Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 79
Filtrar
1.
Cell ; 187(17): 4439-4443, 2024 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-39178827

RESUMO

It is said that all models are wrong, but some are useful. In vitro human cell-based models are a prime example of this maxim. We asked researchers: when is your model system useful? How can it be made more useful? What are its limitations?


Assuntos
Modelos Biológicos , Humanos , Técnicas de Cultura de Células/métodos
2.
Nat Rev Mol Cell Biol ; 22(9): 608-624, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34079104

RESUMO

Liver regeneration is a complex process involving the crosstalk of multiple cell types, including hepatocytes, hepatic stellate cells, endothelial cells and inflammatory cells. The healthy liver is mitotically quiescent, but following toxic damage or resection the cells can rapidly enter the cell cycle to restore liver mass and function. During this process of regeneration, epithelial and non-parenchymal cells respond in a tightly coordinated fashion. Recent studies have described the interaction between inflammatory cells and a number of other cell types in the liver. In particular, macrophages can support biliary regeneration, contribute to fibrosis remodelling by repressing hepatic stellate cell activation and improve liver regeneration by scavenging dead or dying cells in situ. In this Review, we describe the mechanisms of tissue repair following damage, highlighting the close relationship between inflammation and liver regeneration, and discuss how recent findings can help design novel therapeutic approaches.


Assuntos
Cirrose Hepática/patologia , Cirrose Hepática/terapia , Regeneração Hepática/fisiologia , Transplante de Células , Células Epiteliais/citologia , Células Epiteliais/transplante , Células Estreladas do Fígado/metabolismo , Hepatócitos/citologia , Hepatócitos/patologia , Hepatócitos/transplante , Humanos , Inflamação , Macrófagos/citologia , Macrófagos/patologia , Macrófagos/transplante , Transdução de Sinais
3.
Cell ; 160(1-2): 299-312, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25533785

RESUMO

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.


Assuntos
Fígado/citologia , Técnicas de Cultura de Órgãos , Animais , Instabilidade Genômica , Hepatócitos/citologia , Humanos , Camundongos , Organoides/citologia
4.
Cell ; 160(1-2): 324-38, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25557080

RESUMO

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.


Assuntos
Carcinoma Ductal Pancreático/patologia , Modelos Biológicos , Técnicas de Cultura de Órgãos , Organoides/patologia , Neoplasias Pancreáticas/patologia , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Pâncreas/metabolismo , Pâncreas/patologia
5.
Cell ; 155(2): 357-68, 2013 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-24120136

RESUMO

Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street."


Assuntos
Celulas Principais Gástricas/citologia , Células-Tronco/citologia , Estômago/citologia , Animais , Linhagem da Célula , Celulas Principais Gástricas/química , Mucosa Gástrica/citologia , Camundongos , Organoides/citologia , Receptores do Fator de Necrose Tumoral/análise , Via de Sinalização Wnt
6.
Cell ; 151(7): 1595-607, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23260145

RESUMO

Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in ß cells. Here, a mouse genetics approach shows that removal of TCF4 from ß cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that is acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout mice show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease.


Assuntos
Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Redes e Vias Metabólicas , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Dieta Hiperlipídica , Jejum/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Ativação Transcricional
7.
Development ; 149(20)2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-36255367

RESUMO

In June 2022, the second meeting on 'Engineering Multicellular Systems', organized by the European Molecular Biology Laboratory and the Institute of Bioengineering of Catalonia, took place in Barcelona. Stem cell and systems biologists, physicists and engineers from all over the world gathered to discuss how recent breakthroughs in organoid technologies, engineering and mechanobiology are boosting our understanding of early morphogenesis, organogenesis and organ function with applications in tissue engineering, disease modeling and drug screening. The meeting was organized with sustainability in mind, and included an ethics session and an outreach public activity.


Assuntos
Organoides , Engenharia Tecidual , Bioengenharia , Organogênese , Morfogênese
8.
EMBO Rep ; 24(9): e57181, 2023 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-37522754

RESUMO

Hepatocytes form bile canaliculi that dynamically respond to the signalling activity of bile acids and bile flow. Little is known about their responses to intraluminal pressure. During embryonic development, hepatocytes assemble apical bulkheads that increase the canalicular resistance to intraluminal pressure. Here, we investigate whether they also protect bile canaliculi against elevated pressure upon impaired bile flow in adult liver. Apical bulkheads accumulate upon bile flow obstruction in mouse models and patients with primary sclerosing cholangitis (PSC). Their loss under these conditions leads to abnormally dilated canaliculi, resembling liver cell rosettes described in other hepatic diseases. 3D reconstruction reveals that these structures are sections of cysts and tubes formed by hepatocytes. Mathematical modelling establishes that they positively correlate with canalicular pressure and occur in early PSC stages. Using primary hepatocytes and 3D organoids, we demonstrate that excessive canalicular pressure causes the loss of apical bulkheads and formation of rosettes. Our results suggest that apical bulkheads are a protective mechanism of hepatocytes against impaired bile flow, highlighting the role of canalicular pressure in liver diseases.


