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51. The function of GORASPs in Golgi apparatus organization in vivo

Author

Grond, Rianne, primary, Veenendaal, Tineke, additional, Duran, Juan M., additional, Raote, Ishier, additional, van Es, Johan H., additional, Corstjens, Sebastiaan, additional, Delfgou, Laura, additional, El Haddouti, Benaissa, additional, Malhotra, Vivek, additional, and Rabouille, Catherine, additional

Published
2020
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53. Oral mucosal organoids as a potential platform for personalized cancer therapy

Author

Driehuis, Else, Kolders, Sigrid, Spelier, Sacha, Lohmussaar, Kadi, Willems, Stefan M, Devriese, Lot A, de Bree, Remco, de Ruiter, Emma J, Korving, Jeroen, Begthel, Harry, Van Es, Johan H, Geurts, Veerle, He, Gui-Wei, van Jaarsveld, Richard H, Oka, Rurika, Muraro, Mauro J, Vivie, Judith, Zandvliet, Maurice M J M, Hendrickx, Antoni P A, Iakobachvili, Nino, Sridevi, Priya, Kranenburg, Onno, van Boxtel, Ruben, Kops, Geert J P L, Tuveson, David A, Peters, Peter J, van Oudenaarden, Alexander, Clevers, Hans, Sub Clinical Pharmacology, Sub Oncologie/Cytologie, dCSCA RMSC-2, Sub Developmental Biology, Faculteit Diergeneeskunde, RS: M4I - Nanoscopy, Institute of Nanoscopy (IoN), Hubrecht Institute for Developmental Biology and Stem Cell Research, Sub Clinical Pharmacology, Sub Oncologie/Cytologie, dCSCA RMSC-2, Sub Developmental Biology, and Faculteit Diergeneeskunde

Subjects
0301 basic medicine, medicine.medical_treatment, Cetuximab, Mice, SCID, Carboplatin, Mice, chemistry.chemical_compound, 0302 clinical medicine, Mice, Inbred NOD, HUMAN HEAD, Antineoplastic Combined Chemotherapy Protocols, Precision Medicine, GENE-EXPRESSION, INHIBITOR, Chemoradiotherapy, Organoids, Oncology, Head and Neck Neoplasms, 030220 oncology & carcinogenesis, SQUAMOUS-CELL CARCINOMA, medicine.drug, Xenotransplantation, MODELS, 03 medical and health sciences, stomatognathic system, Journal Article, Carcinoma, medicine, Organoid, otorhinolaryngologic diseases, Animals, Humans, neoplasms, Cisplatin, Squamous Cell Carcinoma of Head and Neck, business.industry, HUMAN-PAPILLOMAVIRUS, Mouth Mucosa, IN-VITRO, medicine.disease, Xenograft Model Antitumor Assays, Head and neck squamous-cell carcinoma, Radiation therapy, stomatognathic diseases, 030104 developmental biology, chemistry, Cancer research, Drug Screening Assays, Antitumor, GROWTH-FACTOR RECEPTOR, business
Abstract

Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)–derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs.Significance:This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice.See related commentary by Hill and D'Andrea, p. 828.This article is highlighted in the In This Issue feature, p. 813

Published
2019

54. Probing the Tumor Suppressor Function of BAP1 in CRISPR-Engineered Human Liver Organoids

Author

Artegiani, Benedetta, Artegiani, Benedetta, van Voorthuijsen, Lisa, Lindeboom, Rik G. H., Seinstra, Danielle, Heo, Inha, Tapia, Pablo, Lopez-Iglesias, Carmen, Postrach, Daniel, Dayton, Talya, Oka, Rurika, Hu, Huili, van Boxtel, Ruben, van Es, Johan H., Offerhaus, Johan, Peters, Peter J., van Rheenen, Jacco, Vermeulen, Michiel, Clevers, Hans, Artegiani, Benedetta, Artegiani, Benedetta, van Voorthuijsen, Lisa, Lindeboom, Rik G. H., Seinstra, Danielle, Heo, Inha, Tapia, Pablo, Lopez-Iglesias, Carmen, Postrach, Daniel, Dayton, Talya, Oka, Rurika, Hu, Huili, van Boxtel, Ruben, van Es, Johan H., Offerhaus, Johan, Peters, Peter J., van Rheenen, Jacco, Vermeulen, Michiel, and Clevers, Hans

Abstract

The deubiquitinating enzyme BAP1 is a tumor suppressor, among others involved in cholangiocarcinoma. BAP1 has many proposed molecular targets, while its Drosophila homolog is known to deubiquitinate histone H2AK119. We introduce BAP1 loss-of-function by CRISPR/Cas9 in normal human cholangiocyte organoids. We find that BAP1 controls the expression of junctional and cytoskeleton components by regulating chromatin accessibility. Consequently, we observe loss of multiple epithelial characteristics while motility increases. Importantly, restoring the catalytic activity of BAP1 in the nucleus rescues these cellular and molecular changes. We engineer human liver organoids to combine four common cholangiocarcinoma mutations (TP53, PTEN, SMAD4, and NF1). In this genetic background, BAP1 loss results in acquisition of malignant features upon xenotransplantation. Thus, control of epithelial identity through the regulation of chromatin accessibility appears to be a key aspect of BAP1's tumor suppressor function. Organoid technology combined with CRISPR/Cas9 provides an experimental platform for mechanistic studies of cancer gene function in a human context.

