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Cold Spring Harbor, NY - Cold Spring Harbor Laboratory (CSHL) has been awarded a research subcontract by Leidos Biomedical Research to lead a Cancer Model Development Center (CMDC) for pancreatic, breast, colorectal, lung, liver and other upper-gastrointestinal cancers. The project is 100% supported by U.S. federal funds (NCI Contract No. HHSN261201500003I, Task Order Number HHSN26100008). CSHL Cancer Center Director Dr. David Tuveson and CSHL Research Director Dr. David Spector will lead the multinational collaborative effort with Dr. Hans Clevers of the Hubrecht Institute, Dr. Aldo Scarpa and Dr. Vincenzo Corbo of the ARC-Net Centre for Applied Research on Cancer at the University of Verona, Italy, and Dr. James Crawford of Northwell Health and Dr. Peter Gregersen of Northwell's Feinstein Institute for Medical Research. The new center will generate three-dimensional organoid culture systems of cancers - next-generation models that improve upon current two-dimensional model systems used to study cancers and develop therapeutics. "CSHL is excited to lead this international team to develop more effective research models for cancer that can be shared broadly with the scientific community in order to accelerate discoveries for improved diagnosis and treatments for cancer patients," said Dr. Tuveson. Dr. Priya Sridevi from CSHL is the lead project manager for this CMDC. Under the contract, the CSHL-led CMDC will establish up to 150 organoid models in one and a half years, contributing to a larger international effort to generate 1,000 new cancer models. The Human Cancer Model Initiative (HCMI) was announced in July 2016 by the National Cancer Institute, Wellcome Trust Sanger Institute in the United Kingdom (UK), Cancer Research UK, and the foundation Hubrecht Organoid Technology. As part of NCI's Precision Medicine Initiative in Oncology, this new project is timed to take advantage of the latest cell culture and genomic sequencing techniques to create models that are representative of patient tumors and annotated with genomic and clinical information. This effort is a first step toward learning how to use these tools to design individualized treatments. Dr. Tuveson, the project's principal investigator, led an effort to develop pancreas cancer organoids, establishing CSHL as an instructional site offering courses in organoid development to the professional scientific community worldwide. Organoids can be established from healthy human tissue as well as from a variety of tumor tissue types. The power of the organoid is that it faithfully recapitulates the tissue from which it is derived. It can be genetically manipulated using technologies like shRNA (short hairpin RNA) that can turn genes on and off, or the revolutionary gene-editing tool CRISPR-Cas9. Moreover, organoid models are amenable to drug screening approaches so they can be used to validate therapeutics. The pioneer of the organoid model system, Dr. Clevers, is a key member of the new CSHL-led center. "We have laid the foundations for this collaborative program through informal exchanges of our young scientists," said Dr. Clevers. "It is very exciting that we can now turn this into a mature, well-funded endeavor that will create next-generation cancer models, as close as possible to what we find in individual patients." In support of the project, the ARC-Net team led by Dr. Scarpa and Dr. Corbo will leverage the biobanking infrastructure coordinated by Dr. Rita T. Lawlor at ARC-Net. "We are very proud to be part of this international research group," said Dr. Corbo. "This collaboration brings together world-leading expertise in cancer genomics and cancer modeling with the potential of accelerating the implementation of personalized medicine. We see an unprecedented opportunity to develop better models of cancers that will enable researchers to interrogate the wealth of genomic information available today for the rational development of cancer therapeutics." "Northwell Health and the Feinstein Institute for Medical Research are very excited to be part of this international effort, as it will help lay the foundation for new standards in clinical care that incorporate ex vivo studies of cancer tissues to guide cancer therapies," said Dr. Gregersen, Professor and Director of the Feinstein Institute's Boas Center for Genomics and Human Genetics. Dr. Crawford, Professor and Chair of the Department of Pathology and Laboratory Medicine at the Hofstra Northwell School of Medicine, noted: "Through this multi-institutional collaboration, Northwell Health will also be well-positioned to help advance the clinical trials necessary for bringing such advances into the realm of clinical care." CSHL entered into a strategic alliance with