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5. Stronger induction of trained immunity by mucosal BCG or MTBVAC vaccination compared to standard intradermal vaccination

Author

Vierboom, Michel P.M., Dijkman, Karin, Sombroek, Claudia C., Hofman, Sam O., Boot, Charelle, Vervenne, Richard A.W., Haanstra, Krista G., van der Sande, Maarten, van Emst, Liesbeth, Domínguez-Andrés, Jorge, Moorlag, Simone J.C.F.M., Kocken, Clemens H.M., Thole, Jelle, Rodríguez, Esteban, Puentes, Eugenia, Martens, Joost H.A., van Crevel, Reinout, Netea, Mihai G., Aguilo, Nacho, Martin, Carlos, and Verreck, Frank A.W.

Published
2021
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6. Description and functional validation of human enteroendocrine cell sensors.

Author

Beumer, Joep, Geurts, Maarten H., Geurts, Veerle, Andersson-Rolf, Amanda, Akkerman, Ninouk, Völlmy, Franziska, Krueger, Daniel, Busslinger, Georg A., Martínez-Silgado, Adriana, Boot, Charelle, Yousef Yengej, Fjodor A., Puschhof, Jens, Van de Wetering, Wiline J., Knoops, Kevin, López-Iglesias, Carmen, Peters, Peter J., Vivié, Judith A., Mooijman, Dylan, van Es, Johan H., and Clevers, Hans

Published
2024
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7. High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells

Author

Beumer, Joep, Puschhof, Jens, Bauzá-Martinez, Julia, Martínez-Silgado, Adriana, Elmentaite, Rasa, James, Kylie R., Ross, Alexander, Hendriks, Delilah, Artegiani, Benedetta, Busslinger, Georg A., Ponsioen, Bas, Andersson-Rolf, Amanda, Saftien, Aurelia, Boot, Charelle, Kretzschmar, Kai, Geurts, Maarten H., Bar-Ephraim, Yotam E., Pleguezuelos-Manzano, Cayetano, Post, Yorick, Begthel, Harry, van der Linden, Franka, Lopez-Iglesias, Carmen, van de Wetering, Willine J., van der Linden, Reinier, Peters, Peter J., Heck, Albert J.R., Goedhart, Joachim, Snippert, Hugo, Zilbauer, Matthias, Teichmann, Sarah A., Wu, Wei, and Clevers, Hans

Published
2020
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8. Improved detection of colibactin-induced mutations by genotoxic E. coli in organoids and colorectal cancer

Author

CMM Groep Van Mil, CMM, Cancer, Hubrecht Institute with UMC, Rosendahl Huber, Axel, Pleguezuelos-Manzano, Cayetano, Puschhof, Jens, Ubels, Joske, Boot, Charelle, Saftien, Aurelia, Verheul, Mark, Trabut, Laurianne T., Groenen, Niels, van Roosmalen, Markus, Ouyang, Kyanna S., Wood, Henry, Quirke, Phil, Meijer, Gerrit, Cuppen, Edwin, Clevers, Hans, van Boxtel, Ruben, CMM Groep Van Mil, CMM, Cancer, Hubrecht Institute with UMC, Rosendahl Huber, Axel, Pleguezuelos-Manzano, Cayetano, Puschhof, Jens, Ubels, Joske, Boot, Charelle, Saftien, Aurelia, Verheul, Mark, Trabut, Laurianne T., Groenen, Niels, van Roosmalen, Markus, Ouyang, Kyanna S., Wood, Henry, Quirke, Phil, Meijer, Gerrit, Cuppen, Edwin, Clevers, Hans, and van Boxtel, Ruben

Published
2024

10. Evaluation of heterologous prime-boost vaccination strategies using chimpanzee adenovirus and modified vaccinia virus for TB subunit vaccination in rhesus macaques

Author

Vierboom, Michel P. M., Chenine, Agnes L., Darrah, Patricia A., Vervenne, Richard A. W., Boot, Charelle, Hofman, Sam O., Sombroek, Claudia C., Dijkman, Karin, Khayum, Mohamed A., Stammes, Marieke A., Haanstra, Krista G., Hoffmann, Chantal, Schmitt, Doris, Silvestre, Nathalie, White, Alexander G., Borish, H. Jacob, Seder, Robert A., Ouaked, Nadia, Leung-Theung-Long, Stephane, Inchauspé, Geneviève, Anantha, Ravi, Limbach, Mary, Evans, Thomas G., Casimiro, Danilo, Lempicki, Maria, Laddy, Dominick J., Bonavia, Aurelio, and Verreck, Frank A. W.

Published
2020
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13. Prevention of tuberculosis infection and disease by local BCG in repeatedly exposed rhesus macaques

Author

Dijkman, Karin, Sombroek, Claudia C., Vervenne, Richard A. W., Hofman, Sam O., Boot, Charelle, Remarque, Edmond J., and Kocken, Clemens H. M.

Subjects
BCG -- Usage, Rhesus monkey -- Research -- Health aspects, Immune response -- Research, Tuberculosis -- Prevention -- Research, Immunoglobulin A, Vaccines, Immunoglobulins, Interleukins, Skin, Immunization, Wildlife conservation, Vaccination, Biological sciences, Health
Abstract

Tuberculosis (TB) remains the deadliest infectious disease.sup.1, and the widely used Bacillus Calmette-Guérin (BCG) vaccine fails to curb the epidemic. An improved vaccination strategy could provide a cost-effective intervention to break the transmission cycle and prevent antimicrobial resistance.sup.2,3. Limited knowledge of the host responses critically involved in protective immunity hampers the development of improved TB vaccination regimens. Therefore, assessment of new strategies in preclinical models to select the best candidate vaccines before clinical vaccine testing remains indispensable. We have previously established in rhesus macaques (Macaca mulatta) that pulmonary mucosal BCG delivery reduces TB disease where standard intradermal injection fails.sup.4,5. Here, we show that pulmonary BCG prevents infection by using a repeated limiting-dose Mycobacterium tuberculosis challenge model and identify polyfunctional T-helper type 17 (T.sub.H17) cells, interleukin-10 and immunoglobulin A as correlates of local protective immunity. These findings warrant further research into mucosal immunization strategies and their translation to clinical application to more effectively prevent the spread of TB. Delivery of the Bacillus Calmette-Guérin vaccine into the lungs but not the skin of rhesus macaques protects animals from infection with Mycobacterium tuberculosis, suggesting that immune responses elicited locally may be required for vaccine efficacy., Author(s): Karin Dijkman [sup.1] , Claudia C. Sombroek [sup.1] , Richard A. W. Vervenne [sup.1] , Sam O. Hofman [sup.1] , Charelle Boot [sup.1] , Edmond J. Remarque [sup.1] , [...]

Published
2019
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14. Colon Tumors in Enterotoxigenic Bacteroides fragilis (ETBF)-Colonized Mice Do Not Display a Unique Mutational Signature but Instead Possess Host-Dependent Alterations in the APC Gene

Author

Allen, Jawara, primary, Rosendahl Huber, Axel, additional, Pleguezuelos-Manzano, Cayetano, additional, Puschhof, Jens, additional, Wu, Shaoguang, additional, Wu, Xinqun, additional, Boot, Charelle, additional, Saftien, Aurelia, additional, O’Hagan, Heather M., additional, Wang, Hao, additional, van Boxtel, Ruben, additional, Clevers, Hans, additional, and Sears, Cynthia L., additional

Published
2022
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15. Colon Tumors in Enterotoxigenic Bacteroides fragilis (ETBF)-Colonized Mice Do Not Display a Unique Mutational Signature but Instead Possess Host-Dependent Alterations in the APC Gene

Author

Allen, Jawara, Rosendahl Huber, Axel, Pleguezuelos-Manzano, Cayetano, Puschhof, Jens, Wu, Shaoguang, Wu, Xinqun, Boot, Charelle, Saftien, Aurelia, O'Hagan, Heather M, Wang, Hao, van Boxtel, Ruben, Clevers, Hans, Sears, Cynthia L, Allen, Jawara, Rosendahl Huber, Axel, Pleguezuelos-Manzano, Cayetano, Puschhof, Jens, Wu, Shaoguang, Wu, Xinqun, Boot, Charelle, Saftien, Aurelia, O'Hagan, Heather M, Wang, Hao, van Boxtel, Ruben, Clevers, Hans, and Sears, Cynthia L

Abstract

Enterotoxigenic Bacteroides fragilis (ETBF) is consistently found at higher frequency in individuals with sporadic and hereditary colorectal cancer (CRC) and induces tumorigenesis in several mouse models of CRC. However, whether specific mutations induced by ETBF lead to colon tumor formation has not been investigated. To determine if ETBF-induced mutations impact the Apc gene, and other tumor suppressors or proto-oncogenes, we performed whole-exome sequencing and whole-genome sequencing on tumors isolated after ETBF and sham colonization of Apcmin/+ and Apcmin/+Msh2fl/flVC mice, as well as whole-genome sequencing of organoids cocultured with ETBF. Our results indicate that ETBF-induced tumor formation results from loss of heterozygosity (LOH) of Apc, unless the mismatch repair system is disrupted, in which case, tumor formation results from new acquisition of protein-truncating mutations in Apc. In contrast to polyketide synthase-positive Escherichia coli (pks+ E. coli), ETBF does not produce a unique mutational signature; instead, ETBF-induced tumors arise from errors in DNA mismatch repair and homologous recombination DNA damage repair, established pathways of tumor formation in the colon, and the same genetic mechanism accounting for sham tumors in these mouse models. Our analysis informs how this procarcinogenic bacterium may promote tumor formation in individuals with inherited predispositions to CRC, such as Lynch syndrome or familial adenomatous polyposis (FAP). IMPORTANCE Many studies have shown that microbiome composition in both the mucosa and the stool differs in individuals with sporadic and hereditary colorectal cancer (CRC). Both human and mouse models have established a strong association between particular microbes and colon tumor induction. However, the genetic mechanisms underlying putative microbe-induced colon tumor formation are not well established. In this paper, we applied whole-exome sequencing and whole-genome sequencing to investigate

Published
2022

16. Differentiation and CRISPR-Cas9-mediated genetic engineering of human intestinal organoids

Author

Martinez-Silgado, Adriana, Yousef Yengej, Fjodor A, Puschhof, Jens, Geurts, Veerle, Boot, Charelle, Geurts, Maarten H, Rookmaaker, Maarten B, Verhaar, Marianne C, Beumer, Joep, Clevers, Hans, Martinez-Silgado, Adriana, Yousef Yengej, Fjodor A, Puschhof, Jens, Geurts, Veerle, Boot, Charelle, Geurts, Maarten H, Rookmaaker, Maarten B, Verhaar, Marianne C, Beumer, Joep, and Clevers, Hans

Abstract

Intestinal organoids are three-dimensional cultures that resemble key aspects of the epithelium of origin. Here, we describe how to differentiate human small intestinal organoids by combining growth media variations and genetic engineering. We detail the differentiation of human intestinal organoids in the presence and absence of BMP agonists to recapitulate a broader scope of functional cell states found in vivo. Using transient overexpression of the transcription factor Neurogenin-3, we describe the enhancement of differentiation toward rare enteroendocrine cells. For complete details on the use and execution of this protocol, please refer to Beumer et al. (2022).

Published
2022

17. Mapping prohormone processing by proteases in human enteroendocrine cells using genetically engineered organoid models

Author

Hubrecht Institute with UMC, CMM, Cancer, Beumer, Joep, Bauzá-Martinez, Julia, Veth, Tim S., Geurts, Veerle, Boot, Charelle, Gilliam-Vigh, Hannah, Poulsen, Steen S., Knop, Filip K., Wu, Wei, Clevers, Hans, Hubrecht Institute with UMC, CMM, Cancer, Beumer, Joep, Bauzá-Martinez, Julia, Veth, Tim S., Geurts, Veerle, Boot, Charelle, Gilliam-Vigh, Hannah, Poulsen, Steen S., Knop, Filip K., Wu, Wei, and Clevers, Hans

Published
2022

18. Colon Tumors in Enterotoxigenic Bacteroides fragilis (ETBF)Colonized Mice Do Not Display a Unique Mutational Signature but Instead Possess Host-Dependent Alterations in the APC Gene

Author

CMM, Cancer, Hubrecht Institute with UMC, Allen, Jawara, Huber, Axel Rosendahl, Pleguezuelos-Manzano, Cayetano, Puschhof, Jens, Wu, Shaoguang, Wu, Xinqun, Boot, Charelle, Saftien, Aurelia, O’Hagan, Heather M., Wang, Hao, van Boxtel, Ruben, Clevers, Hans, Sears, Cynthia L., CMM, Cancer, Hubrecht Institute with UMC, Allen, Jawara, Huber, Axel Rosendahl, Pleguezuelos-Manzano, Cayetano, Puschhof, Jens, Wu, Shaoguang, Wu, Xinqun, Boot, Charelle, Saftien, Aurelia, O’Hagan, Heather M., Wang, Hao, van Boxtel, Ruben, Clevers, Hans, and Sears, Cynthia L.

Published
2022

19. Differentiation and CRISPR-Cas9-mediated genetic engineering of human intestinal organoids

Author

Nefro Vasculaire Geneeskunde, MS Nefrologie, Regenerative Medicine and Stem Cells, Circulatory Health, CMM, Cancer, Hubrecht Institute with UMC, Martinez-Silgado, Adriana, Yousef Yengej, Fjodor A., Puschhof, Jens, Geurts, Veerle, Boot, Charelle, Geurts, Maarten H., Rookmaaker, Maarten B., Verhaar, Marianne C., Beumer, Joep, Clevers, Hans, Nefro Vasculaire Geneeskunde, MS Nefrologie, Regenerative Medicine and Stem Cells, Circulatory Health, CMM, Cancer, Hubrecht Institute with UMC, Martinez-Silgado, Adriana, Yousef Yengej, Fjodor A., Puschhof, Jens, Geurts, Veerle, Boot, Charelle, Geurts, Maarten H., Rookmaaker, Maarten B., Verhaar, Marianne C., Beumer, Joep, and Clevers, Hans

Published
2022

20. BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states

Author

Nefro Vasculaire Geneeskunde, Beumer, Joep, Puschhof, Jens, Yengej, Fjodor Yousef, Zhao, Lianzheng, Martinez-Silgado, Adriana, Blotenburg, Marloes, Begthel, Harry, Boot, Charelle, van Oudenaarden, Alexander, Chen, Ye Guang, Clevers, Hans, Nefro Vasculaire Geneeskunde, Beumer, Joep, Puschhof, Jens, Yengej, Fjodor Yousef, Zhao, Lianzheng, Martinez-Silgado, Adriana, Blotenburg, Marloes, Begthel, Harry, Boot, Charelle, van Oudenaarden, Alexander, Chen, Ye Guang, and Clevers, Hans

Published
2022

21. The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells

Author

Tanner, Rachel, primary, Hoogkamer, Emily, additional, Bitencourt, Julia, additional, White, Andrew, additional, Boot, Charelle, additional, Sombroek, Claudia C., additional, Harris, Stephanie A., additional, O'Shea, Matthew K., additional, Wright, Daniel, additional, Wittenberg, Rachel, additional, Sarfas, Charlotte, additional, Satti, Iman, additional, Verreck, Frank A.W., additional, Sharpe, Sally A., additional, Fletcher, Helen A., additional, and McShane, Helen, additional

Published
2021
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22. Intestinal organoid cocultures with microbes

