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 ReversaRESUMO
RNF43/ZNRF3 negatively regulate WNT signalling. Both genes are mutated in several types of cancers, however, their contribution to liver disease is unknown. Here we describe that hepatocyte-specific loss of Rnf43/Znrf3 results in steatohepatitis and in increase in unsaturated lipids, in the absence of dietary fat supplementation. Upon injury, Rnf43/Znrf3 deletion results in defective hepatocyte regeneration and liver cancer, caused by an imbalance between differentiation/proliferation. Using hepatocyte-, hepatoblast- and ductal cell-derived organoids we demonstrate that the differentiation defects and lipid alterations are, in part, cell-autonomous. Interestingly, ZNRF3 mutant liver cancer patients present poorer prognosis, altered hepatic lipid metabolism and steatohepatitis/NASH signatures. Our results imply that RNF43/ZNRF3 predispose to liver cancer by controlling the proliferative/differentiation and lipid metabolic state of hepatocytes. Both mechanisms combined facilitate the progression towards malignancy. Our findings might aid on the management of those RNF43/ZNRF3 mutated individuals at risk of developing fatty liver and/or liver cancer.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Regeneração Hepática , Fígado/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adulto , Animais , Carcinoma Hepatocelular/patologia , Diferenciação Celular , Proliferação de Células , Fígado Gorduroso/patologia , Deleção de Genes , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatomegalia/patologia , Humanos , Hiperplasia , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/genética , Lipidômica , Fígado/patologia , Neoplasias Hepáticas/patologia , Camundongos , PrognósticoRESUMO
Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/patologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias Hepáticas/patologia , Organoides/patologia , Cultura Primária de Células/métodos , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/uso terapêutico , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Proliferação de Células , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Medicina de Precisão , Transcriptoma , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent data suggest that SRC family kinases (SFKs) could represent potential therapeutic targets for rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. Here, we assessed the effect of a recently developed selective SFK inhibitor (a pyrazolo[3,4-d]pyrimidine derivative, called SI221) on RMS cell lines. SI221, which showed to be mainly effective against the SFK member YES, significantly reduced cell viability and induced apoptosis, without affecting non-tumor cells, such as primary human skin fibroblasts and differentiated C2C12 cells. Moreover, SI221 decreased in vitro cell migration and invasion and reduced tumor growth in a RMS xenograft model. SFK inhibition also induced muscle differentiation in RMS cells by affecting the NOTCH3 receptor-p38 mitogen-activated protein kinase (MAPK) axis, which regulates the balance between proliferation and differentiation. Overall, our findings suggest that SFK inhibition, besides reducing RMS cell growth and invasive potential, could also represent a differentiation therapeutic strategy for RMS.
Assuntos
Antineoplásicos/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Rabdomiossarcoma/patologia , Quinases da Família src/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Diferenciação Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Imunofluorescência , Humanos , Camundongos , Reação em Cadeia da Polimerase , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The liver is capable of full regeneration following several types and rounds of injury, ranging from hepatectomy to toxin-mediated damage. The source of this regenerative capacity has long been a hotly debated topic. The damage response that occurs when hepatocyte proliferation is impaired is thought to be mediated by oval/dedifferentiated progenitor cells, which replenish the hepatocyte and ductal compartments of the liver. Recently, reports have questioned whether these oval/progenitor cells truly serve as the facultative stem cell of the liver following toxin-mediated damage. In this issue of the JCI, Kordes and colleagues use lineage tracing to follow transplanted rat hepatic stellate cells, a resident liver mesenchymal cell population, in hosts that have suffered liver damage. Transplanted stellate cells repopulated the damaged rat liver by contributing to the oval cell response. These data establish yet another cell type of mesenchymal origin as the progenitor for the oval/ductular response in the rat. The lack of uniformity between different damage models, the extent of the injury to the liver parenchyma, and potential species-specific differences might be at the core of the discrepancy between different studies. Taken together, these data imply a considerable degree of plasticity in the liver, whereby several cell types can contribute to regeneration.