Northwell Health in April 2015, with the objective of providing CSHL researchers access to Northwell's growing network of clinical services encompassing more than 16,000 new cancer cases annually. For CSHL and Northwell Health, this CMDC project demonstrates the power of their strategic affiliation to establish closer links between research and the clinic for the benefit of cancer patients. The multinational HCMI effort aims to speed up development of new models and to make research more efficient by avoiding unnecessary duplication of scientific efforts. Genetic sequencing data from the tumors and derived models will be available to researchers, along with clinical data about the patients and their tumors. All information related to the models will be shared in a way that protects patient privacy. The goal is to give scientists around the world access to the best resources to be able to easily study all types of cancer. These new cell models could transform how we study cancer and could help to develop better treatments for patients,Scientists will make the models using tissue from patients with different types of cancer, potentially including rare and pediatric cancers, which are often under-represented or not available at all in existing cell-line collections. The new models will have the potential to reflect the biology of tumors more accurately and better represent the overall cancer patient population. The Hubrecht Institute, founded in 1916, is a research institute of the Royal Netherlands Academy of Arts and Sciences (KNAW), situated on Utrecht Science Park "The Uithof" of the largest university in the Netherlands (Utrecht). Research at the Hubrecht Institute focuses on developmental biology and stem cells at the organismal, cellular, and molecular level. A variety of biological processes are being studied, mainly concerning embryonic development and development and homeostasis of organs. Presently there are 19 research groups, including the research group of Hans Clevers, with a total of about 220 employees. Prof. Dr. Hans Clevers discovered methods to grow stem cell-derived human epithelial 'mini-organs' (organoids) from tissues of patients with various diseases including cancer. Clevers' international reputation has brought him numerous grants and prestigious awards. For more information, visit https:/ ARC-Net, Applied Research on Cancer Network, is a university research centre that was established in 2007 through a joint initiative between the University of Verona, the University Hospital Trust of Verona and the Cariverona Foundation. ARC-Net represents Italy in the International Cancer Genome Consortium where the Centre coordinates the effort of the Italian Pancreatic Cancer Genome Project to the molecular characterization of rare pancreatic tumors. ARC-Net is organized into 5 core facilities platforms, which include a cancer tissue biobank that collects biological material and associated clinical, pathological and epidemiological data. To date the biobank has material from over 5,000 consented patients and has produced over 150 patients-derived xenografts of pancreatic cancer and other cancer models. For more information, visit http://www. Northwell Health is New York State's largest health care provider and private employer, with 21 hospitals and over 550 outpatient facilities. We care for more than two million people annually in the metro New York area and beyond, thanks to philanthropic support from our communities. Our 61,000 employees - 15,000+ nurses and nearly 3,400 physicians, including nearly 2,700 members of Northwell Health Physician Partners -- are working to change health care for the better. We're making breakthroughs in medicine at the Feinstein Institute. We're training the next generation of medical professionals at the visionary Hofstra Northwell School of Medicine and the School of Graduate Nursing and Physician Assistant Studies. And we offer health insurance through CareConnect. For information on our more than 100 medical specialties, visit Northwell.edu. Founded in 1890, Cold Spring Harbor Laboratory has shaped contemporary biomedical research and education with programs in cancer, neuroscience, plant biology and quantitative biology. CSHL has been a National Cancer Institute designated Cancer Center since 1987. Home to eight Nobel Prize winners, the private, not-for-profit Laboratory employs 1,100 people including 600 scientists, students and technicians. The Meetings & Courses Program hosts more than 12,000 scientists from around the world each year on its campuses in Long Island and in Suzhou, China. The Laboratory's education arm also includes an academic publishing house, a graduate school and programs for middle and high school students and teachers. For more information, visit http://www.