Published
2019

55. Oral mucosal organoids as a potential platform for personalized cancer therapy

Author

Sub Clinical Pharmacology, Sub Oncologie/Cytologie, dCSCA RMSC-2, Sub Developmental Biology, Faculteit Diergeneeskunde, Driehuis, Else, Kolders, Sigrid, Spelier, Sacha, Lohmussaar, Kadi, Willems, Stefan M, Devriese, Lot A, de Bree, Remco, de Ruiter, Emma J, Korving, Jeroen, Begthel, Harry, Van Es, Johan H, Geurts, Veerle, He, Gui-Wei, van Jaarsveld, Richard H, Oka, Rurika, Muraro, Mauro J, Vivie, Judith, Zandvliet, Maurice M J M, Hendrickx, Antoni P A, Iakobachvili, Nino, Sridevi, Priya, Kranenburg, Onno, van Boxtel, Ruben, Kops, Geert J P L, Tuveson, David A, Peters, Peter J, van Oudenaarden, Alexander, Clevers, Hans, Sub Clinical Pharmacology, Sub Oncologie/Cytologie, dCSCA RMSC-2, Sub Developmental Biology, Faculteit Diergeneeskunde, Driehuis, Else, Kolders, Sigrid, Spelier, Sacha, Lohmussaar, Kadi, Willems, Stefan M, Devriese, Lot A, de Bree, Remco, de Ruiter, Emma J, Korving, Jeroen, Begthel, Harry, Van Es, Johan H, Geurts, Veerle, He, Gui-Wei, van Jaarsveld, Richard H, Oka, Rurika, Muraro, Mauro J, Vivie, Judith, Zandvliet, Maurice M J M, Hendrickx, Antoni P A, Iakobachvili, Nino, Sridevi, Priya, Kranenburg, Onno, van Boxtel, Ruben, Kops, Geert J P L, Tuveson, David A, Peters, Peter J, van Oudenaarden, Alexander, and Clevers, Hans

Published
2019

56. Enteroendocrine and tuft cells support Lgr5 stem cells on Paneth cell depletion

Author

van Es, Johan H, Wiebrands, Kay, López-Iglesias, Carmen, van de Wetering, Marc, Zeinstra, Laura, van den Born, Maaike, Korving, Jeroen, Sasaki, Nobuo, Peters, Peter J, van Oudenaarden, Alexander, Clevers, Hans, van Es, Johan H, Wiebrands, Kay, López-Iglesias, Carmen, van de Wetering, Marc, Zeinstra, Laura, van den Born, Maaike, Korving, Jeroen, Sasaki, Nobuo, Peters, Peter J, van Oudenaarden, Alexander, and Clevers, Hans

Abstract

Cycling intestinal Lgr5+ stem cells are intermingled with their terminally differentiated Paneth cell daughters at crypt bottoms. Paneth cells provide multiple secreted (e.g., Wnt, EGF) as well as surface-bound (Notch ligand) niche signals. Here we show that ablation of Paneth cells in mice, using a diphtheria toxin receptor gene inserted into the P-lysozyme locus, does not affect the maintenance of Lgr5+ stem cells. Flow cytometry, single-cell sequencing, and histological analysis showed that the ablated Paneth cells are replaced by enteroendocrine and tuft cells. As these cells physically occupy Paneth cell positions between Lgr5 stem cells, they serve as an alternative source of Notch signals, which are essential for Lgr5+ stem cell maintenance. Our combined in vivo results underscore the adaptive flexibility of the intestine in maintaining normal tissue homeostasis.

Published
2019

57. Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping

Author

Gehart, Helmuth, van Es, Johan H, Hamer, Karien, Beumer, Joep, Kretzschmar, Kai, Dekkers, Johanna F, Rios, Anne, Clevers, Hans, Gehart, Helmuth, van Es, Johan H, Hamer, Karien, Beumer, Joep, Kretzschmar, Kai, Dekkers, Johanna F, Rios, Anne, and Clevers, Hans

Abstract

Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.

Published
2019

58. Oral mucosal organoids as a potential platform for personalized cancer therapy

Author

Pathologie Patiëntenadministratie, UMC Utrecht, Pathologie Pathologen staf, Cancer, MS Medische Oncologie, MS Hoofd-Hals Chirurgische Oncologie, Pathologie Groep Moelans, Hubrecht Institute with UMC, MMB Infectiepreventie, Infection & Immunity, CMM Groep Coffer, MS CGO, Regenerative Medicine and Stem Cells, CMM Groep Cuppen, CMM Sectie Molecular Cancer Research, Child Health, Driehuis, Else, Kolders, Sigrid, Spelier, Sacha, Lõhmussaar, Kadi, Willems, Stefan M., Devriese, Lot A., de Bree, Remco, de Ruiter, Emma J., Korving, Jeroen, Begthel, Harry, van Es, Johan H., Geurts, Veerle, He, Gui Wei, van Jaarsveld, Richard H., Oka, Rurika, Muraro, Mauro J., Vivié, Judith, Zandvliet, Maurice M.J.M., Hendrickx, Antoni P.A., Iakobachvili, Nino, Sridevi, Priya, Kranenburg, Onno, van Boxtel, Ruben, Kops, Geert J.P.L., Tuveson, David A., Peters, Peter J., van Oudenaarden, Alexander, Clevers, Hans, Pathologie Patiëntenadministratie, UMC Utrecht, Pathologie Pathologen staf, Cancer, MS Medische Oncologie, MS Hoofd-Hals Chirurgische Oncologie, Pathologie Groep Moelans, Hubrecht Institute with UMC, MMB Infectiepreventie, Infection & Immunity, CMM Groep Coffer, MS CGO, Regenerative Medicine and Stem Cells, CMM Groep Cuppen, CMM Sectie Molecular Cancer Research, Child Health, Driehuis, Else, Kolders, Sigrid, Spelier, Sacha, Lõhmussaar, Kadi, Willems, Stefan M., Devriese, Lot A., de Bree, Remco, de Ruiter, Emma J., Korving, Jeroen, Begthel, Harry, van Es, Johan H., Geurts, Veerle, He, Gui Wei, van Jaarsveld, Richard H., Oka, Rurika, Muraro, Mauro J., Vivié, Judith, Zandvliet, Maurice M.J.M., Hendrickx, Antoni P.A., Iakobachvili, Nino, Sridevi, Priya, Kranenburg, Onno, van Boxtel, Ruben, Kops, Geert J.P.L., Tuveson, David A., Peters, Peter J., van Oudenaarden, Alexander, and Clevers, Hans

