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intestinal organoids

Shuji Hibiya, Kiichiro Tsuchiya, Ryohei Hayashi, Keita Fukushima, Nobukatsu Horita, Sho Watanabe, Tomoaki Shirasaki, Ryu Nishimura, Natsuko Kimura, Tatsunori Nishimura, Noriko Gotoh, Shigeru Oshima, Ryuichi Okamoto, Tetsuya Nakamura, Mamoru Watanabe
BACKGROUND & AIMS: Patients with ulcerative colitis (UC) are at an increased risk of developing colitis-associated cancer (CAC), suggesting that continuous inflammation in the colon promotes the transformation of colonic epithelial cells. However, the mechanisms underlying cell transformation in UC remain unknown. We therefore aimed to investigate the effect of long-term inflammation on intestinal epithelial cells (IECs) using organoid culture. METHODS: IECs were isolated from mice colon, and were cultured according to a method for a three-dimensional (3D) organoid culture...
October 11, 2016: Journal of Crohn's & Colitis
Fangkun Liu, Jing Huang, Bo Ning, Zhixiong Liu, Shen Chen, Wei Zhao
Patient-derived cell lines and animal models have proven invaluable for the understanding of human intestinal diseases and for drug development although both inherently comprise disadvantages and caveats. Many genetically determined intestinal diseases occur in specific tissue microenvironments that are not adequately modeled by monolayer cell culture. Likewise, animal models incompletely recapitulate the complex pathologies of intestinal diseases of humans and fall short in predicting the effects of candidate drugs...
2016: Frontiers in Pharmacology
Dustin J Flanagan, Renate H M Schwab, Bang M Tran, Toby J Phesse, Elizabeth Vincan
The discovery of Lgr5 as a marker of adult stem cells meant that stem cell populations could be purified and studied in isolation. Importantly, when cultured under the appropriate conditions these stem cells form organoids in tissue culture that retain many features of the tissue of origin. The organoid cultures are accessible to genetic and biochemical manipulation, bridging the gap between in vivo mouse models and conventional tissue culture. Here we describe robust protocols to establish organoids from gastrointestinal tissues (stomach, intestine, liver) and Cre-recombinase mediated gene manipulation in vitro...
October 5, 2016: Methods in Molecular Biology
Francis Blokzijl, Joep de Ligt, Myrthe Jager, Valentina Sasselli, Sophie Roerink, Nobuo Sasaki, Meritxell Huch, Sander Boymans, Ewart Kuijk, Pjotr Prins, Isaac J Nijman, Inigo Martincorena, Michal Mokry, Caroline L Wiegerinck, Sabine Middendorp, Toshiro Sato, Gerald Schwank, Edward E S Nieuwenhuis, Monique M A Verstegen, Luc J W van der Laan, Jeroen de Jonge, Jan N M IJzermans, Robert G Vries, Marc van de Wetering, Michael R Stratton, Hans Clevers, Edwin Cuppen, Ruben van Boxtel
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...
October 3, 2016: Nature
Kai Kretzschmar, Hans Clevers
Organoids are three-dimensional in-vitro-grown cell clusters with near-native microanatomy that arise from self-organizing mammalian pluripotent or adult stem cells. Although monolayer stem cell cultures were established more than 40 years ago, organoid technology has recently emerged as an essential tool for both fundamental and biomedical research. For developmental biologists, organoids provide powerful means for ex vivo modeling of tissue morphogenesis and organogenesis. Here we discuss how organoid cultures of the intestine and other tissues have been established and how they are utilized as an in vitro model system for stem cell research and developmental biology...
September 26, 2016: Developmental Cell
Jeffrey M Beekman
Cystic fibrosis transmembrane conductance regulator (CFTR) modulators that target the mutant CFTR protein are being introduced for treatment of cystic fibrosis. Stratification of subjects based on their CFTR genotype has been proven essential to demonstrate clinical efficacy of these novel treatments. Despite this stratification, considerable heterogeneity between subjects receiving CFTR modulators is still observed which remains largely uncharacterized. The CFTR genotype, and additional genetic and environmental factors that impact either tissue-specific CFTR protein characteristics or the pharmacokinetic properties of treatments will likely determine the individual response to therapy...