News Article | May 19, 2017
Site: www.sciencedaily.com

Scientists have made an important step in understanding the organisation of nerve cells embedded within the gut that control its function -- a discovery that could give insight into the origin of common gastrointestinal diseases, including irritable bowel syndrome and chronic constipation. The findings, published in Science, reveal how the enteric nervous system -- a chaotic network of half a billion nerve cells and many more supporting cells inside the gut wall -- is formed during mouse development. The research was led by the Francis Crick Institute, in collaboration with the University of Leuven, Stanford University, the Hubrecht Institute and the Quadram Institute Bioscience. The work was funded by the Francis Crick Institute, the Medical Research Council and the UK Biotechnology and Biological Sciences Research Council. Often known as the 'second brain' for its vast number of neurons and complex connectivity, the enteric nervous system has a crucial role in maintaining a healthy gut. Therefore, understanding how this neural mosaic is organised could help scientists find treatments for common gastrointestinal disorders. "The gut wall is home to many types of nerve cells which appear to be distributed randomly," says Vassilis Pachnis, Group Leader at the Francis Crick Institute. "But despite this chaos, the neural networks of the gut are responsible for well organised and stereotypic functions such as production of stomach acid, movement of food along the gut, communication with immune cells and bacteria, and relay of information to the brain. We wanted to find out how organised activity emerges from such a chaotic system." During development, a unique and dynamic population of cells known as progenitor cells divide to produce copies of themselves, which can then generate many other types of cells. Using genetic tools, the team labelled individual progenitor cells of the enteric nervous system with unique colours and followed their descendants -- also marked with the same colour -- through development and into the adult animal. By examining the type of cells produced by single progenitors, they could understand their properties. They found that some progenitors only produced nerve cells, others only produced nerve-supporting cells called glia, and some produced both. Neurons and glia originating from the same parent stayed close to each other, forming relatively tight groups of cells. Cell groups that descended from different but neighbouring parent cells overlapped like a Venn diagram that could be viewed on the gut surface. Interestingly, this close relationship was maintained by the descendants of single progenitors down through all layers of the gut wall thereby forming overlapping columns of cells. "We uncovered a set of rules that control the organisation of the 'second brain' not just along a single gut layer but across the 3D space of the gut wall," says Reena Lasrado, first author of the paper and researcher in Vassilis's lab at the Crick. The team explored whether this intricate structure of the enteric nervous system also contributes to nerve cell activity in the gut. "A subtle electrical stimulation to the enteric nervous system showed that nerve cells generated by the same parent cell responded in synchrony," says Vassilis. "This suggests that developmental relationships between cells of the enteric nervous system of mammals are fundamental for the neural regulation of gut function." "Now that we have a better understanding of how the enteric nervous system is built and works, we can start to look at what happens when things go wrong particularly during the critical stages of embryo development or early life. Perhaps mistakes in the blueprint used to build the neural networks of the gut are the basis of common gastrointestinal problems."


News Article | May 19, 2017
Site: www.eurekalert.org

Scientists have made an important step in understanding the organisation of nerve cells embedded within the gut that control its function -- a discovery that could give insight into the origin of common gastrointestinal diseases, including irritable bowel syndrome and chronic constipation. The findings, published in Science, reveal how the enteric nervous system -- a chaotic network of half a billion nerve cells and many more supporting cells inside the gut wall -- is formed during mouse development. The research was led by the Francis Crick Institute, in collaboration with the University of Leuven, Stanford University, the Hubrecht Institute and the Quadram Institute Bioscience. The work was funded by the Francis Crick Institute, the Medical Research Council and the UK Biotechnology and Biological Sciences Research Council. Often known as the 'second brain' for its vast number of neurons and complex connectivity, the enteric nervous system has a crucial role in maintaining a healthy gut. Therefore, understanding how this neural mosaic is organised could help scientists find treatments for common gastrointestinal disorders. "The gut wall is home to many types of nerve cells which appear to be distributed randomly," says Vassilis Pachnis, Group Leader at the Francis Crick Institute. "But despite this chaos, the neural networks of the gut are responsible for well organised and stereotypic functions such as production of stomach acid, movement of food along the gut, communication with immune cells and bacteria, and relay of information to the brain. We wanted to find out how organised activity emerges from such a chaotic system." During development, a unique and dynamic population of cells known as progenitor cells divide to produce copies of themselves, which can then generate many other types of cells. Using genetic tools, the team labelled individual progenitor cells of the enteric nervous system with unique colours and followed their descendants -- also marked with the same colour -- through development and into the adult animal. By examining the type of cells produced by single progenitors, they could understand their properties. They found that some progenitors only produced nerve cells, others only produced nerve-supporting cells called glia, and some produced both. Neurons and glia originating from the same parent stayed close to each other, forming relatively tight groups of cells. Cell groups that descended from different but neighbouring parent cells overlapped like a Venn diagram that could be viewed on the gut surface. Interestingly, this close relationship was maintained by the descendants of single progenitors down through all layers of the gut wall thereby forming overlapping columns of cells. "We uncovered a set of rules that control the organisation of the 'second brain' not just along a single gut layer but across the 3D space of the gut wall," says Reena Lasrado, first author of the paper and researcher in Vassilis's lab at the Crick. The team explored whether this intricate structure of the enteric nervous system also contributes to nerve cell activity in the gut. "A subtle electrical stimulation to the enteric nervous system showed that nerve cells generated by the same parent cell responded in synchrony," says Vassilis. "This suggests that developmental relationships between cells of the enteric nervous system of mammals are fundamental for the neural regulation of gut function." "Now that we have a better understanding of how the enteric nervous system is built and works, we can start to look at what happens when things go wrong particularly during the critical stages of embryo development or early life. Perhaps mistakes in the blueprint used to build the neural networks of the gut are the basis of common gastrointestinal problems."


