RESUMO
The study of intestinal stem cells is a prerequisite for the development of therapies aimed at regenerating the gut. To enable investigation of adult slow-cycling H2B-GFP-retaining putative small intestinal (SI) stem cells in vitro, we have developed a three-dimensional (3D) SI organoid culture model based on the Tet-Op histone 2 B (H2B)-green fluorescent protein (GFP) fusion protein (Tet-Op-H2B-GFP) transgenic mouse. SI crypts were isolated from 6- to 12-week-old Tet-Op-H2B-GFP transgenic mice and cultured with appropriate growth factors and an animal-derived matrix (Matrigel). For in vitro transgene expression, doxycycline was added to the culture medium for 24 h. By pulse-chase experiments, H2B-GFP expression and retention were assessed through direct GFP fluorescence observations, both by confocal and fluorescence microscopy and by immunohistochemistry. The percentages of H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring relevant GFP-positive cells. Our results indicate that 24 h exposure to doxycycline (pulse) induced ubiquitous expression of H2B-GFP in the SI organoids. During subsequent culture, in the absence of doxycycline (chase), there was a gradual loss (due to cell division) of H2B-GFP. At 6-day chase, slow-cycling H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells were detected in the SI organoids. The developed culture model allows detection of slow-cycling H2B-GFP-retaining putative SI stem cells and will enable the study of self-renewal and regeneration for further characterization of these cells.
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The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/- cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.
Assuntos
Proteínas de Ligação a DNA , Neoplasias Ovarianas , Humanos , Feminino , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Células HeLa , Medicina de Precisão , Proteína BRCA2/metabolismo , Reparo do DNA , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Linhagem Celular TumoralRESUMO
Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ development, function and disease. However, the full potential of organoids in research and therapy has remained unrealized, owing to the poorly defined animal-derived matrices in which they are grown. Here we used modular synthetic hydrogel networks to define the key extracellular matrix (ECM) parameters that govern intestinal stem cell (ISC) expansion and organoid formation, and show that separate stages of the process require different mechanical environments and ECM components. In particular, fibronectin-based adhesion was sufficient for ISC survival and proliferation. High matrix stiffness significantly enhanced ISC expansion through a yes-associated protein 1 (YAP)-dependent mechanism. ISC differentiation and organoid formation, on the other hand, required a soft matrix and laminin-based adhesion. We used these insights to build a fully defined culture system for the expansion of mouse and human ISCs. We also produced mechanically dynamic matrices that were initially optimal for ISC expansion and subsequently permissive to differentiation and intestinal organoid formation, thus creating well-defined alternatives to animal-derived matrices for the culture of mouse and human stem-cell-derived organoids. Our approach overcomes multiple limitations of current organoid cultures and greatly expands their applicability in basic and clinical research. The principles presented here can be extended to identify designer matrices that are optimal for long-term culture of other types of stem cells and organoids.
Assuntos
Técnicas de Cultura de Células/métodos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Intestinos/citologia , Organoides/citologia , Organoides/crescimento & desenvolvimento , Células-Tronco/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Adesão Celular , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Forma Celular , Fibronectinas/metabolismo , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/síntese química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Camundongos , Proteólise , Nicho de Células-TroncoRESUMO
A long-standing question in biology is whether multipotent somatic stem and progenitor cells (SSPCs) feature molecular properties that could guide their system-independent identification. Population-based transcriptomic studies have so far not been able to provide a definite answer, given the rarity and heterogeneous nature of these cells. Here, we exploited the resolving power of single-cell RNA-sequencing to develop a computational model that is able to accurately distinguish SSPCs from differentiated cells across tissues. The resulting classifier is based on the combined expression of 23 genes including known players in multipotency, proliferation, and tumorigenesis, as well as novel ones, such as Lcp1 and Vgll4 that we functionally validate in intestinal organoids. We show how this approach enables the identification of stem-like cells in still ambiguous systems such as the pancreas and the epidermis as well as the exploration of lineage commitment hierarchies, thus facilitating the study of biological processes such as cellular differentiation, tissue regeneration, and cancer. Stem Cells 2017;35:2390-2402.