Published
2019

59. Probing the Tumor Suppressor Function of BAP1 in CRISPR-Engineered Human Liver Organoids

Author

CMM Groep Cuppen, Hubrecht Institute with UMC, Pathologie Groep Moelans, UMC Utrecht Holding, Pathologie Pathologen staf, Cancer, CMM Sectie Molecular Cancer Research, Child Health, Regenerative Medicine and Stem Cells, Artegiani, Benedetta, van Voorthuijsen, Lisa, Lindeboom, Rik G.H., Seinstra, Daniëlle, Heo, Inha, Tapia, Pablo, López-Iglesias, Carmen, Postrach, Daniel, Dayton, Talya, Oka, Rurika, Hu, Huili, van Boxtel, Ruben, van Es, Johan H., Offerhaus, Johan, Peters, Peter J., van Rheenen, Jacco, Vermeulen, Michiel, Clevers, Hans, CMM Groep Cuppen, Hubrecht Institute with UMC, Pathologie Groep Moelans, UMC Utrecht Holding, Pathologie Pathologen staf, Cancer, CMM Sectie Molecular Cancer Research, Child Health, Regenerative Medicine and Stem Cells, Artegiani, Benedetta, van Voorthuijsen, Lisa, Lindeboom, Rik G.H., Seinstra, Daniëlle, Heo, Inha, Tapia, Pablo, López-Iglesias, Carmen, Postrach, Daniel, Dayton, Talya, Oka, Rurika, Hu, Huili, van Boxtel, Ruben, van Es, Johan H., Offerhaus, Johan, Peters, Peter J., van Rheenen, Jacco, Vermeulen, Michiel, and Clevers, Hans

Published
2019

60. Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping

Author

Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Child Health, Regenerative Medicine and Stem Cells, Cancer, Gehart, Helmuth, van Es, Johan H., Hamer, Karien, Beumer, Joep, Kretzschmar, Kai, Dekkers, Johanna F., Rios, Anne, Clevers, Hans, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Child Health, Regenerative Medicine and Stem Cells, Cancer, Gehart, Helmuth, van Es, Johan H., Hamer, Karien, Beumer, Joep, Kretzschmar, Kai, Dekkers, Johanna F., Rios, Anne, and Clevers, Hans

Published
2019

61. Enteroendocrine and tuft cells support Lgr5 stem cells on Paneth cell depletion

Author

Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Child Health, Regenerative Medicine and Stem Cells, Van Es, Johan H., Wiebrands, Kay, López-Iglesias, Carmen, Van De Wetering, Marc, Zeinstra, Laura, Van Den Born, Maaike, Korving, Jeroen, Sasaki, Nobuo, Peters, Peter J., Van Oudenaarden, Alexander, Clevers, Hans, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Child Health, Regenerative Medicine and Stem Cells, Van Es, Johan H., Wiebrands, Kay, López-Iglesias, Carmen, Van De Wetering, Marc, Zeinstra, Laura, Van Den Born, Maaike, Korving, Jeroen, Sasaki, Nobuo, Peters, Peter J., Van Oudenaarden, Alexander, and Clevers, Hans

Published
2019

64. Probing the Tumor Suppressor Function of BAP1 in CRISPR-Engineered Human Liver Organoids

Author

Artegiani, Benedetta, primary, van Voorthuijsen, Lisa, additional, Lindeboom, Rik G.H., additional, Seinstra, Daniëlle, additional, Heo, Inha, additional, Tapia, Pablo, additional, López-Iglesias, Carmen, additional, Postrach, Daniel, additional, Dayton, Talya, additional, Oka, Rurika, additional, Hu, Huili, additional, van Boxtel, Ruben, additional, van Es, Johan H., additional, Offerhaus, Johan, additional, Peters, Peter J., additional, van Rheenen, Jacco, additional, Vermeulen, Michiel, additional, and Clevers, Hans, additional

Published
2019
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66. Enteroendocrine cells switch hormone expression along the crypt-to-villus BMP signalling gradient

Author

Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Child Health, Regenerative Medicine and Stem Cells, Cancer, Beumer, Joep, Artegiani, Benedetta, Post, Yorick, Reimann, Frank, Gribble, Fiona, Nguyen, Thuc Nghi, Zeng, Hongkui, Van den Born, Maaike, Van Es, Johan H., Clevers, Hans, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Child Health, Regenerative Medicine and Stem Cells, Cancer, Beumer, Joep, Artegiani, Benedetta, Post, Yorick, Reimann, Frank, Gribble, Fiona, Nguyen, Thuc Nghi, Zeng, Hongkui, Van den Born, Maaike, Van Es, Johan H., and Clevers, Hans