No statistical methods were used to predetermine sample size. YapΔ/Δ and TazΔ/Δ mice were generated by crossing Yap or Taz floxed mice30 with the villin-cre line (Jackson Laboratory), the villin-creERT2 line (S. Robine, Institut Curie-CNRS) or the Lgr5-creERT line (Jackson Laboratory). The Rosa26-lox-STOP-lox-rtta-IRES-EGFP and Rosa26 lacZ mouse lines were obtained from Jackson Laboratory. The YapTg transgenic line described in this study was generated by introducing a HA-tagged wild-type Yap cDNA downstream of 7 Tet-repressor elements in the pTRE2 vector (J. Whitsett, Cincinnati Children’s Hospital Medical Center). The transgenic Yap construct was linearized and microinjected in ICR embryos. As shown in Extended Data Fig. 3a, activation of Cre deletes a neo cassette and allows for expression of the rtTA gene. In the presence of doxycycline the rtTA activates transcription of HA-Yap. Apc floxed mice were obtained from O. Sansom (Beatson Institute). Lats1 and Lats2 floxed alleles were obtained from R. Johnson (MD Anderson Cancer Center) and crossed with villin-creERT2 mice to obtain Lats1Δ/Δ;Lats2Δ/Δ mice. To measure polyp formation, YapΔ/Δ mice were backcrossed to a Bl/6 background for 4 generations before crossing to ApcMin/+ mice. Polyps from Yap+/+ (Yap+/+;villin-cre;ApcMin/+), Yap+/Δ (Yapfl/+;villin-cre;ApcMin/+) and YapΔ/Δ (Yapfl/fl;villin-cre;ApcMin/+) mice were counted 16 weeks after birth or when animals appeared moribund. Survival of ApcMin mice was measured by the number of days before mice were euthanized due to poor health. In vivo assays comparing control and Yap mutant animals were performed between age- and sex-matched pairs. No method of randomization was followed and no animals were excluded in this study. The investigators were not blinded to allocation during experiments and outcome assessment. Inducible Cre-mediated deletion of genes was performed by intraperitoneal injections of >5-week-old mice with 200 μl tamoxifen in corn oil at 10 mg ml−1. To create mosaic expression of Yap, Yapfl/fl;villin-creERT2 mice were induced with a single injection of 200 μl of tamoxifen at a suboptimal dose typically between 0.5 and 2.0 mg ml−1. For in vivo regeneration assays, mice were given a single dose of 10 or 12 Gy using a GammaCell 40 irradiator. Animals were maintained and handled under procedures approved by the Canadian Council on Animal Care. The immunohistochemistry stainings and standard colorimetric in situ hybridization were carried out according to methods described elsewhere31. Staining experiments were repeated on independent tissue sections prepared from separate mice as indicated by n values in figure legends. The following primary antibodies were used for immunostaining: rat anti-Ki67 (Dako, Cat. no. M7249, 1:1,000), rabbit anti-Yap/Taz (Cell Signaling, Cat. no. 8418, 1:100), rabbit anti-Yap (Cell Signaling, Cat. no. 14074, 1:300), mouse anti-Yap (Santa Cruz, Cat. no. sc-101199, 1:100), rabbit anti-Lef (Cell Signaling, Cat. no. 2230, 1:300), phosphor-Egfr (Tyr1092) (Abcam, Cat. no. ab40815, 1:300), anti-cleaved caspase-3 (Cell Signaling, Cat. no. 9664, 1:300) and anti-lysozyme (Dako, Cat. no. A0099, 1:1,000). Detection of primary antibodies was achieved using the Dako Envision plus system. Multi-colour fluorescence in situ hybridization with tyramide signal amplification (TSA) was done essentially as described elsewhere32, 33, 34. In brief, RNA probes from hybridized sections were detected using appropriate hapten-specific HRP-conjugated antibodies (anti-digoxigenin-HRP (Roche, Cat. no. 11207733910, 1:500), anti-dinitrophenyl-HRP (PerkinElmer, Cat. no. NEL747A001KT, 1:300), and anti-fluorescein-HRP (Life Technologies, A21253, 1:500)). After overnight incubation with antibodies at 4°C (or 2 h at room temperature for anti-fluorescein-HRP detection of cryptin1) sections were washed in PBS, and rinsed twice in 100 mM borate pH 8.5 plus 0.1% BSA. TSA reaction was performed by applying 300 μl per slide of the following mixture: 100 mM borate pH 8.5, 2% dextran sulfate, 0.1% Tween-20 and 0.003% H O , 450 μg ml−1 