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Células-Tronco Multipotentes/metabolismo , Células-Tronco/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Diferenciação Celular/fisiologia , Genômica , Humanos , Células-Tronco Multipotentes/citologia , Células-Tronco/citologiaRESUMO
The Wnt/ß-catenin signalling and autophagy pathways each play important roles during development, adult tissue homeostasis and tumorigenesis. Here we identify the Wnt/ß-catenin signalling pathway as a negative regulator of both basal and stress-induced autophagy. Manipulation of ß-catenin expression levels in vitro and in vivo revealed that ß-catenin suppresses autophagosome formation and directly represses p62/SQSTM1 (encoding the autophagy adaptor p62) via TCF4. Furthermore, we show that during nutrient deprivation ß-catenin is selectively degraded via the formation of a ß-catenin-LC3 complex, attenuating ß-catenin/TCF-driven transcription and proliferation to favour adaptation during metabolic stress. Formation of the ß-catenin-LC3 complex is mediated by a W/YXXI/L motif and LC3-interacting region (LIR) in ß-catenin, which is required for interaction with LC3 and non-proteasomal degradation of ß-catenin. Thus, Wnt/ß-catenin represses autophagy and p62 expression, while ß-catenin is itself targeted for autophagic clearance in autolysosomes upon autophagy induction. These findings reveal a regulatory feedback mechanism that place ß-catenin at a key cellular integration point coordinating proliferation with autophagy, with implications for targeting these pathways for cancer therapy.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Neoplasias do Colo/patologia , Lisossomos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Western Blotting , Imunoprecipitação da Cromatina , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Sequestossoma-1 , Fator de Transcrição 4 , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Proteínas Wnt/genética , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
BACKGROUND: Cyclooxygenase-2 (COX-2) overexpression in colorectal cancer increases levels of its pro-tumorigenic product prostaglandin E2 (PGE(2)). The recently identified colorectal tumour suppressor 15-prostaglandin dehydrogenase (15-PGDH) catalyses prostaglandin turnover and is downregulated at a very early stage in colorectal tumorigenesis; however, the mechanism responsible remains unclear. As Wnt/ß-catenin signalling is also deregulated early in colorectal neoplasia, a study was undertaken to determine whether ß-catenin represses 15-PGDH expression. METHODS: The effect of modulating Wnt/ß-catenin signalling (using ß-catenin siRNA, mutant TCF4, Wnt3A or GSK3 inhibition) on 15-PGDH mRNA, protein expression and promoter activity was determined in colorectal cell lines by immunoblotting, qRT-PCR and reporter assays. The effect of ß-catenin deletion in vivo was addressed by 15-PGDH immunostaining of ß-catenin(-/lox)-villin-creERT2 mouse tissue. 15-PGDH promoter occupancy was determined using chromatin immunoprecipitation and PGE(2) levels by ELISA. RESULTS: The study shows for the first time that ß-catenin knockdown upregulates 15-PGDH in colorectal adenoma and carcinoma cells without affecting COX-2 protein levels. A dominant negative mutant form of TCF4 (dnTCF4), unable to bind ß-catenin, also upregulated 15-PGDH; conversely, increasing ß-catenin activity using Wnt3A or GSK3 inhibition downregulated 15-PGDH. Importantly, inducible ß-catenin deletion in vivo also upregulated intestinal epithelial 15-PGDH. 15-PGDH regulation occurred at the protein, mRNA and promoter activity levels and chromatin immunoprecipitation indicated ß-catenin/TCF4 binding to the 15-PGDH promoter. ß-catenin knockdown decreased PGE(2) levels, and this was significantly rescued by 15-PGDH siRNA. CONCLUSION: These data suggest a novel role for ß-catenin in promoting colorectal tumorigenesis through very early 15-PGDH suppression leading to increased PGE(2) levels, possibly even before COX-2 upregulation.
Assuntos
Adenoma/enzimologia , Neoplasias Colorretais/enzimologia , Hidroxiprostaglandina Desidrogenases/metabolismo , Mucosa Intestinal/enzimologia , beta Catenina/fisiologia , Animais , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Regulação para Baixo , Repressão Enzimática , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Hidroxiprostaglandina Desidrogenases/biossíntese , Hidroxiprostaglandina Desidrogenases/genética , Immunoblotting , Imuno-Histoquímica , Camundongos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Regulação para Cima , beta Catenina/genéticaRESUMO
The intestine is a prime example of self-renewal where stem cells give rise to progenitor cells called transit-amplifying cells which differentiate into more specialized cells. There are two intestinal lineages: the absorptive (enterocytes and microfold cells) and the secretory (Paneth cells, enteroendocrine, goblet cells, and tuft cells). Each of these differentiated cell types has a role in creating an "ecosystem" to maintain intestinal homeostasis. Here, we summarize the main roles of each cell type.