Published
2018

67. Profiling proliferative cells and their progeny in damaged murine hearts

Author

TN Onderwijs, Hubrecht Institute with UMC, Onderzoek Molecular Cardiology, Circulatory Health, Regenerative Medicine and Stem Cells, CMM Sectie Molecular Cancer Research, Cancer, Child Health, Brain, Kretzschmar, Kai, Post, Yorick, Bannier-Hélaouët, Marie, Mattiotti, Andrea, Drost, Jarno, Basak, Onur, Li, Vivian S.W., van den Born, Maaike, Gunst, Quinn D., Versteeg, Danielle, Kooijman, Lieneke, van der Elst, Stefan, van Es, Johan H., van Rooij, Eva, van den Hoff, Maurice J.B., Clevers, Hans, TN Onderwijs, Hubrecht Institute with UMC, Onderzoek Molecular Cardiology, Circulatory Health, Regenerative Medicine and Stem Cells, CMM Sectie Molecular Cancer Research, Cancer, Child Health, Brain, Kretzschmar, Kai, Post, Yorick, Bannier-Hélaouët, Marie, Mattiotti, Andrea, Drost, Jarno, Basak, Onur, Li, Vivian S.W., van den Born, Maaike, Gunst, Quinn D., Versteeg, Danielle, Kooijman, Lieneke, van der Elst, Stefan, van Es, Johan H., van Rooij, Eva, van den Hoff, Maurice J.B., and Clevers, Hans

Published
2018

68. Troy+ brain stem cells cycle through quiescence and regulate their number by sensing niche occupancy

Author

TN Onderwijs, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Brain, Basak, Onur, Krieger, Teresa G., Muraro, Mauro J., Wiebrands, Kay, Stange, Daniel E., Frias-Aldeguer, Javier, Rivron, Nicolas C., van de Wetering, Marc, van Es, Johan H., van Oudenaarden, Alexander, Simons, Benjamin D., Clevers, Hans, TN Onderwijs, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Brain, Basak, Onur, Krieger, Teresa G., Muraro, Mauro J., Wiebrands, Kay, Stange, Daniel E., Frias-Aldeguer, Javier, Rivron, Nicolas C., van de Wetering, Marc, van Es, Johan H., van Oudenaarden, Alexander, Simons, Benjamin D., and Clevers, Hans

Published
2018

69. Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation

Author

Massachusetts Institute of Technology. Department of Biology, Koch Institute for Integrative Cancer Research at MIT, Mana, Miyeko, Yilmaz, Omer, Sabatini, David, Dominguez-Brauer, Carmen, Hao, Zhenyue, Elia, Andrew J., Fortin, Jérôme M., Nechanitzky, Robert, Brauer, Patrick M., Sheng, Yi, Chio, Iok In Christine, Haight, Jillian, Pollett, Aaron, Cairns, Robert, Tworzyanski, Leanne, Inoue, Satoshi, Reardon, Colin, Marques, Ana, Silvester, Jennifer, Cox, Maureen A., Wakeham, Andrew, van Es, Johan H., Clevers, Hans, Sato, Toshiro, Mak, Tak W., Massachusetts Institute of Technology. Department of Biology, Koch Institute for Integrative Cancer Research at MIT, Mana, Miyeko, Yilmaz, Omer, Sabatini, David, Dominguez-Brauer, Carmen, Hao, Zhenyue, Elia, Andrew J., Fortin, Jérôme M., Nechanitzky, Robert, Brauer, Patrick M., Sheng, Yi, Chio, Iok In Christine, Haight, Jillian, Pollett, Aaron, Cairns, Robert, Tworzyanski, Leanne, Inoue, Satoshi, Reardon, Colin, Marques, Ana, Silvester, Jennifer, Cox, Maureen A., Wakeham, Andrew, van Es, Johan H., Clevers, Hans, Sato, Toshiro, and Mak, Tak W.

Abstract

The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche.

Published
2018

72. A Single-Cell RNA Sequencing Study Reveals Cellular and Molecular Dynamics of the Hippocampal Neurogenic Niche

Author

Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Child Health, Regenerative Medicine and Stem Cells, Artegiani, Benedetta, Lyubimova, Anna, Muraro, Mauro J., van Es, Johan H., van Oudenaarden, Alexander, Clevers, Hans, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Child Health, Regenerative Medicine and Stem Cells, Artegiani, Benedetta, Lyubimova, Anna, Muraro, Mauro J., van Es, Johan H., van Oudenaarden, Alexander, and Clevers, Hans

Published
2017

73. The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent '+4' cell markers

Author

Muñoz, Javier, Stange, Daniel E., Schepers, Arnout G., van de Wetering, Marc, Koo, Bon-Kyoung, Itzkovitz, Shalev, Volckmann, Richard, Kung, Kevin S., Koster, Jan, Radulescu, Sorina, Myant, Kevin, Versteeg, Rogier, Sansom, Owen J., van Es, Johan H., Barker, Nick, van Oudenaarden, Alexander, Mohammed, Shabaz, Heck, Albert J. R., Clevers, Hans, Hubrecht Institute for Developmental Biology and Stem Cell Research, Oncogenomics, and CCA -Cancer Center Amsterdam

Abstract

Two types of stem cells are currently defined in small intestinal crypts: cycling crypt base columnar (CBC) cells and quiescent '+4' cells. Here, we combine transcriptomics with proteomics to define a definitive molecular signature for Lgr5(+) CBC cells. Transcriptional profiling of FACS-sorted Lgr5(+) stem cells and their daughters using two microarray platforms revealed an mRNA stem cell signature of 384 unique genes. Quantitative mass spectrometry on the same cell populations identified 278 proteins enriched in intestinal stem cells. The mRNA and protein data sets showed a high level of correlation and a combined signature of 510 stem cell-enriched genes was defined. Spatial expression patterns were further characterized by mRNA in-situ hybridization, revealing that approximately half of the genes were expressed in a gradient with highest levels at the crypt bottom, while the other half was expressed uniquely in Lgr5(+) stem cells. Lineage tracing using a newly established knock-in mouse for one of the signature genes, Smoc2, confirmed its stem cell specificity. Using this resource, we find-and confirm by independent approaches-that the proposed quiescent/'+4' stem cell markers Bmi1, Tert, Hopx and Lrig1 are robustly expressed in CBC cells. The EMBO Journal (2012) 31, 3079-3091. doi:10.1038/emboj.2012.166; Published online 12 June 2012