4-iodophenol: 1:250 Tyramide product (that is, DyLight633-tyramide, Dylight488-tyramide, Dylight 555-tyramide). The TSA reaction was allowed to proceed for 20 min and then terminated by washing slides in 100 mM glycine pH 2.0 for 15 min. Sections were washed further in PBS for the next round of detection. To synthesize tyramide products, the following succinimidyl esters were used for conjugation with tyramine: DyLight 633 NHS-Ester (Thermo Scientific Cat#46414), DyLight 550 NHS-Ester (Thermo Scientific Cat#62262), DyLight 488 NHS-Ester (Thermo Scientific Cat. no. 46402). The synthesis reaction was carried out as described previously32. The following in situ hybridization probes were obtained from the collection of MGC clones at the Lunenfeld Tanenbaum Research Institute: TweakR (BC025860), Ly6c1 (BC092082), Edn1 (BC029547), Areg (BC009138), Ereg (BC027838), Il1rn (BC042532), Il33 (BC003847), Msln (BC023753) and Cyr61 (BC066019). The Olfm4 and cryptdin1 probes were a gift from H. Clevers (Hubrecht Institute). Before fixing organoids, 10 μM Edu was added to the culture media for 1 h. Then organoids were fixed in 10% buffered formalin for 30 min, permeabilized in 0.5% Triton for 20 min and blocked in 2% BSA. Incorporated Edu was detected using the ClickIt EDU Imaging kit (Invitrogen) according to the manufacturer’s instructions. The primary antibodies used for immunostaining were mouse anti-Yap (Santa Cruz, Cat # sc-101199, 1:100), mouse anti-HA (Sigma-Aldrich, Cat. no. H9658, 1:1,000), and chicken anti-β-gal (Abcam, Cat. no. ab9361, 1:300). The secondary antibodies used in immunostaining were: CF555-donkey anti-mouse (Biotium, Cat. no. 20037, 1:400) and CF647 donkey anti-rabbit (Biotium, Cat. no. 20047, 1:400). Organoids were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich) before mounting onto slides for visualization. Images were acquired using a 20×/NA oil immersion objective lens (HCX PL APO, Leica), an EM-CCD camera (ImagEM, Hamamatsu) on an inverted microscope (DMIRE2, Leica) with a spinning disk confocal scanner (CSU10, Yokogawa) and Volocity. De novo crypts were scored as any protrusions, typically containing Paneth cells, budding from the initial sphere formed after seeding isolated crypts. Crypts were counted from bright-field images using Image J. At least four independent cultures derived from four different mice per genotype were used for quantification. Survival of crypts in Fig. 1 was determined by Ki67 staining of cross-sections of proximal portions of the small intestine at 3 days post-irradiation (10 Gy or 12 Gy). Values in Fig. 1b represent average number of fully labelled Ki67+ crypts per intestinal circumference based on counts from at least two sections per mouse and assays were repeated in 6 independent mice per genotype for both 10 Gy and 12 Gy treatments. The percentage of surviving Yap-positive versus negative Lgr5+ ISCs in Fig. 1d was performed by counting 587 β-gal+ crypts from a total of 7 untreated YapΔLgr5-cre mice and 394 β-gal+ crypts from a total of 9 irradiated YapΔLgr5-cre mice. In Yap;ApcΔLgr5-cre mice tumour initiating cells were visualized by staining for the Wnt target gene, Lef. As shown in Extended Data Fig. 10b, Lef is undetected in wild-type crypts and highly upregulated in Apc-null cells and thus serves as a robust marker of Apc deletion31. The percentage of Paneth cells in Lef+ foci (Fig. 4a) was assessed by preparing consecutive sections stained for Lyz, Lef and Yap, respectively. Lysozyme-positive Paneth cells from a total of 207 Yap wild-type and 201 Yap mutant Lef+ foci were counted from 5 Yap;ApcΔLgr5-cre mice (10–16 days after tamoxifen injection) using Image J and the percentage of total cells within the boundaries of a given Lef+ lesion was calculated. Relative activation of Egfr was quantified in consecutive sections from 5 Yap;ApcΔLgr5-cre mice stained for Lef, Yap and phospho-Egfr. For assessing Phospho-Egfr, staining intensity in Lef+ foci was assessed in a blinded fashion. For this, consecutive sections stained for Yap were masked from the observer scoring