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Enterócitos , Células Epiteliais , Diferenciação Celular , Células M , Células-TroncoRESUMO
Three-dimensional (3D) culture models are more physiologically relevant than two-dimensional (2D) cell culture models. 2D approaches cannot reproduce the complexity of the tumor microenvironment and are less able to translate biological insights; and drug response studies have many limitations to be extrapolated to the clinics. Here, we use the Caco-2 colon cancer cell line, which is an immortalized human epithelial cell line that under specific conditions can polarize and differentiate into a villus-like phenotype. We describe cell differentiation and cell growth in both 2D and 3D culture conditions, concluding that cell morphology, polarity, proliferation and differentiation are highly dependent on the type of cell culture system.
Assuntos
Técnicas de Cultura de Células em Três Dimensões , Intestinos , Humanos , Células CACO-2 , Fenótipo , Diferenciação CelularRESUMO
The technique electric cell-substrate impedance sensing (ECIS) can be used to detect and monitor the behavior of intestinal cells. The methodology presented was designed to achieve results within a short time frame, and it was tailored to use a colonic cancer cell line. Differentiation of intestinal cancer cells has previously been reported to be regulated by retinoic acid (RA). Here, colonic cancer cells were cultured in the ECIS array before being treated with RA, and any changes in response to RA were monitored after treatment. The ECIS recorded changes in impedance in response to the treatment and vehicle. This methodology poses as a novel way to record the behavior of colonic cells and opens new avenues for in vitro research.
Assuntos
Neoplasias do Colo , Intestinos , Humanos , Impedância Elétrica , Diferenciação Celular , Tretinoína/farmacologiaRESUMO
The intestine consists of epithelial cells surrounded by a complex environment as mesenchymal cells and the gut microbiota. With its impressive stem cell regeneration capability, the intestine is able to constantly replenish cells lost through apoptosis or abrasion by food passing through. Over the past decade, researchers have identified signaling pathways involved in stem cell homeostasis such as retinoids pathway. Retinoids are also involved in cell differentiation of healthy and cancer cells. In this study, we describe several approaches in vitro and in vivo to further investigate the effect of retinoids on stem cells, progenitors, and differentiated intestinal cells.
Assuntos
Apoptose , Bioensaio , Diferenciação Celular , Intestinos , Retinoides/farmacologiaRESUMO
Based on the crystal structures of human vitamin D receptor (hVDR) bound to 1α,25-dihydroxy-vitamin D(3) (1,25 D) and superagonist ligands, we previously designed new superagonist ligands with a tetrahydrofuran ring at the side chain that optimize the aliphatic side-chain conformation through an entropy benefit. Following a similar strategy, four novel vitamin D analogues with aromatic furan side chains (3a, 3b, 4a, 4b) have now been developed. The triene system has been constructed by an efficient stereoselective intramolecular cyclization of an enol triflate (A-ring precursor) followed by a Suzuki-Miyaura coupling of the resulting intermediate with an alkenyl boronic ester (CD-side chain, upper fragment). The furan side chains have been constructed by gold chemistry. These analogues exhibit significant pro-differentiation effects and transactivation potency. The crystal structure of 3a in a complex with the ligand-binding domain of hVDR revealed that the side-chain furanic ring adopts two conformations.
Assuntos
Furanos/química , Furanos/farmacologia , Receptores de Calcitriol/metabolismo , Vitamina D/análogos & derivados , Vitamina D/farmacologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Ativação Transcricional/efeitos dos fármacosRESUMO
The Triple Negative Breast Cancer (TNBC) subtype is known to have a more aggressive clinical course compared to other breast cancer subtypes. Targeted therapies for this type of breast cancer are limited and patients are mostly treated with conventional chemo- and radio-therapies which are not specific and do not target resistant cells. Therefore, one of the major clinical challenges is to find compounds that target the drug-resistant cell populations which are responsible for reforming secondary tumours. The molecular profiling of the different TNBC subtypes holds a promise for better defining these resistant cells specific to each tumour. To this end, a better understanding of TNBC heterogeneity and cancer stemness is required, and extensive genomic analysis can help to understand the disease complexity and distinguish new molecular drivers that can be targeted in the clinics. The use of persister cancer cell-targeting therapies combined with other therapies may provide a big advance to improve TNBC patients' survival.