October 2016: Pediatric Pulmonology
Sung Noh Hong, James C Y Dunn, Matthias Stelzner, Martín G Martín
: : Intestinal failure is a rare life-threatening condition that results in the inability to maintain normal growth and hydration status by enteral nutrition alone. Although parenteral nutrition and whole organ allogeneic transplantation have improved the survival of these patients, current therapies are associated with a high risk for morbidity and mortality. Development of methods to propagate adult human intestinal stem cells (ISCs) and pluripotent stem cells raises the possibility of using stem cell-based therapy for patients with monogenic and polygenic forms of intestinal failure...
September 16, 2016: Stem Cells Translational Medicine
Jacquelien Noordhoek, Vincent Gulmans, Kors van der Ent, Jeffrey M Beekman
PURPOSE OF REVIEW: New therapeutics have been introduced for cystic fibrosis that modulate cystic fibrosis transmembrane conductance regulator (CFTR) function in a mutation-specific fashion. Despite CFTR genotype-based stratification of treatments, treatment efficacy is variable between study participants suggesting that individual factors further contribute to drug efficacy. Moreover, these treatments are licensed for a limited amount of CFTR mutations, and study participants with rare mutations that can potentially benefit from available treatments may be missed...
November 2016: Current Opinion in Pulmonary Medicine
Thomas E Wallach, James R Bayrer
The development of sustainable intestinal organoid cell culture has emerged as a new modality for the study of intestinal function and cellular processes. Organoid culture is providing a new test-bed for therapeutic research and development. Intestinal organoids, self-renewing three-dimensional structures comprised of intestinal stem cells and their differentiated epithelial progeny allow for more facile and robust exploration of cellular activity, cell organization and structure, genetic manipulation, and vastly more physiologic modeling of intestinal response to stimuli as compared to traditional two dimensional cell line cultures...
September 14, 2016: Journal of Pediatric Gastroenterology and Nutrition
Giulia Nigro, Melissa Hanson, Cindy Fevre, Marc Lecuit, Philippe J Sansonetti
The gut, particularly the colon, is the host of approximately 1000 bacterial species, the so-called gut microbiota. The relationship between the gut microbiota and the host is symbiotic and mutualistic, influencing many aspects of the biology of the host. This homeostatic balance can be disrupted by enteric pathogens, such as Shigella flexneri or Listeria monocytogenes, which are able to invade the epithelial layer and consequently subvert physiological functions. To study the host-microbe interactions in vitro, the crypt culture model, known as intestinal organoids, is a powerful tool...
September 15, 2016: Methods in Molecular Biology
Jarno Drost, Benedetta Artegiani, Hans Clevers
We established an in vitro culture model in which intestinal epithelial stem cells can grow into three-dimensional, ever-expanding epithelial organoids that retain their original organ identity and genetic stability. Moreover, organoids can easily be genetically modified using different genome modification strategies, including viral delivery of transgenes and CRISPR/Cas9 technology. These combined characteristics make them a useful in vitro model system to study many biological processes including the contribution of cellular signaling pathways to tissue homeostasis and disease...
2016: Methods in Molecular Biology
Jessica L Forbester, Nicholas Hannan, Ludovic Vallier, Gordon Dougan
Intestinal human organoids (iHOs) provide an effective system for studying the intestinal epithelium and its interaction with various stimuli. By using combinations of different signaling factors, human induced pluripotent stem cells (hIPSCs) can be driven to differentiate down the intestinal lineage. Here, we describe the process for this differentiation, including the derivation of hindgut from hIPSCs, embedding hindgut into a pro-intestinal culture system and passaging the resulting iHOs. We then describe how to carry out microinjections to introduce bacteria to the apical side of the intestinal epithelial cells (IECs)...