phospho-Egfr staining intensity. Lef+ foci were scored as ‘+’ if phospho-Egfr expression was elevated compared to wild-type adjacent crypts at comparable levels within the crypt–villus axis (see Extended Data Fig. 10f, panels xi and xii). Lef+ foci were scored as ‘++’ if staining intensity was very strong even relative to the stem cell compartment in normal crypts and/or displayed prominent apical staining (see Yap-positive foci in Fig. 4c, panel iv, and Extended Data Fig. 10f, panels v and vi). Lef+ foci were scored as ‘–’ if staining intensity was undetected or unchanged relative to adjacent wild-type crypts (see Yap mutant foci in Fig. 4c and Extended Data Fig. 10f). In Extended Data Fig. 1, caspase 3 and BrdU positive crypt cells were counted from at least six sections per mouse in 4 independent mice per genotype and expressed as a percentage of total crypt cells. All data are presented as average values with s.e.m. Mann–Whitney (two-tailed) U-test was used to determine statistical significance. Calculations were performed using GraphPad Prism 5 software. RNA was isolated from organoids cultured for 24 h after seeding in Matrigel. RNA samples were pooled from at least three organoid cultures derived from at least three independent mice per genotype (Yapfl/+;villin-cre, Yapfl/fl;villin-cre and YapTg). Quality of RNA was verified by running samples on a Bioanalyzer. High-throughput sequencing was performed using the Illumina HiSeq 2000 at the Lunenfeld Tanenbaum Research Institute (LTRI) sequencing facility. Raw sequencing reads in Fastq formats were mapped onto mouse genome (mm9) using Tophat 1.4.1 and the RPKMs (reads per kilobase of exon model per million mapped reads) were calculated using a customized script. RNA-seq data are presented in Supplementary Table 1. Combined fold change presented in Extended Data Fig. 3d and Supplementary Table 1 was calculated using the following formula: combination fold change = log [(YapΔ/Δ/Yap+/Δ)/(Dox+/Dox−)]. R, Cluster 3.0 and Java TreeView were used for data visualization. Gut organoids were cultured according to a previously described protocol established by Sato and Clevers7. Briefly, crypts were harvested by incubating opened small intestines in PBS containing 2 mM EDTA. The epithelium was released by vigorous shaking and crypts separated using a 70 μm cell strainer. Crypts were seeded in growth factor reduced Matrigel (BD Biosciences) and grown in Advanced DMEM/F12 (Invitrogen) supplemented with 2 mM GlutaMax (Invitrogen), 100 U ml−1 Penicillin/100 μg ml−1 Streptomycin (Invitrogen), N2 Supplement (Invitrogen), B-27 Supplement (Invitrogen Cat), mouse recombinant Egf (R&D Systems), 100 ng ml−1 mouse recombinant Noggin (Peprotech), 150 ng ml−1 human Rsp1 (R&D Systems). Apc-deficient organoids were harvested from Yapfl/+;Apcfl/fl;villin-creERT, Yapfl/fl;Apcfl/fl;villin-creERT or YapTg;Apcfl/fl;villin-creERT mice injected with tamoxifen and seeded 48 h later in basal growth medium without Egf, Rsp1 or Noggin. To induce Yap expression in YapTg organoids, 1.5 μg ml−1 doxycycline was added to the culture medium on day 0. Egf (R&D Systems, Cat. no. AF2028), Areg (R&D Systems, Cat. no. AF989) and Ereg (R&D Systems, Cat. no. 1068-EP-050) were added to the culture medium at a final concentration of 0.5 μg ml−1. The following inhibitors were used: PD153053 (0.5 μM, Tocris Bioscience), U0126 (10 μM, Merck Millipore). To examine pErk1/2 levels, organoids were harvested at day 2 in cold PBS containing 5 mM EDTA, 1 mM NaVO , 1.5 mM NaF and protease inhibitors. Organoids were incubated at 4°C for 30 min to dissolve Matrigel and then lysed in TNTE buffer (50 mM Tris/HCl pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA) containing standard protease and phosphatase inhibitors. Protein concentrations were measured and samples were subjected to SDS–PAGE. Total RNA was extracted by removing culture medium and directly lysing organoids in wells using RTL buffer of the Rneasy Mini Kit (Qiagen). RNA was purified using columns and genomic DNA was removed by treatment with RNase-Free DNase (Qiagen).