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Several non-hypercalcemic analogs of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) show antitumor activity in a subset of cancer patients. High vitamin D receptor (VDR) expression, which is associated with good prognosis but is lost during tumor progression. We show that the SNAIL transcription factor represses VDR gene expression in human colon cancer cells and blocks the antitumor action of EB1089, a 1,25(OH)(2)D(3) analog, in xenografted mice. In human colon cancers, elevated SNAIL expression correlates with downregulation of VDR.
Assuntos
Calcitriol/análogos & derivados , Calcitriol/antagonistas & inibidores , Neoplasias do Colo/metabolismo , Proteínas de Ligação a DNA/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores de Calcitriol/metabolismo , Fatores de Transcrição/farmacologia , Animais , Antineoplásicos/antagonistas & inibidores , Caderinas/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Imunoprecipitação , Camundongos , Regiões Promotoras Genéticas/genética , Receptores de Calcitriol/genética , Fatores de Transcrição da Família SnailRESUMO
Organoids allow the recapitulation of intestinal homeostasis and cancerogenesis in vitro; however, RNA sequencing (RNA-seq)-based methods for drug screens are missing. We develop targeted organoid sequencing (TORNADO-seq), a high-throughput, high-content drug discovery platform that uses targeted RNA-seq to monitor the expression of large gene signatures for the detailed evaluation of cellular phenotypes in organoids. TORNADO-seq is a fast, highly reproducible time- and cost-effective ($5 per sample) method that can probe cell mixtures and their differentiation state in the intestinal system. We apply this method to isolate drugs that enrich for differentiated cell phenotypes and show that these drugs are highly efficacious against cancer compared to wild-type organoids. Furthermore, TORNADO-seq facilitates in-depth insight into the mode of action of these drugs. Our technology can easily be adapted to many other systems and will allow for more systematic, large-scale, and quantitative approaches to study the biology of complex cellular systems.
Assuntos
Antineoplásicos/farmacologia , Detecção Precoce de Câncer/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Organoides/efeitos dos fármacos , Medicamentos sob Prescrição/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antineoplásicos/química , Antineoplásicos/classificação , Diferenciação Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Enterócitos/patologia , Redes Reguladoras de Genes , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Ensaios de Triagem em Larga Escala , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Organoides/metabolismo , Organoides/patologia , Celulas de Paneth/efeitos dos fármacos , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Medicamentos sob Prescrição/química , Medicamentos sob Prescrição/classificação , RNA-Seq , Análise de Sequência de RNA , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/classificaçãoRESUMO
Recent advances in intestinal organoid technologies have paved the way for in vitro recapitulation of the homeostatic renewal of adult tissues, tissue or organ morphogenesis during development, and pathogenesis of many disorders. In vitro modelling of individual patient diseases using organoid systems have been considered key in establishing rational design of personalized treatment strategies and in improving therapeutic outcomes. In addition, the transplantation of organoids into diseased tissues represents a novel approach to treat currently incurable diseases. Emerging evidence from intensive studies suggests that organoid systems' development and functional maturation depends on the presence of an extracellular matrix with suitable biophysical properties, where advanced synthetic hydrogels open new avenues for theoretical control of organoid phenotypes and potential applications of organoids in therapeutic purposes. In this review, we discuss the status, applications, challenges and perspectives of intestinal organoid systems emphasising on hydrogels and their properties suitable for intestinal organoid culture. We provide an overview of hydrogels used for intestinal organoid culture and key factors regulating their biological activity. The comparison of different hydrogels would be a theoretical basis for establishing design principles of synthetic niches directing intestinal cell fates and functions. STATEMENT OF SIGNIFICANCE: Intestinal organoid is an in vitro recapitulation of the gut, which self-organizes from intestinal stem cells and maintains many features of the native tissue. Since the development of this technology, intestinal organoid systems have made significant contribution to rapid progress in intestinal biology. Prevailing methodology for organoid culture, however, depends on animal-derived matrices and suffers from variability and potential risk for contamination of pathogens, limiting their therapeutic application. Synthetic scaffold matrices, hydrogels, might provide solutions to these issues and deepen our understanding on how intestinal cells sense and respond to key biophysical properties of the surrounding matrices. This review provides an overview of developing intestinal models and biomaterials, thereby leading to better understanding of current intestinal organoid systems for both biologists and materials scientists.