Assuntos
Bile , Hepatopatias , Camundongos , Animais , Fígado , Canalículos Biliares , Hepatócitos
9.
Gut ; 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39266051

RESUMO

BACKGROUND: Cholangiocarcinoma (CCA) is a very difficult-to-treat cancer. Chemotherapies are little effective and response to immune checkpoint inhibitors is limited. Therefore, new therapeutic strategies need to be identified. OBJECTIVE: We characterised the enzyme protein arginine-methyltransferase 5 (PRMT5) as a novel therapeutic target in CCA. DESIGN: We evaluated the expression of PRMT5, its functional partner MEP50 and methylthioadenosine phosphorylase (MTAP)-an enzyme that modulates the sensitivity of PRMT5 to pharmacological inhibitors-in human CCA tissues. PRMT5-targeting drugs, currently tested in clinical trials for other malignancies, were assessed in human CCA cell lines and organoids, as well as in two immunocompetent CCA mouse models. Transcriptomic, proteomic and functional analyses were performed to explore the underlying antitumoural mechanisms. RESULTS: PRMT5 and MEP50 proteins were correlatively overexpressed in most CCA tissues. MTAP was absent in 25% of intrahepatic CCA. PRMT5-targeting drugs markedly inhibited CCA cell proliferation, synergising with cisplatin and gemcitabine and hindered the growth of cholangiocarcinoma organoids. PRMT5 inhibition blunted the expression of oncogenic genes involved in chromatin remodelling and DNA repair, consistently inducing the formation of RNA loops and promoting DNA damage. Treatment with PRMT5-targeting drugs significantly restrained the growth of experimental CCA without adverse effects and concomitantly induced the recruitment of CD4 and CD8 T cells to shrinking tumourous lesions. CONCLUSION: PRMT5 and MEP50 are frequently upregulated in human CCA, and PRMT5-targeting drugs have significant antitumoural efficacy in clinically relevant CCA models. Our findings support the evaluation of PRMT5 inhibitors in clinical trials, including their combination with cytotoxic and immune therapies.

10.
Chem Res Toxicol ; 37(2): 234-247, 2024 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-38232180

RESUMO

Human tissue three-dimensional (3D) organoid cultures have the potential to reproduce in vitro the physiological properties and cellular architecture of the organs from which they are derived. The ability of organoid cultures derived from human stomach, liver, kidney, and colon to metabolically activate three dietary carcinogens, aflatoxin B1 (AFB1), aristolochic acid I (AAI), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was investigated. In each case, the response of a target tissue (liver for AFB1; kidney for AAI; colon for PhIP) was compared with that of a nontarget tissue (gastric). After treatment cell viabilities were measured, DNA damage response (DDR) was determined by Western blotting for p-p53, p21, p-CHK2, and γ-H2AX, and DNA adduct formation was quantified by mass spectrometry. Induction of the key xenobiotic-metabolizing enzymes (XMEs) CYP1A1, CYP1A2, CYP3A4, and NQO1 was assessed by qRT-PCR. We found that organoids from different tissues can activate AAI, AFB1, and PhIP. In some cases, this metabolic potential varied between tissues and between different cultures of the same tissue. Similarly, variations in the levels of expression of XMEs were observed. At comparable levels of cytotoxicity, organoids derived from tissues that are considered targets for these carcinogens had higher levels of adduct formation than a nontarget tissue.


Assuntos
Adutos de DNA , Neoplasias , Humanos , Carcinógenos/toxicidade , Carcinógenos/metabolismo , Fígado/metabolismo , Organoides/metabolismo
11.
Nat Rev Genet ; 24(6): 348, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36697957
12.
Development ; 146(12)2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31142540

