Organoïdes de modèle pour les maladies infectieuses: la culture des droits humains et murins organoïdes d'estomac et microinjection de Helicobacter pylori
Summary
Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.
Abstract
Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.
Introduction
L'étude des agents pathogènes repose sur des systèmes modèles adéquats pour imiter l'infection in vivo. Pour certains agents infectieux, des systèmes de modèles adéquats font défaut tandis que certains des systèmes utilisés sont loin d'être optimale. Un exemple est la bactérie gastrique Helicobacter pylori (H. pylori), qui est un lien de causalité liée au développement d'un cancer gastrique. Cependant, en l'absence d'un système de culture cellulaire approprié plus, de nombreuses études qui ont pour but d'analyser les mécanismes moléculaires sous-jacents des lignées cellulaires de cancer utilisation de développement de cancer, qui représentent le point d'extrémité de la cascade cancéreuse. Les cellules primaires, non-transformé serait un meilleur modèle pour ces études. Cependant, les cellules primaires sont disponibles uniquement à partir d'un petit nombre de donateurs et ne peuvent pas être cultivées sur des périodes de temps plus longues. Au cours des dernières années, la recherche sur les cellules souches a fait d'importants progrès pour fournir de nouvelles sources de cultures de cellules primaires pour l'étude de la biologie de l'infection.
Cultures detrois sources de cellules souches ont été utilisées: des cellules souches embryonnaires (ESC), des cellules pluripotentes induites souches (CISP) ou des cellules souches adultes. Ils ont été utilisés pour modéliser les infections par des virus, tels que le 1,2 cytomégalovirus ou le virus de l'hépatite C 3-7, des parasites tels que Plasmodium falciparum 8 ou 9 Toxoplasma gondii, et les bactéries, telles que Bacterioides thetaiotaomicron 10 ou 11 Salmonella enterica. Plus récemment, plusieurs approches ont été publiés pour modéliser infection à H. pylori avec organoïdes dérivées de cellules iPS ESC ou 12, les cellules souches adultes de souris 21,22 ou humaines cellules souches adultes 13 – 15.
Le développement des cultures organoïdes de cellules souches adultes est issue d'une étude, dans laquelle les cellules souches isolées à partir de simples épithélium intestinal murin ont été ensemencées dans une matrice en 3 dimensions etincorporé dans le milieu qui a imité environnement des cellules souches intestinales contenant EGF comme mitogene, R-spondine pour améliorer la signalisation Wnt et Noggin pour inhiber la protéine morphogénique osseuse (BMP) 16 signalisation. Notamment ces cultures ne nécessitent pas de co-culture avec des cellules mésenchymateuses. Dans ces conditions, les cellules souches prolifèrent et forment de petites structures avec des domaines hébergeant des cellules des cryptes intestinales, et les domaines qui contiennent les cellules de la villosité intestinale. Les organoïdes ainsi auto-organisent pour simuler la situation in vivo. Aujourd'hui, les cellules souches adultes de nombreux tissus murins et humains peut être cultivé in vitro et l'auto-organiser en organites qui ressemblent à leur homologue in vivo, tels que l'intestin grêle et le côlon 17, 13,18 estomac, le foie 19,20, du pancréas et 21 22 prostate.
Ici, nous fournissons un protocole de vidéo sur la souris de culture ou organoïdes gastriques humaines du cel souches adultesls et les micro-injection avec H. pylori. Ce protocole est basé sur les rapports précédents 13,18. Ce procédé peut être adapté pour la culture et infecter d'autres cultures telles que les organites organoïdes intestinaux.
Protocol
Representative Results
Discussion
This protocol describes the use of ever-expanding, untransformed primary organoids from adult stem cells for infection biology. Critical steps are i) the isolation of viable glands, ii) expansion of organoids and iii) the microinjection. Below are some suggestions for modifications, troubleshooting and technical considerations.
Compared to other isolation methods, which use vigorous shaking or pipetting to release glands and can be equally successful, the technique presented here has the adva…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by EU Marie Curie Fellowship (EU/300686-InfO) to S.B. and a Research Prize from the United European Gastroenterology Foundation to H.C. We would like to thank Harry Begthel, Jeroen Korving and the Hubrecht Imaging Center for technical assistance, Meritxell Huch for help with initial organoid culture and Yana Zavros for discussion.