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Materiais Biocompatíveis , Organoides , Animais , Humanos , Hidrogéis , Células-Tronco , TecnologiaRESUMO
The genomes of many human CRCs have been sequenced, revealing a large number of genetic alterations. However, the molecular mechanisms underlying the accumulation of these alterations are still being debated. In this study, we examined colorectal tumours that developed in mice with Apclox/lox, LSL-KrasG12D, and Tp53lox/lox targetable alleles. Organoids were derived from single cells and the spectrum of mutations was determined by exome sequencing. The number of single nucleotide substitutions (SNSs) correlated with the age of the tumour, but was unaffected by the number of targeted cancer-driver genes. Thus, tumours that expressed mutant Apc, Kras, and Tp53 alleles had as many SNSs as tumours that expressed only mutant Apc. In contrast, the presence of large-scale (>10 Mb) copy number alterations (CNAs) correlated strongly with Tp53 inactivation. Comparison of the SNSs and CNAs present in organoids derived from the same tumour revealed intratumoural heterogeneity consistent with genomic lesions accumulating at significantly higher rates in tumour cells compared to normal cells. The rate of acquisition of SNSs increased from the early stages of cancer development, whereas large-scale CNAs accumulated later, after Tp53 inactivation. Thus, a significant fraction of the genomic instability present in cancer cells cannot be explained by aging processes occurring in normal cells before oncogenic transformation.
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The presence of the proteins mouse R-Spondin1 (mRSpo1) and mouse Noggin (mNoggin) in a 3D-organoid culture allows for the maintenance of intestinal stem cells. Here, we describe a transient gene expression method for the production of these proteins from human embryo kidney 293 (HEK293) cells cultivated in suspension using orbitally shaken bioreactors. Plasmid DNA was delivered into cells using the cationic polymer polyethylenimine (PEI). The 7-day production cultures were performed in the presence of valproic acid (VPA), an enhancer of recombinant gene expression. Both proteins were secreted from the transfected cells. mRSpo1 was produced as a secreted Fc fusion protein (mRSpo1-Fc) and purified by protein A-based affinity chromatography. mNoggin was produced as a secreted histidine-tagged protein (mNoggin-His) and purified by immobilized metal affinity chromatography (IMAC). This transient transfection system supports a high production efficiency.
Assuntos
Organoides/citologia , Proteínas Recombinantes/metabolismo , Células-Tronco/citologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromatografia de Afinidade , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Organoides/metabolismo , Polietilenoimina/química , Proteínas Recombinantes/genética , Células-Tronco/metabolismoRESUMO
Intestinal stem cells are responsible for tissue renewal. The study of stem cell properties has become a major challenge in the field. We describe here a method based on Cre recombinase inducible lentivirus vectors that permits delivery of transgenes, either for overexpression or knockdown, in primary stem cells that can be cultured in an 3D intestinal organoid system. This method is an excellent approach for genetic manipulation and can complement in vivo transgenic experiments.
Assuntos
Integrases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Integrases/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Organoides/citologia , Organoides/metabolismo , Receptores Acoplados a Proteínas G/genética , Recombinação Genética/genética , Recombinação Genética/fisiologiaRESUMO
In many tumor types, only a minor pool of cancer cells-the so-called cancer stem cells-is able to colonize distant organs and give rise to secondary tumors. In humans, the liver is one of the main target organs for many metastatic tumor types, including colorectal cancer. However, mouse tumour models only rarely spontaneously metastasize to the liver. Therefore, reliable in vivo experimental metastasis assays are crucial to study cell seeding capacity and the mechanisms controlling these metastatic stem cell properties. Here, we describe an intrasplenic injection model that mimics the process of liver metastasis occurring in cancer patients.