Patent
Hubrecht Institute and Konicklijke Nederlandse Akademie Van Wetenschappen | Date: 2010-05-10

The invention relates to the fields of biochemistry, pharmacy and oncology. The invention particularly relates to the use of novel stem cell markers for the isolation of stem cells. The invention further relates to the obtained stem cells and their use in for example research or treatment, for example, for the preparation of a medicament for the treatment of damaged or diseased tissue. In one of the embodiments, the invention provides a method for obtaining (or isolating) stem cells comprising optionally preparing a cell suspension from a tissue or organ sample, contacting said cell suspension with an Lgr 6 or 5 binding compound, identify the cells bound to said binding compound, and optionally isolating the stem cells from said binding compound. The invention further relates to means suitable for cancer treatment and even more specific for the treatment of cancer by eradicating cancer stem cells.


Barker N.,Hubrecht Institute | Clevers H.,Hubrecht Institute
Gastroenterology | Year: 2010

Molecular markers are used to characterize and track adult stem cells. Colon cancer research has led to the identification of 2 related receptors, leucine-rich repeat-containing, G-protein-coupled receptors (Lgr)5 and Lgr6, that are expressed by small populations of cells in a variety of adult organs. Genetic mouse models have allowed the visualization, isolation, and genetic marking of Lgr5+ve and Lgr6+ve cells and provided evidence that they are stem cells. The Lgr5+ve cells were found to occupy locations not commonly associated with stem cells in the stomach, small intestine, colon, and hair follicles. A multipotent population of skin stem cells express Lgr6. Single Lgr5+ve stem cells from the small intestine and the stomach can be cultured into long-lived organoids. Further studies of these markers might reveal adult stem cell populations in additional tissues. Identification of the ligands for Lgr5 and 6 will help elucidate stem cell functions and modes of intracellular signaling. © 2010 AGA Institute.