Published
2016

74. Dual regulatory switch through interactions of Tcf7l2/Tcf4 with stage-specific partners propels oligodendroglial maturation

Author

Zhao, Chuntao, Deng, Yaqi, Liu, Lei, Yu, Kun, Zhang, Liguo, Wang, Haibo, He, Xuelian, Wang, Jincheng, Lu, Changqing, Wu, Laiman N., Weng, Qinjie, Mao, Meng, Li, Jianrong, Van Es, Johan H., Xin, Mei, Parry, Lee, Goldman, Steven A., Clevers, Hans, Lu, Q. Richard, Zhao, Chuntao, Deng, Yaqi, Liu, Lei, Yu, Kun, Zhang, Liguo, Wang, Haibo, He, Xuelian, Wang, Jincheng, Lu, Changqing, Wu, Laiman N., Weng, Qinjie, Mao, Meng, Li, Jianrong, Van Es, Johan H., Xin, Mei, Parry, Lee, Goldman, Steven A., Clevers, Hans, and Lu, Q. Richard

Published
2016

75. Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters

Author

Tetteh, Paul W., Basak, Onur, Farin, Henner F., Wiebrands, Kay, Kretzschmar, Kai, Begthel, Harry, van den Born, Maaike, Korving, Jeroen, De Sauvage, Frederic, Van Es, Johan H., Van Oudenaarden, Alexander, Clevers, Hans, Tetteh, Paul W., Basak, Onur, Farin, Henner F., Wiebrands, Kay, Kretzschmar, Kai, Begthel, Harry, van den Born, Maaike, Korving, Jeroen, De Sauvage, Frederic, Van Es, Johan H., Van Oudenaarden, Alexander, and Clevers, Hans

Published
2016

76. Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research

Author

Tetteh, Paul W, Kretzschmar, Kai, Begthel, Harry, van den Born, Maaike, Korving, Jeroen, Morsink, Folkert H M, Farin, Henner, van Es, Johan H, Offerhaus, G Johan A, Clevers, Hans, Tetteh, Paul W, Kretzschmar, Kai, Begthel, Harry, van den Born, Maaike, Korving, Jeroen, Morsink, Folkert H M, Farin, Henner, van Es, Johan H, Offerhaus, G Johan A, and Clevers, Hans

Abstract

Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1(CreER) knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1(CreER) KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1(CreER) KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.

Published
2016

77. Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters

Author

Tetteh, Paul W, Basak, Onur, Farin, Henner F, Wiebrands, Kay, Kretzschmar, Kai, Begthel, Harry, van den Born, Maaike, Korving, Jeroen, de Sauvage, Frederic, van Es, Johan H, van Oudenaarden, Alexander, Clevers, Hans, Tetteh, Paul W, Basak, Onur, Farin, Henner F, Wiebrands, Kay, Kretzschmar, Kai, Begthel, Harry, van den Born, Maaike, Korving, Jeroen, de Sauvage, Frederic, van Es, Johan H, van Oudenaarden, Alexander, and Clevers, Hans

Abstract

Intestinal crypts display robust regeneration upon injury. The relatively rare secretory precursors can replace lost stem cells, but it is unknown if the abundant enterocyte progenitors that express the Alkaline phosphate intestinal (Alpi) gene also have this capacity. We created an Alpi-IRES-CreERT2 (Alpi(CreER)) knockin allele for lineage tracing. Marked clones consist entirely of enterocytes and are all lost from villus tips within days. Genetic fate-mapping of Alpi(+) cells before or during targeted ablation of Lgr5-expressing stem cells generated numerous long-lived crypt-villus "ribbons," indicative of dedifferentiation of enterocyte precursors into Lgr5(+) stems. By single-cell analysis of dedifferentiating enterocytes, we observed the generation of Paneth-like cells and proliferative stem cells. We conclude that the highly proliferative, short-lived enterocyte precursors serve as a large reservoir of potential stem cells during crypt regeneration.

Published
2016

78. Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon

Author

Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Sasaki, Nobuo, Sachs, Norman, Wiebrands, Kay, Ellenbroek, Saskia I J, Fumagalli, Arianna, Lyubimova, Anna, Begthel, Harry, Van Born, Maaike Den, Van Es, Johan H., Karthaus, Wouter R., Li, Vivian S W, López-Iglesias, Carmen, Peters, Peter J., Van Rheenen, Jacco, Van Oudenaarden, Alexander, Clevers, Hans, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Sasaki, Nobuo, Sachs, Norman, Wiebrands, Kay, Ellenbroek, Saskia I J, Fumagalli, Arianna, Lyubimova, Anna, Begthel, Harry, Van Born, Maaike Den, Van Es, Johan H., Karthaus, Wouter R., Li, Vivian S W, López-Iglesias, Carmen, Peters, Peter J., Van Rheenen, Jacco, Van Oudenaarden, Alexander, and Clevers, Hans

Published
2016

79. Dual regulatory switch through interactions of Tcf7l2/Tcf4 with stage-specific partners propels oligodendroglial maturation

Author

Hubrecht Institute with UMC, Zhao, Chuntao, Deng, Yaqi, Liu, Lei, Yu, Kun, Zhang, Liguo, Wang, Haibo, He, Xuelian, Wang, Jincheng, Lu, Changqing, Wu, Laiman N., Weng, Qinjie, Mao, Meng, Li, Jianrong, Van Es, Johan H., Xin, Mei, Parry, Lee, Goldman, Steven A., Clevers, Hans, Lu, Q. Richard, Hubrecht Institute with UMC, Zhao, Chuntao, Deng, Yaqi, Liu, Lei, Yu, Kun, Zhang, Liguo, Wang, Haibo, He, Xuelian, Wang, Jincheng, Lu, Changqing, Wu, Laiman N., Weng, Qinjie, Mao, Meng, Li, Jianrong, Van Es, Johan H., Xin, Mei, Parry, Lee, Goldman, Steven A., Clevers, Hans, and Lu, Q. Richard