Materials
| Medium | |||
| HEPES | Invitrogen | 15630-056 | |
| Advanced DMEM/F12 | Invitrogen | 12634-028 | |
| Matrigel, GFR, phenol free | BD | 356231 | |
| GlutaMAX | Invitrogen | 35050-079 | Stock concentration 200 mM, final concentration 2 mM |
| B27 | Invitrogen | 17504-044 | Stock concentration 50 x, final concentration 1x |
| N-Acetylcysteine | Sigma-Aldrich | A9165-5G | Stock concentration 500 mM, final concentration 1 mM |
| Murine recombinant EGF | Invitrogen | PMG8043 | Stock concentration 500 µg/mL, final concentration 50 ng/mL |
| Human recombinant FGF10 | Peprotech | 100-26 | Stock concentration 100 µg/mL, final concentration 200 ng/mL |
| TGFβi A-83-01 | Tocris | 2939 | Stock concentration 500 µM, final concentration 2 µM |
| Nicotinamide | Sigma-Aldrich | N0636 | Stock concentration 1 M, final concentration 10 mM |
| [Leu15]-Gastrin | Sigma-Aldrich | G9145 | Stock concentration 100 µM, final concentration 1 nM |
| RHOKi Y-27632 | Sigma-Aldrich | Y0503 | Stock concentration 10 mM, final concentration 10 µM |
| Wnt3A conditioned medium | Stable cell line generated in the Clevers Lab. Final concentration 50%. Cells can be obtained from Hans Clevers. | ||
| R-spondin1 conditioned medium | Stable cell line generated in the Kuo Lab. Final concentration 10%. Cell line can be obtained from Calvin Kuo, Stanford. | ||
| Noggin conditioned medium | Stable cell line generated in the Clevers Lab. Final concentration 10%. Cells can be obtained from Hans Clevers. | ||
| R-spondin3 | R&D | 3500-RS/CF | Alternative source for R-spondin. This has been tested on human intestine organoids (1 µg/mL), but not yet on gastric organoids. |
| Noggin | Peprotech | 120-10 | Alternative source for noggin. This has been tested on human intestine organoids (100 ng/mL), but not yet on gastric organoids. |
| TrypLE express | Life Technologies | 12605036 | Enzymatic dissociation solution |
| CoolCell® Alcohol-Free Cell Freezing Containers | biocision | BCS-405 | |
| Recovery Cell Culture Freezing Medium | Invitrogen | 12648-010 | |
| Antibiotics | |||
| Primocin | Invivogen | ant-pm-1 | An antibiotics composition agains bacteria and fungi. It is helpful after initiation of a culture. For long term culture you can switch to other antibiotics or none. |
| Penicillin/Streptomycin | Invitrogen | 15140-122 | Stock concentration 10000/10000 U/mL, final concentration 100/100 U/mL. Can be used alternatively to Primocin in long term culture. |
| Other | |||
| Tweezers | Neolabs | 2-1033 | Tweezers with fine tips are helpful for the removal of muscle layer from the tissue. |
| 4 Well Multidishes | Thermo Scientific | 144444 | You can use other Multidishes. These were particularly helpful for microinjections because they have a low outer rim and allow more mobility for the manipulator. |
| Micromanipulator | Narishige | M-152 | |
| Microinjector | Narishige | IM-5B | |
| Stereomicroscope | Leica | MZ75 | |
| Workbench | Clean Air | Custom made to fit the stereomicroscope in ML2 condition | |
| Cappillaries | Harvard Apparatus | GC100T-10 | 1 mm outer diameter, 0,78 mm inner diameter. |
| Micropipette Puller | Sutter Instruments | Flaming Brown Micropipette Puller | |
| anti Cag A antibody | Santa Cruz | sc-25766 |
References
- Aiuto, L., et al. Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells. PLoS ONE. 7 (11), e49700 (2012).
- Penkert, R. R., Kalejta, R. F. Human Embryonic Stem Cell Lines Model Experimental Human Cytomegalovirus Latency. mBio. 4 (3), e00298-13-e00298-13 (2013).
- Roelandt, P., et al. Human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis C virus. J Hepatol. 57 (2), 246-251 (2012).
- Schwartz, R. E., Trehan, K., et al. Modeling hepatitis C virus infection using human induced pluripotent stem cells. Proc Natl Acad Sci USA. 109 (7), 2544-2548 (2012).
- Shlomai, A., et al. Modeling host interactions with hepatitis B virus using primary and induced pluripotent stem cell-derived hepatocellular systems. Proc Natl Acad Sci USA. 111 (33), 12193-12198 (2014).
- Wu, X., et al. Productive Hepatitis C Virus Infection of Stem Cell-Derived Hepatocytes Reveals a Critical Transition to Viral Permissiveness during Differentiation. PLoS Pathogens. 8 (4), e1002617 (2012).