August 31, 2016: Methods in Molecular Biology
Nobuo Sasaki, Norman Sachs, Kay Wiebrands, Saskia I J Ellenbroek, Arianna Fumagalli, Anna Lyubimova, Harry Begthel, Maaike van den Born, Johan H van Es, Wouter R Karthaus, Vivian S W Li, Carmen López-Iglesias, Peter J Peters, Jacco van Rheenen, Alexander van Oudenaarden, Hans Clevers
Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5(+) stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5(+) stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus...
September 13, 2016: Proceedings of the National Academy of Sciences of the United States of America
Kevin P O'Rourke, Sarah Ackerman, Lukas E Dow, Scott W Lowe
In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro.
February 20, 2016: Bio-protocol
Kevin P O'Rourke, Lukas E Dow, Scott W Lowe
Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, permeabilize, stain and mount intestinal organoids for analysis by immunofluorescent confocal microscopy.
February 20, 2016: Bio-protocol
Julia Matheson, Claudia Bühnemann, Emma J Carter, David Barnes, Hans-Jürgen Hoppe, Jennifer Hughes, Stephen Cobbold, James Harper, Hans Morreau, Mirvat Surakhy, A Bassim Hassan
Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to β-catenin. Here, we evaluate whether E-cadherin binding can inhibit β-catenin when there is loss of Adenomatous polyposis coli (APC) from the β-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear β-catenin localization, suggesting that E-cadherin inhibits β-catenin...
August 23, 2016: Oncotarget
Jessica Tsalikis, Qun Pan, Ivan Tattoli, Charles Maisonneuve, Benjamin J Blencowe, Dana J Philpott, Stephen E Girardin
BACKGROUND: The intestinal epithelium plays a critical role in nutrient absorption and innate immune defense. Recent studies showed that metabolic stress pathways, in particular the integrated stress response (ISR), control intestinal epithelial cell fate and function. Here, we used RNA-seq to analyze the global transcript level and alternative splicing responses of primary murine enteroids undergoing two distinct ISR-triggering stresses, endoplasmic reticulum (ER) stress and nutrient starvation...
2016: BMC Genomics
Laura Broutier, Amanda Andersson-Rolf, Christopher J Hindley, Sylvia F Boj, Hans Clevers, Bon-Kyoung Koo, Meritxell Huch
Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro...
September 2016: Nature Protocols
Shinya Imada, Yoji Murata, Takenori Kotani, Masaki Hatano, Chunxiao Sun, Tasuku Konno, Jung-Ha Park, Yasuaki Kitamura, Yasuyuki Saito, Hideki Ohdan, Takashi Matozaki
Proper regulation of epithelial cell turnover is important for the structural integrity and homeostasis of various tissues including the intestine. Here we show that ablation of Csk, a negative regulator of Src family kinases (SFKs), specifically in intestinal epithelial cells (IECs) resulted in the development of hyperplasia throughout the intestinal epithelium of mice. Such conditional ablation of Csk also increased the proliferative activity and turnover of IECs, disturbed the differentiation of Paneth and goblet cells, reduced the number of intestinal stem cells, and attenuated the expression of Wnt target genes in the intestine...
August 22, 2016: Molecular and Cellular Biology
Dong-Hun Woo, Qijun Chen, Ting-Lin B Yang, M Rebecca Glineburg, Carla Hoge, Nicolae A Leu, F Brad Johnson, Christopher J Lengner
Patients with dyskeratosis congenita (DC) suffer from stem cell failure in highly proliferative tissues, including the intestinal epithelium. Few therapeutic options exist for this disorder, and patients are treated primarily with bone marrow transplantation to restore hematopoietic function. Here, we generate isogenic DC patient and disease allele-corrected intestinal tissue using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene correction in induced pluripotent stem cells and directed differentiation...
September 1, 2016: Cell Stem Cell
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