Published
2016

80. Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters

Author

Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Brain, Tetteh, Paul W., Basak, Onur, Farin, Henner F., Wiebrands, Kay, Kretzschmar, Kai, Begthel, Harry, van den Born, Maaike, Korving, Jeroen, De Sauvage, Frederic, Van Es, Johan H., Van Oudenaarden, Alexander, Clevers, Hans, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Brain, Tetteh, Paul W., Basak, Onur, Farin, Henner F., Wiebrands, Kay, Kretzschmar, Kai, Begthel, Harry, van den Born, Maaike, Korving, Jeroen, De Sauvage, Frederic, Van Es, Johan H., Van Oudenaarden, Alexander, and Clevers, Hans

Published
2016

81. Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation

Author

Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Dominguez-Brauer, Carmen, Hao, Zhenyue, Elia, Andrew J., Fortin, Jérôme M., Nechanitzky, Robert, Brauer, Patrick M., Sheng, Yi, Mana, Miyeko D., Chio, Iok In Christine, Haight, Jillian, Pollett, Aaron, Cairns, Robert, Tworzyanski, Leanne, Inoue, Satoshi, Reardon, Colin, Marques, Ana, Silvester, Jennifer, Cox, Maureen A., Wakeham, Andrew, Yilmaz, Omer H., Sabatini, David M., van Es, Johan H., Clevers, Hans, Sato, Toshiro, Mak, Tak W., Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Dominguez-Brauer, Carmen, Hao, Zhenyue, Elia, Andrew J., Fortin, Jérôme M., Nechanitzky, Robert, Brauer, Patrick M., Sheng, Yi, Mana, Miyeko D., Chio, Iok In Christine, Haight, Jillian, Pollett, Aaron, Cairns, Robert, Tworzyanski, Leanne, Inoue, Satoshi, Reardon, Colin, Marques, Ana, Silvester, Jennifer, Cox, Maureen A., Wakeham, Andrew, Yilmaz, Omer H., Sabatini, David M., van Es, Johan H., Clevers, Hans, Sato, Toshiro, and Mak, Tak W.

Published
2016

82. Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research

Author

Hubrecht Institute with UMC, Pathologie, Pathologie patiënten zorg, Cancer, CMM Sectie Molecular Cancer Research, Regenerative Medicine and Stem Cells, Child Health, Tetteh, Paul W., Kretzschmar, Kai, Begthel, Harry, Van Den Born, Maaike, Korving, Jeroen, Morsink, Folkert, Farin, Henner, Van Es, Johan H., Offerhaus, G. Johan A, Clevers, Hans, Hubrecht Institute with UMC, Pathologie, Pathologie patiënten zorg, Cancer, CMM Sectie Molecular Cancer Research, Regenerative Medicine and Stem Cells, Child Health, Tetteh, Paul W., Kretzschmar, Kai, Begthel, Harry, Van Den Born, Maaike, Korving, Jeroen, Morsink, Folkert, Farin, Henner, Van Es, Johan H., Offerhaus, G. Johan A, and Clevers, Hans

Published
2016

83. Niche-independent high-purity cultures of Lgr5[superscript +] intestinal stem cells and their progeny

Author

Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Chemical Engineering, Koch Institute for Integrative Cancer Research at MIT, Yin, Xiaolei, Langer, Robert, Karp, Jeffrey Michael, Clevers, Hans, Farin, Henner F., van Es, Johan H., Langer, Robert S, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Chemical Engineering, Koch Institute for Integrative Cancer Research at MIT, Yin, Xiaolei, Langer, Robert, Karp, Jeffrey Michael, Clevers, Hans, Farin, Henner F., van Es, Johan H., and Langer, Robert S

Abstract

Although Lgr5[superscript +] intestinal stem cells have been expanded in vitro as organoids, homogeneous culture of these cells has not been possible thus far. Here we show that two small molecules, CHIR99021 and valproic acid, synergistically maintain self-renewal of mouse Lgr5[superscript +] intestinal stem cells, resulting in nearly homogeneous cultures. The colony-forming efficiency of cells from these cultures is ~100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid, and multilineage differentiation ability is preserved. We made use of these homogeneous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, goblet cells and Paneth cells. Expansion in these culture conditions may be feasible for Lgr5[superscript +] cells from the mouse stomach and colon and from the human small intestine. These methods provide new tools for the study and application of multiple intestinal epithelial cell types., National Institutes of Health (U.S.) (Grant DE013023), Harvard Institute of Translational Immunology/Helmsley Trust Pilot Grant in Crohn's Disease

Published
2016

84. Reg4 + deep crypt secretory cells function as epithelial niche for Lgr5 + stem cells in colon

Author

Sasaki, Nobuo, primary, Sachs, Norman, additional, Wiebrands, Kay, additional, Ellenbroek, Saskia I. J., additional, Fumagalli, Arianna, additional, Lyubimova, Anna, additional, Begthel, Harry, additional, van den Born, Maaike, additional, van Es, Johan H., additional, Karthaus, Wouter R., additional, Li, Vivian S. W., additional, López-Iglesias, Carmen, additional, Peters, Peter J., additional, van Rheenen, Jacco, additional, van Oudenaarden, Alexander, additional, and Clevers, Hans, additional

Published
2016
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85. Dual regulatory switch through interactions of Tcf7l2/Tcf4 with stage-specific partners propels oligodendroglial maturation

Author

Zhao, Chuntao, primary, Deng, Yaqi, additional, Liu, Lei, additional, Yu, Kun, additional, Zhang, Liguo, additional, Wang, Haibo, additional, He, Xuelian, additional, Wang, Jincheng, additional, Lu, Changqing, additional, Wu, Laiman N, additional, Weng, Qinjie, additional, Mao, Meng, additional, Li, Jianrong, additional, van Es, Johan H, additional, Xin, Mei, additional, Parry, Lee, additional, Goldman, Steven A, additional, Clevers, Hans, additional, and Lu, Q. Richard, additional

Published
2016
Full Text
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86. Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters

Author

Tetteh, Paul W., primary, Basak, Onur, additional, Farin, Henner F., additional, Wiebrands, Kay, additional, Kretzschmar, Kai, additional, Begthel, Harry, additional, van den Born, Maaike, additional, Korving, Jeroen, additional, de Sauvage, Frederic, additional, van Es, Johan H., additional, van Oudenaarden, Alexander, additional, and Clevers, Hans, additional

Published
2016
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88. An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking

Author

Onderzoek, Faculteit Diergeneeskunde, Schneeberger, Kerstin, Vogel, Georg F, Teunissen, Hans, van Ommen, Domenique D, Begthel, Harry, El Bouazzaoui, Layla, van Vugt, Anke H M, Beekman, Jeffrey M, Klumperman, Judith, Müller, Thomas, Janecke, Andreas, Gerner, Patrick, Huber, Lukas A, Hess, Michael W, Clevers, Hans, van Es, Johan H, Nieuwenhuis, Edward E S, Middendorp, Sabine, Onderzoek, Faculteit Diergeneeskunde, Schneeberger, Kerstin, Vogel, Georg F, Teunissen, Hans, van Ommen, Domenique D, Begthel, Harry, El Bouazzaoui, Layla, van Vugt, Anke H M, Beekman, Jeffrey M, Klumperman, Judith, Müller, Thomas, Janecke, Andreas, Gerner, Patrick, Huber, Lukas A, Hess, Michael W, Clevers, Hans, van Es, Johan H, Nieuwenhuis, Edward E S, and Middendorp, Sabine

Published
2015

89. Generation and analysis of mouse intestinal tumors and organoids harboring APC and K-Ras mutations

Author

van Es, Johan H, Clevers, Hans, van Es, Johan H, and Clevers, Hans

Abstract

Genetically engineered mouse models of intestinal cancer are experimental systems in which mice are genetically manipulated to develop malignancies in the gastrointestinal tract. These models enable researchers to study the mechanisms of onset, progression, and metastasis of the disease. They also provide a valuable biological system which is suitable for testing (novel) drugs in vivo. Recently, an in vitro culture model has been established in which intestinal epithelial stem cells can grow into three-dimensional, ever-expanding epithelial organoids that retain their original organ identity and genetic stability. This culture system has been applied to diseased epithelia, such as adenoma, adenocarcinoma, and Barrett's epithelium. These organoids can be particularly useful for studying the mechanisms of intestinal tumors and to test (novel) drugs in vitro. Here, we describe our current laboratory protocols to generate and analyze intestinal tumors and organoids harboring APC and K-Ras double mutations.

Published
2015

90. Targeting development of incretin-producing cells increases insulin secretion

Author

Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Child Health, Petersen, Natalia, Reimann, Frank, van Es, Johan H, van den Berg, Bernard M, Kroone, Chantal, Pais, Ramona, Jansen, Erik, Clevers, Hans, Gribble, Fiona M, de Koning, Eelco J P, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Child Health, Petersen, Natalia, Reimann, Frank, van Es, Johan H, van den Berg, Bernard M, Kroone, Chantal, Pais, Ramona, Jansen, Erik, Clevers, Hans, Gribble, Fiona M, and de Koning, Eelco J P

Published
2015

91. Porcupine inhibitor suppresses paracrine Wnt-driven growth of Rnf43;Znrf3-mutant neoplasia

Author

Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Koo, Bon-Kyoung, van Es, Johan H, van den Born, Maaike, Clevers, Hans, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Koo, Bon-Kyoung, van Es, Johan H, van den Born, Maaike, and Clevers, Hans

Published
2015

92. Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer

Author

Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Dow, Lukas E, O'Rourke, Kevin P, Simon, Janelle, Tschaharganeh, Darjus F, van Es, Johan H, Clevers, Hans, Lowe, Scott W, Hubrecht Institute with UMC, CMM Sectie Molecular Cancer Research, Cancer, Regenerative Medicine and Stem Cells, Child Health, Dow, Lukas E, O'Rourke, Kevin P, Simon, Janelle, Tschaharganeh, Darjus F, van Es, Johan H, Clevers, Hans, and Lowe, Scott W

Published
2015

93. An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking

Author

Regenerative Medicine and Stem Cells, Beekman, Child Health, Infection & Immunity, CMM Sectie Celbiologie, Cancer, CMM Sectie Molecular Cancer Research, Hubrecht Institute with UMC, Zorg en O&O, MDL onderzoek 2, Schneeberger, Kerstin, Vogel, Georg F., Teunissen, Hans, Zomer-van Ommen, Domenique, Begthel, Harry, El Bouazzaoui, Layla, van Vugt, Anke H. M., Beekman, JM, Klumperman, Judith, Mueller, Thomas, Janecke, Andreas, Gerner, Patrick, Huber, Lukas A., Hess, Michael W., Clevers, Hans, van Es, Johan H., Nieuwenhuis, Edward E. S., Middendorp, Sabine, Regenerative Medicine and Stem Cells, Beekman, Child Health, Infection & Immunity, CMM Sectie Celbiologie, Cancer, CMM Sectie Molecular Cancer Research, Hubrecht Institute with UMC, Zorg en O&O, MDL onderzoek 2, Schneeberger, Kerstin, Vogel, Georg F., Teunissen, Hans, Zomer-van Ommen, Domenique, Begthel, Harry, El Bouazzaoui, Layla, van Vugt, Anke H. M., Beekman, JM, Klumperman, Judith, Mueller, Thomas, Janecke, Andreas, Gerner, Patrick, Huber, Lukas A., Hess, Michael W., Clevers, Hans, van Es, Johan H., Nieuwenhuis, Edward E. S., and Middendorp, Sabine