RESUMO

During mouse embryogenesis, progenitors within the liver known as hepatoblasts give rise to adult hepatocytes and cholangiocytes. Hepatoblasts, which are specified at E8.5-E9.0, have been regarded as a homogeneous progenitor population that initiate differentiation from E13.5. Recently, scRNA-seq analysis has identified sub-populations of transcriptionally distinct hepatoblasts at E11.5. Here, we show that hepatoblasts are not only transcriptionally but also functionally heterogeneous, and that a subpopulation of E9.5-E10.0 hepatoblasts exhibit a previously unidentified early commitment to cholangiocyte fate. Importantly, we also identify a subpopulation constituting 2% of E9.5-E10.0 hepatoblasts that express the adult stem cell marker Lgr5, and generate both hepatocyte and cholangiocyte progeny that persist for the lifespan of the mouse. Combining lineage tracing and scRNA-seq, we show that Lgr5 marks E9.5-E10.0 bipotent liver progenitors residing at the apex of a hepatoblast hierarchy. Furthermore, isolated Lgr5+ hepatoblasts can be clonally expanded in vitro into embryonic liver organoids, which can commit to either hepatocyte or cholangiocyte fates. Our study demonstrates functional heterogeneity within E9.5 hepatoblasts and identifies Lgr5 as a marker for a subpopulation of bipotent liver progenitors.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos/citologia , Fígado/embriologia , Receptores Acoplados a Proteínas G/metabolismo , Alelos , Animais , Sequência de Bases , Contagem de Células , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Desenvolvimento Embrionário , Células Epiteliais/citologia , Feminino , Hepatócitos/metabolismo , Homeostase , Masculino , Camundongos , Microscopia Confocal , Células-Tronco/citologia
13.
Hepatology ; 73(6): 2380-2396, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33222246

RESUMO

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.


Assuntos
Neoplasias dos Ductos Biliares , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma , DNA (Citosina-5-)-Metiltransferase 1 , Inibidores Enzimáticos/farmacologia , Antígenos de Histocompatibilidade , Histona-Lisina N-Metiltransferase , Animais , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/fisiologia , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade/metabolismo , Código das Histonas/efeitos dos fármacos , Código das Histonas/fisiologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos , Resultado do Tratamento , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
14.
Nature ; 538(7624): 260-264, 2016 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-27698416

RESUMO

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.


Assuntos
Células-Tronco Adultas/metabolismo , Envelhecimento/genética , Acúmulo de Mutações , Taxa de Mutação , Especificidade de Órgãos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , Pré-Escolar , Colo/metabolismo , Análise Mutacional de DNA , Feminino , Genes Neoplásicos/genética , Humanos , Incidência , Intestino Delgado/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Células-Tronco Multipotentes/metabolismo , Neoplasias/epidemiologia , Neoplasias/genética , Organoides/metabolismo , Mutação Puntual/genética , Adulto Jovem
15.
BMC Biol ; 19(1): 37, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627108

RESUMO

BACKGROUND: Organoids are morphologically heterogeneous three-dimensional cell culture systems and serve as an ideal model for understanding the principles of collective cell behaviour in mammalian organs during development, homeostasis, regeneration, and pathogenesis. To investigate the underlying cell organisation principles of organoids, we imaged hundreds of pancreas and cholangiocarcinoma organoids in parallel using light sheet and bright-field microscopy for up to 7 days. RESULTS: We quantified organoid behaviour at single-cell (microscale), individual-organoid (mesoscale), and entire-culture (macroscale) levels. At single-cell resolution, we monitored formation, monolayer polarisation, and degeneration and identified diverse behaviours, including lumen expansion and decline (size oscillation), migration, rotation, and multi-organoid fusion. Detailed individual organoid quantifications lead to a mechanical 3D agent-based model. A derived scaling law and simulations support the hypotheses that size oscillations depend on organoid properties and cell division dynamics, which is confirmed by bright-field microscopy analysis of entire cultures. CONCLUSION: Our multiscale analysis provides a systematic picture of the diversity of cell organisation in organoids by identifying and quantifying the core regulatory principles of organoid morphogenesis.


Assuntos
Divisão Celular , Colangiocarcinoma/fisiopatologia , Morfogênese , Organoides/crescimento & desenvolvimento , Pâncreas/fisiologia , Animais , Epitélio/crescimento & desenvolvimento , Humanos , Camundongos , Microscopia
16.
Int J Mol Sci ; 24(1)2022 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-36614051

RESUMO

Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.


