Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium

Sato, Toshiro; Stange, Daniel E.; Ferrante, Marc; Vries, Robert G.J.; Es, Johan H. van; Brink, Stieneke van den; Houdt, Winan J. van; Pronk, Apollo; Gorp, Joost van; Siersema, Peter D.; Clevers, Hans

doi:10.1053/j.gastro.2011.07.050

Cited by 2,987 publications

(3,523 citation statements)

References 40 publications

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“…33 We were unable to establish organoids from intestines of mice induced for BRAF V600K expression for 3 or 4 days, further supporting our finding that ISCs are lost by the treatment. Organoids prepared from non-induced BRAF V600K transgenic mice that were maintained without doxycycline were organized into radial crypts and a central differentiated villus domain containing debris of extruded cells, as expected.…”

Section: Above)supporting

confidence: 71%

“…As the ISC compartment is maintained by Wnt and Notch signals, 1 we tested the effects of the ligands Wnt3a and Jagged1, which are known to activate the Wnt and Notch pathways in organotypic culture. 33 We found that Wnt3a, but not ); GDC: GDC0941 500 nM). n ⩾ 4 wells in multiple experiments, significance calculated by Student's t-test.…”

Section: Above)mentioning

confidence: 64%

“…For RNA analysis, villi were scraped from the inner intestinal surface after initial washing steps, whereas crypts were isolated by filtering (70 μm, BD) after phosphate-buffered saline/EDTA treatment, as described in Sato et al 33 For total tissue analysis, small pieces of the intestine were homogenized in Trizol (Life Technologies) using a Tissue Lyser machine (Qiagen, Hilden, Germany). Single-cell suspensions for FACS were prepared by digesting crypts of compound transgenic mice in 500 μg/ml trypsin and 0.8 μ/ml DNAseI for approximately 20 min at room temperature.…”

Section: Qrt-pcr and Microarray Analysismentioning

confidence: 99%

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Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity

et al. 2014

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Colon cancer cells frequently carry mutations that activate the β-catenin and mitogen-activated protein kinase (MAPK) signaling cascades. Yet how oncogenic alterations interact to control cellular hierarchies during tumor initiation and progression is largely unknown. We found that oncogenic BRAF modulates gene expression associated with cell differentiation in colon cancer cells. We therefore engineered a mouse with an inducible oncogenic BRAF transgene, and analyzed BRAF effects on cellular hierarchies in the intestinal epithelium in vivo and in primary organotypic culture. We demonstrate that transgenic expression of oncogenic BRAF in the mouse strongly activated MAPK signal transduction, resulted in the rapid development of generalized serrated dysplasia, but unexpectedly also induced depletion of the intestinal stem cell (ISC) pool. Histological and gene expression analyses indicate that ISCs collectively converted to short-lived progenitor cells after BRAF activation. As Wnt/β-catenin signals encourage ISC identity, we asked whether β-catenin activity could counteract oncogenic BRAF. Indeed, we found that intestinal organoids could be partially protected from deleterious oncogenic BRAF effects by Wnt3a or by small-molecule inhibition of GSK3β. Similarly, transgenic expression of stabilized β-catenin in addition to oncogenic BRAF partially prevented loss of stem cells in the mouse intestine. We also used BRAF V637E knock-in mice to follow changes in the stem cell pool during serrated tumor progression and found ISC marker expression reduced in serrated hyperplasia forming after BRAF activation, but intensified in progressive dysplastic foci characterized by additional mutations that activate the Wnt/β-catenin pathway. Our study suggests that oncogenic alterations activating the MAPK and Wnt/β-catenin pathways must be consecutively and coordinately selected to assure stem cell maintenance during colon cancer initiation and progression. Notably, loss of stem cell identity upon induction of BRAF/MAPK activity may represent a novel fail-safe mechanism protecting intestinal tissue from oncogene activation.

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Section: Above)supporting

confidence: 71%

Section: Above)mentioning

confidence: 64%

Section: Qrt-pcr and Microarray Analysismentioning

confidence: 99%

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Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity

et al. 2014

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“…Murine colonic organoids were produced from intestinal crypts of a female C57 Bl6/J mouse according to previous reports [22] with some modifications. Briefly, murine colonic crypts were dissociated with Gentle Cell Dissociation reagent (Stemcell Technology Inc., Vancouver, Canada) and further incubated in trypsin (ThermoFischer).…”

Section: Methodsmentioning

confidence: 99%

Characterization of Trichuris muris secreted proteins and extracellular vesicles provides new insights into host–parasite communication

Eichenberger

Talukder

Field

et al. 2018

J of Extracellular Vesicle

110

148

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Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Owing to the difficulty in obtaining parasite material, the mouse whipworm Trichuris muris has been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterization of the excretory/secretory products of T. muris. We identify 148 proteins secreted by T. muris and show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep® gradient to purify the EVs, highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterize the proteomic and genomic content of the EVs, identifying >350 proteins, 56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping to T. muris gene models. Many of the miRNAs putatively mapped to mouse genes are involved in regulation of inflammation, implying a role in parasite-driven immunomodulation. In addition, for the first time to our knowledge, colonic organoids have been used to demonstrate the internalization of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterization of the proteins and EVs secreted by T. muris provides important information on whipworm–host communication and forms the basis for future studies.

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“…This landmark paper established a novel experimental paradigm for generating tissue organoids. Subsequent studies worldwide confirmed the generalizability of such approach to human stem cells, and produced organoids of a variety of organ types including colon,11 intestine,12 prostate,13, 14 fallopian tube,15 stomach,16 liver,17, 18 kidney,19 lung,20 and brain 8…”

Section: Organogenesis In a Dish: An Overviewmentioning

confidence: 91%

Translational potential of human brain organoids

Sun

Tan

2018

Ann Clin Transl Neurol

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The recent technology of 3D cultures of cellular aggregates derived from human stem cells have led to the emergence of tissue‐like structures of various organs including the brain. Brain organoids bear molecular and structural resemblance with developing human brains, and have been demonstrated to recapitulate several physiological and pathological functions of the brain. Here we provide an overview of the development of brain organoids for the clinical community, focusing on the current status of the field with an critical evaluation of its translational value.

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Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium

Cited by 2,987 publications

References 40 publications

Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity

Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity

Characterization of Trichuris muris secreted proteins and extracellular vesicles provides new insights into host–parasite communication

Translational potential of human brain organoids

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