Published
2015

94. Niche-independent high-purity cultures of Lgr5[superscript +] intestinal stem cells and their progeny

Author

Yin, Xiaolei, Clevers, Hans, Farin, Henner F., van Es, Johan H., Karp, Jeffrey Michael, Langer, Robert S, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Chemical Engineering, Koch Institute for Integrative Cancer Research at MIT, Yin, Xiaolei, Langer, Robert, and Karp, Jeffrey Michael

Abstract

Although Lgr5[superscript +] intestinal stem cells have been expanded in vitro as organoids, homogeneous culture of these cells has not been possible thus far. Here we show that two small molecules, CHIR99021 and valproic acid, synergistically maintain self-renewal of mouse Lgr5[superscript +] intestinal stem cells, resulting in nearly homogeneous cultures. The colony-forming efficiency of cells from these cultures is ~100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid, and multilineage differentiation ability is preserved. We made use of these homogeneous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, goblet cells and Paneth cells. Expansion in these culture conditions may be feasible for Lgr5[superscript +] cells from the mouse stomach and colon and from the human small intestine. These methods provide new tools for the study and application of multiple intestinal epithelial cell types., National Institutes of Health (U.S.) (Grant DE013023), Harvard Institute of Translational Immunology/Helmsley Trust Pilot Grant in Crohn's Disease

Published
2013

95. Paneth cells

Author

van Es, Johan H., Clevers, Hans, and Hubrecht Institute for Developmental Biology and Stem Cell Research

Subjects
Paneth Cells, Crohn Disease, Agricultural and Biological Sciences(all), Biochemistry, Genetics and Molecular Biology(all), Intestine, Small, Humans, General Agricultural and Biological Sciences, General Biochemistry, Genetics and Molecular Biology
Published
2014

97. Paneth cells

Author

van Es, Johan H, Clevers, Hans, van Es, Johan H, and Clevers, Hans

Published
2014

98. Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny

Author

Yin, Xiaolei, Farin, Henner F, van Es, Johan H, Clevers, Hans, Langer, Robert, Karp, Jeffrey M, Yin, Xiaolei, Farin, Henner F, van Es, Johan H, Clevers, Hans, Langer, Robert, and Karp, Jeffrey M

Abstract

Although Lgr5(+) intestinal stem cells have been expanded in vitro as organoids, homogeneous culture of these cells has not been possible thus far. Here we show that two small molecules, CHIR99021 and valproic acid, synergistically maintain self-renewal of mouse Lgr5(+) intestinal stem cells, resulting in nearly homogeneous cultures. The colony-forming efficiency of cells from these cultures is ~100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid, and multilineage differentiation ability is preserved. We made use of these homogeneous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, goblet cells and Paneth cells. Expansion in these culture conditions may be feasible for Lgr5(+) cells from the mouse stomach and colon and from the human small intestine. These methods provide new tools for the study and application of multiple intestinal epithelial cell types.

Published
2014

99. Biased competition between Lgr5 intestinal stem cells driven by oncogenic mutation induces clonal expansion

Author

Snippert, Hugo J, Schepers, Arnout G, van Es, Johan H, Simons, Benjamin D, Clevers, Hans, Snippert, Hugo J, Schepers, Arnout G, van Es, Johan H, Simons, Benjamin D, and Clevers, Hans

Abstract

The concept of 'field cancerization' describes the clonal expansion of genetically altered, but morphologically normal cells that predisposes a tissue to cancer development. Here, we demonstrate that biased stem cell competition in the mouse small intestine can initiate the expansion of such clones. We quantitatively analyze how the activation of oncogenic K-ras in individual Lgr5(+) stem cells accelerates their cell division rate and creates a biased drift towards crypt clonality. K-ras mutant crypts then clonally expand within the epithelium through enhanced crypt fission, which distributes the existing Paneth cell niche over the two new crypts. Thus, an unequal competition between wild-type and mutant intestinal stem cells initiates a biased drift that leads to the clonal expansion of crypts carrying oncogenic mutations.

Published
2014

100. GRASP65 controls the cis Golgi integrity in vivo

Author

Veenendaal, Tineke, Jarvela, Tim, Grieve, Adam G, van Es, Johan H, Linstedt, Adam D, Rabouille, Catherine, Veenendaal, Tineke, Jarvela, Tim, Grieve, Adam G, van Es, Johan H, Linstedt, Adam D, and Rabouille, Catherine

Abstract

GRASP65 and GRASP55 are peripheral Golgi proteins localized to cis and medial/trans cisternae, respectively. They are implicated in diverse aspects of protein transport and structure related to the Golgi complex, including the stacking of the Golgi stack and/or the linking of mammalian Golgi stacks into the Golgi ribbon. Using a mouse model, we interfered with GRASP65 by homologous recombination and confirmed its absence of expression. Surprisingly, the mice were healthy and fertile with no apparent defects in tissue, cellular or subcellular organization. Immortalized MEFs derived from the mice did not show any growth or morphological defects. However, despite the normal appearance of the Golgi ribbon, a fluorescence recovery after photobleaching assay revealed functional discontinuities specific to the cis cisternal membrane network. This leads to a strong change in the plasma membrane GSII lectin staining that was also observed in certain mutant tissues. These findings substantiate the role of GRASP65 in continuity of the cis Golgi network required for proper glycosylation, while showing that neither this continuity nor GRASP65 itself are essential for the viability of a complex organism.

Published
2014

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