Assuntos
Benzo(a)pireno , Adutos de DNA , Organoides , Humanos , Ativação Metabólica , Benzo(a)pireno/toxicidade , Citocromo P-450 CYP1A1/metabolismo , Adutos de DNA/metabolismo , Fígado/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo
17.
BMC Dev Biol ; 20(1): 4, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32098630

RESUMO

BACKGROUND: Pancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human. Mouse pancreatic organoids exhibit unlimited expansion potential, while previously reported human pancreas organoid (hPO) cultures do not expand efficiently long-term in a chemically defined, serum-free medium. We sought to generate a 3D culture system for long-term expansion of human pancreas ductal cells as hPOs to serve as the basis for studies of human pancreas ductal epithelium, exocrine pancreatic diseases and the development of a genomically stable replacement cell therapy for diabetes mellitus. RESULTS: Our chemically defined, serum-free, human pancreas organoid culture medium supports the generation and expansion of hPOs with high efficiency from both fresh and cryopreserved primary tissue. hPOs can be expanded from a single cell, enabling their genetic manipulation and generation of clonal cultures. hPOs expanded for months in vitro maintain their ductal morphology, biomarker expression and chromosomal integrity. Xenografts of hPOs survive long-term in vivo when transplanted into the pancreas of immunodeficient mice. Notably, mouse orthotopic transplants show no signs of tumorigenicity. Crucially, our medium also supports the establishment and expansion of hPOs in a chemically defined, modifiable and scalable, biomimetic hydrogel. CONCLUSIONS: hPOs can be expanded long-term, from both fresh and cryopreserved human pancreas tissue in a chemically defined, serum-free medium with no detectable tumorigenicity. hPOs can be clonally expanded, genetically manipulated and are amenable to culture in a chemically defined hydrogel. hPOs therefore represent an abundant source of pancreas ductal cells that retain the characteristics of the tissue-of-origin, which opens up avenues for modelling diseases of the ductal epithelium and increasing understanding of human pancreas exocrine biology as well as for potentially producing insulin-secreting cells for the treatment of diabetes.


Assuntos
Organoides/citologia , Pâncreas/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Citometria de Fluxo , Instabilidade Genômica/fisiologia , Humanos , Técnicas In Vitro , Lentivirus/genética , Masculino , Técnicas de Cultura de Órgãos , Organoides/metabolismo , Pâncreas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Development ; 144(6): 938-941, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28292837

RESUMO

The recent increase in organoid research has been met with great enthusiasm, as well as expectation, from the scientific community and the public alike. There is no doubt that this technology opens up a world of possibilities for scientific discovery in developmental biology as well as in translational research, but whether organoids can truly live up to this challenge is, for some, still an open question. In this Spotlight article, Meritxell Huch and Juergen Knoblich begin by discussing the exciting promise of organoid technology and give concrete examples of how this promise is starting to be realised. In the second part, Matthias Lutolf and Alfonso Martinez-Arias offer a careful and considered view of the state of the organoid field and its current limitations, and lay out the approach they feel is necessary to maximise the potential of organoid technology.


Assuntos
Organoides/fisiologia , Pesquisa Translacional Biomédica , Crescimento e Desenvolvimento , Humanos , Engenharia Tecidual
19.
Nature ; 513(7518): 422-425, 2014 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-25043003

RESUMO

The somatic mutations present in the genome of a cell accumulate over the lifetime of a multicellular organism. These mutations can provide insights into the developmental lineage tree, the number of divisions that each cell has undergone and the mutational processes that have been operative. Here we describe whole genomes of clonal lines derived from multiple tissues of healthy mice. Using somatic base substitutions, we reconstructed the early cell divisions of each animal, demonstrating the contributions of embryonic cells to adult tissues. Differences were observed between tissues in the numbers and types of mutations accumulated by each cell, which likely reflect differences in the number of cell divisions they have undergone and varying contributions of different mutational processes. If somatic mutation rates are similar to those in mice, the results indicate that precise insights into development and mutagenesis of normal human cells will be possible.


Assuntos
Linhagem da Célula/genética , Células Clonais/citologia , Células Clonais/metabolismo , Genoma/genética , Mutagênese/genética , Mutação/genética , Animais , Relógios Biológicos/genética , Divisão Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Taxa de Mutação , Organoides/citologia , Organoides/metabolismo , Filogenia , Análise de Sequência de DNA , Cauda/citologia
20.
Gut ; 68(12): 2228-2237, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300517

RESUMO

Organoid cultures have emerged as an alternative in vitro system to recapitulate tissues in a dish. While mouse models and cell lines have furthered our understanding of liver biology and associated diseases, they suffer in replicating key aspects of human liver tissue, in particular its complex architecture and metabolic functions. Liver organoids have now been established for multiple species from induced pluripotent stem cells, embryonic stem cells, hepatoblasts and adult tissue-derived cells. These represent a promising addition to our toolbox to gain a deeper understanding of this complex organ. In this perspective we will review the advances in the liver organoid field, its limitations and potential for biomedical applications.


Assuntos
Pesquisa Biomédica/métodos , Fígado/citologia , Modelos Biológicos , Organoides/fisiologia , Medicina Regenerativa/métodos , Animais , Técnicas de Cultura de Células , Linhagem Celular , Humanos , Organoides/citologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA