos , Fígado/imunologia , Masculino , Camundongos , Organoides/metabolismo , RNA Citoplasmático Pequeno/genética , Reprodutibilidade dos Testes , Células-Tronco/imunologia
19.
Cell Mol Life Sci ; 76(20): 4043-4070, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31317205

RESUMO

Stem cells give rise to all cells and build the tissue structures in our body, and heterogeneity and plasticity are the hallmarks of stem cells. Epigenetic modification, which is associated with niche signals, determines stem cell differentiation and somatic cell reprogramming. Stem cells play a critical role in the development of tumors and are capable of generating 3D organoids. Understanding the properties of stem cells will improve our capacity to maintain tissue homeostasis. Dissecting epigenetic regulation could be helpful for achieving efficient cell reprograming and for developing new drugs for cancer treatment. Stem cell-derived organoids open up new avenues for modeling human diseases and for regenerative medicine. Nevertheless, in addition to the achievements in stem cell research, many challenges still need to be overcome for stem cells to have versatile application in clinics.


Assuntos
Epigênese Genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Organoides/metabolismo , Células-Tronco/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Diferenciação Celular , Transdiferenciação Celular , Reprogramação Celular , Transição Epitelial-Mesenquimal , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Células-Tronco Neoplásicas/patologia , Organoides/patologia , Medicina Regenerativa/métodos , Nicho de Células-Tronco/genética , Transplante de Células-Tronco/métodos , Células-Tronco/classificação , Células-Tronco/citologia
20.
PLoS One ; 14(7): e0219944, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31339921

RESUMO

Precision medicine might be the response to the recent questioning of the use of metformin as an anticancer drug in colorectal cancer (CRC). Thus, in order to establish properly its benefits, metformin application needs to be assayed on the different progression stages of CRC. In this way, intestinal organoids imply a more physiological tool, representing a new therapeutic opportunity for CRC personalized treatment to assay tumor stage-dependent drugs. The previously reported lipid metabolism-related axis, Acyl-CoA synthetases/ Stearoyl-CoA desaturase (ACSLs/SCD), stimulates colon cancer progression and metformin is able to rescue the invasive and migratory phenotype conferred to cancer cells upon this axis overexpression. Therefore, we checked ACSL/SCD axis status, its regulatory miRNAs and the effect of metformin treatment in intestinal organoids with the most common acquired mutations in a sporadic CRC (CRC-like organoids) as a model for specific and personalized treatment. Despite ACSL4 expression is upregulated progressively in CRC-like organoids, metformin is able to downregulate its expression, especially in the first two stages (I, II). Besides, organoids are clearly more sensitive in the first stage (Apc mutated) to metformin than current chemotherapeutic drugs such as fluorouracil (5-FU). Metformin performs an independent "Warburg effect" blockade to cancer progression and is able to reduce crypt stem cell markers expression such as LGR5+. These results suggest a putative increased efficiency of the use of metformin in early stages of CRC than in advanced disease.


Assuntos
Neoplasias Colorretais/metabolismo , Metabolismo dos Lipídeos , Organoides/metabolismo , Animais , Antineoplásicos/farmacologia , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Neoplasias Colorretais/patologia , Regulação para Baixo , Fluoruracila/farmacologia , Glicólise , Hipoglicemiantes/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Metformina/farmacologia , Camundongos , Organoides/efeitos dos fármacos
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