Author

Hubrecht Institute with UMC, Puschhof, Jens, Pleguezuelos-Manzano, Cayetano, Martinez-Silgado, Adriana, Akkerman, Ninouk, Saftien, Aurelia, Boot, Charelle, de Waal, Amy, Beumer, Joep, Dutta, Devanjali, Heo, Inha, Clevers, Hans, Hubrecht Institute with UMC, Puschhof, Jens, Pleguezuelos-Manzano, Cayetano, Martinez-Silgado, Adriana, Akkerman, Ninouk, Saftien, Aurelia, Boot, Charelle, de Waal, Amy, Beumer, Joep, Dutta, Devanjali, Heo, Inha, and Clevers, Hans

Published
2021

23. High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells

Author

CMM Groep Snippert, Cancer, CMM Sectie Molecular Cancer Research, Child Health, Regenerative Medicine and Stem Cells, Hubrecht Institute with UMC, Beumer, Joep, Puschhof, Jens, Bauzá-Martinez, Julia, Martínez-Silgado, Adriana, Elmentaite, Rasa, James, Kylie R, Ross, Alexander, Hendriks, Delilah, Artegiani, Benedetta, Busslinger, Georg A, Ponsioen, Bas, Andersson-Rolf, Amanda, Saftien, Aurelia, Boot, Charelle, Kretzschmar, Kai, Geurts, Maarten H, Bar-Ephraim, Yotam E, Pleguezuelos-Manzano, Cayetano, Post, Yorick, Begthel, Harry, van der Linden, Franka, Lopez-Iglesias, Carmen, van de Wetering, Willine J, van der Linden, Reinier, Peters, Peter J, Heck, Albert J R, Goedhart, Joachim, Snippert, Hugo, Zilbauer, Matthias, Teichmann, Sarah A, Wu, Wei, Clevers, Hans, CMM Groep Snippert, Cancer, CMM Sectie Molecular Cancer Research, Child Health, Regenerative Medicine and Stem Cells, Hubrecht Institute with UMC, Beumer, Joep, Puschhof, Jens, Bauzá-Martinez, Julia, Martínez-Silgado, Adriana, Elmentaite, Rasa, James, Kylie R, Ross, Alexander, Hendriks, Delilah, Artegiani, Benedetta, Busslinger, Georg A, Ponsioen, Bas, Andersson-Rolf, Amanda, Saftien, Aurelia, Boot, Charelle, Kretzschmar, Kai, Geurts, Maarten H, Bar-Ephraim, Yotam E, Pleguezuelos-Manzano, Cayetano, Post, Yorick, Begthel, Harry, van der Linden, Franka, Lopez-Iglesias, Carmen, van de Wetering, Willine J, van der Linden, Reinier, Peters, Peter J, Heck, Albert J R, Goedhart, Joachim, Snippert, Hugo, Zilbauer, Matthias, Teichmann, Sarah A, Wu, Wei, and Clevers, Hans

Published
2020

24. Disparate Tuberculosis Disease Development in Macaque Species Is Associated With Innate Immunity

Author

Dijkman, Karin, primary, Vervenne, Richard A. W., additional, Sombroek, Claudia C., additional, Boot, Charelle, additional, Hofman, Sam O., additional, van Meijgaarden, Krista E., additional, Ottenhoff, Tom H. M., additional, Kocken, Clemens H. M., additional, Haanstra, Krista G., additional, Vierboom, Michel P. M., additional, and Verreck, Frank A. W., additional

Published
2019
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25. Intestinal organoid cocultures with microbes

Author

Puschhof, Jens, Pleguezuelos-Manzano, Cayetano, Martinez-Silgado, Adriana, Akkerman, Ninouk, Saftien, Aurelia, Boot, Charelle, de Waal, Amy, Beumer, Joep, Dutta, Devanjali, Heo, Inha, and Clevers, Hans

Subjects
stem-cells, expansion, host, responses, rna, in-vitro
Abstract

This protocol comprises various methods to coculture organoids (particularly human small intestinal and colon organoids) with microbes, including microinjection into the lumen and periphery of 3D organoids and exposure of organoids to microbes in a 2D layer., Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host-microbe interactions with great experimental control. This protocol comprises methods to coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.

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