Barker N.,Hubrecht Institute | Barker N.,University Utrecht | Bartfeld S.,Max Planck Institute for Infection Biology | Clevers H.,Hubrecht Institute | Clevers H.,University Utrecht
Cell Stem Cell | Year: 2010

The epithelial lining of the intestine, stomach, and skin is continuously exposed to environmental assault, imposing a requirement for regular self-renewal. Resident adult stem cell populations drive this renewal, and much effort has been invested in revealing their identity. Reliable adult stem cell biomarkers would accelerate our understanding of stem cell roles in tissue homeostasis and cancer. Membrane-expressed markers would also facilitate isolation of these adult stem cell populations for exploitation of their regenerative potential. Here, we review recent advances in adult stem cell biology, highlighting the promise and pitfalls of the candidate biomarkers of the various stem cell populations. © 2010 Elsevier Inc.


Van Impel A.,Hubrecht Institute | Schulte-Merker S.,Hubrecht Institute
Advances in Anatomy Embryology and Cell Biology | Year: 2014

Zebrafish have been widely used to study vasculogenesis and angiogenesis, and the vascular system is one of the most intensively studied organ systems in teleosts. It is a little surprising, therefore, that the development of the zebrafish lymphatic network has only been investigated in any detail for less than a decade now. In those last few years, however, significant progress has been made. Due to favorable imaging possibilities within the early zebrafish embryo, we have a very good understanding of what cellular behavior accompanies the formation of the lymphatic system and which cells within the vasculature are destined to contribute to lymphatic vessels. The migration routes of future lymphatic endothelial cells have been monitored in great detail, and a number of transgenic lines have been developed that help to distinguish between arterial, venous, and lymphatic fates in vivo. Furthermore, both forward and reverse genetic tools have been systematically employed to unravel which genes are involved in the process. Not surprisingly, a number of known players were identified (such as vegfc and flt4), but work on zebrafish has also distinguished genes and proteins that had not previously been connected to lymphangiogenesis. Here, we will review these topics and also compare the equivalent stages of lymphatic development in zebrafish and mice. We will, in addition, highlight some of those studies in zebrafish that have helped to identify and to further characterize human disease conditions. © Springer-Verlag Wien 2014.


Welling M.,Hubrecht Institute | Geijsen N.,Hubrecht Institute | Geijsen N.,University Utrecht
Trends in Cell Biology | Year: 2013

Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass (ICM) of blastocyst embryos. Although first characterized over 30 years ago, the ontology of these cells remains elusive. Identifying the in vivo counterpart of murine ESCs will be essential for the derivation of stable ESC lines from other species. Several hypotheses exist concerning the ontology of murine ESCs. Recent data demonstrate that ESCs emerge from a subpopulation of ICM cells that transit through a Blimp1-positive state, suggesting that perhaps a germ cell developmental program underlies ESC derivation and maintenance. Alternatively, the common dependence of ESCs and diapause embryos on the cytokine LIF (leukemia inhibitory factor) has been thought to signify that murine ESCs employ a diapause-like program for their maintenance of pluripotency. Here we review different hypotheses regarding the nature of murine ESCs and discuss their implications for human pluripotent stem cell biology. © 2013 Elsevier Ltd.


Gommans W.M.,Hubrecht Institute | Berezikov E.,Hubrecht Institute
Methods in Molecular Biology | Year: 2012

Our understanding of the importance of noncoding RNA molecules is steadily growing. One such important class of RNA molecules are microRNAs (miRNAs). These tiny RNAs fulfill important functions in cellular behavior by influencing the protein output levels of a high variety of genes through the regulation of target messenger RNAs. Moreover, miRNAs have been implicated in a wide range of diseases. In pathological conditions, the miRNA expression levels can be altered due to changes in the transcriptional or posttranscriptional regulation of miRNA expression. On the other side, mRNA molecules might be able to escape the regulation by miRNAs. In this review, we give an overview on how miRNA biogenesis can be altered in disease as well as how mRNAs can avoid the regulation by miRNAs. The interplay between these two processes defines the final protein output in a cell, and thus the normal or pathological cellular phenotype. © 2012 Springer Science+Business Media, LLC.

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