- Yoshida, T., et al. Use of human hepatocyte-like cells derived from induced pluripotent stem cells as a model for hepatocytes in hepatitis C virus infection. Biochem Biophys Res Commun. 416 (1-2), 119-124 (2011).
- Ng, S., et al. Human iPSC-Derived Hepatocyte-like Cells Support Plasmodium Liver-Stage Infection In Vitro. Stem Cell Report. 4 (2), (2015).
- Klotz, C., Aebischer, T., Seeber, F. Stem cell-derived cell cultures and organoids for protozoan parasite propagation and studying host-parasite interaction. Int J Med Microbiol. 302 (4-5), 203-209 (2012).
- Engevik, M. A., et al. Loss of NHE3 alters gut microbiota composition and influences Bacteroides thetaiotaomicron growth. AJP: GI. 305 (10), G697-G711 (2013).
- Wilson, S. S., Tocchi, A., Holly, M. K., Parks, W. C., Smith, J. G. A small intestinal organoid model of non-invasive enteric pathogen-epithelial cell interactions. Mucosal Immunol. 8 (2), 352-361 (2015).
- McCracken, K. W., et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature. 516 (7531), 400-404 (2014).
- Bartfeld, S., et al. In Vitro Expansion of Human Gastric Epithelial Stem Cells and Their Responses to Bacterial Infection. Gastroenterology. 148 (1), (2014).
- Schlaermann, P., Toelle, B., et al. A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro. Gut. , (2014).
- Bertaux-Skeirik, N., et al. CD44 Plays a Functional Role in Helicobacter pylori-induced Epithelial Cell Proliferation. PLOS Pathogens. 11 (2), e1004663 (2015).
- Sato, T., et al. Single Lgr5 stem cells build crypt villus structures in vitro without a mesenchymal niche. Nature. 459 (7244), 262-265 (2009).
- Sato, T., et al. Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett’s Epithelium. Gastroenterology. 141 (5), 1762-1772 (2011).
- Barker, N., et al. Lgr5+ve Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In Vitro. Cell Stem Cell. 6 (1), 25-36 (2010).
- Huch, M., et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature. 494 (7436), 247-250 (2013).
- Huch, M., et al. Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver. Cell. 160 (1-2), 299-312 (2015).
- Boj, S. F., et al. Organoid Models of Human and Mouse Ductal Pancreatic Cancer. Cell. 160 (1-2), 324-338 (2015).
- Karthaus, W. R., et al. Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell. 159 (1), 163-175 (2014).
- Bartfeld, S., et al. High-throughput and single-cell imaging of NF-kappaB oscillations using monoclonal cell lines. BMC cell. 11, 21 (2010).
- Blanchard, T. G., Nedrud, J. G. Laboratory Maintenance of Helicobacter Species. Curr Protoc Microbiol. , (2006).
- Van Es, J. H., de Geest, N., van de Born, M., Clevers, H., Hassan, B. A. Intestinal stem cells lacking the Math1 tumour suppressor are refractory to Notch inhibitors. Nat Commun. 1 (2), 1-5 (2010).
- Andersson-Rolf, A., Fink, J., Mustata, R. C., Koo, B. -. K. A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture. J Vis Exp. (90), (2014).
- Stange, D. E., Koo, B. -. K., et al. Differentiated Troy+ Chief Cells Act as Reserve Stem Cells to Generate All Lineages of the Stomach Epithelium. Cell. 155 (2), 357-368 (2013).
- Van de Wetering, M., Sancho, E., et al. The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell. 111 (2), 241-250 (2002).
- Schumacher, M. A., Aihara, E., et al. The use of murine-derived fundic organoids in studies of gastric physiology. Journal Physiol. 593 (8), 1809-1827 (2015).
- Schwank, G., Andersson-Rolf, A., Koo, B. -. K., Sasaki, N., Clevers, H. Generation of BAC Transgenic Epithelial Organoids. PLoS ONE. 8 (10), e76871 (2013).
- Koo, B. -. K., et al. Controlled gene expression in primary Lgr5 organoid cultures. Nat Meth. 9 (1), 81-83 (2012).
- Schwank, G., et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell. 13 (6), 653-658 (2013).
- Li, V. S. W., Ng, S. S., et al. Wnt Signaling through Inhibition of β-Catenin Degradation in an Intact Axin1 Complex. Cell. 149 (6), 1245-1256 (2012).
- Van de Wetering, M., et al. Prospective derivation of a ‘Living Organoid Biobank’ of colorectal cancer patients. Cell. 161 (4), 933-945 (2015).
