Summary

Enfeksiyon Hastalıkları Modeli Olarak Organoids: Helicobacter Pylori ve İnsan Kültür ve kemirgen Mide Organoids ve Mikroenjeksiyon

Published: November 12, 2015
doi:

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

Patojenlerin çalışma, in vivo enfeksiyon taklit etmek için yeterli bir model sistemler kullanır. Kullanılan sistemlerin bazı optimum uzak iken bazı enfektif ajanlar için yeterli model sistemler eksiktir. Bir örnek nedensel mide kanseri gelişimi ile ilgilidir mide bakterisi Helicobacter pylori (H. pylori) 'dir. Yine daha uygun bir hücre kültürü sisteminde yokluğunda amacı birçok çalışma, kanserli kaskadının son nokta temsil kanser gelişimi kullanımı kanser hücre çizgisi altında yatan moleküler mekanizmaların, analiz etmek. Primer, dönüştürülmemiş hücreler bu çalışmalar için daha iyi bir model olabilir. Bununla birlikte, birincil hücreler donör az sayıda kullanılabilir ve daha uzun süreler boyunca kültüre olamaz. Son yıllarda, kök hücre araştırmaları enfeksiyon biyoloji çalışma için primer hücre kültürleri için yeni kaynaklar sağlamak için önemli bir ilerleme kaydetmiştir.

Dan KültürlerÜç kök hücre kaynağı kullanılmıştır: Embriyonik kök hücreler (ESC), uyarılmış pluripotent kök hücreler (iPSC) ya da erişkin kök hücreler. Bunlar, Sitomegalovirüs, 1,2 veya Hepatit C virüsü gibi virüsler, enfeksiyonlar modellemek için kullanılmıştır 3-7, örneğin Bacterioides thetaiotaomicron 10 veya Salmonella enterica 11 olarak Plasmodium falciparum 8 veya 9, Toxoplasma gondii ve bakteriler gibi parazitler. En son, çeşitli yaklaşımlar H. enfeksiyonu modellemek için yayınlanmıştır 15 – ESC veya iPS hücrelerinin 12, fare yetişkin kök hücrelerin 21,22 ya da insan erişkin kök hücreleri 13 türetilen organoids ile pylori.

Yetişkin kök hücrelerinden organoid kültürlerinin gelişmesi sıçangil bağırsak epiteli izole tek kök hücre, bir 3-boyutlu bir matris içine ekildi bir çalışmada, gelen veKemik morfojenik protein (BMP) 16 sinyalizasyon inhibe Wnt sinyalizasyon ve Noggin geliştirmek için mitojendir, R-spondin olarak EGF içeren bağırsak kök hücrelerin çevreye taklit ortamda gömülü. Özellikle, bu kültürler mezenkimal hücreler ile birlikte kültür gerektirmez. Bu koşullar altında, kök hücreler çoğalmaya ve etki bağırsak kript hücreleri barındıran küçük yapılar oluşturur, ve bağırsak villuslarının hücreleri içeren alan. Organoids böylece in vivo durumu taklit etmek kendini düzenliyoruz. Bugün birçok fare ve insani dokulardan yetişkin kök hücrelerin in vitro ve yetiştirilebilir gibi ince bağırsak ve kolon 17, mide 13,18, karaciğer 19,20 olarak in vivo meslektaşı benzeyen organoids içine kendini organize pankreas 21 ve Prostat 22.

Burada yetişkin kök cel kültür fare veya insan mide organoids bir video protokol sağlarls H. ile Microinject ve pylori. Bu protokol daha önceki raporlarda 13,18 dayanmaktadır. Bu yöntem, kültürlenmesi ve intestinal organoids gibi diğer organoid kültürleri enfekte edilmesi için adapte edilebilir.

Protocol

Mide organoid Kültür 1. kurulması Not: Bu protokol, fare veya insan dokusundan gastrik bezleri izolasyonu için de kullanılabilir. Yaklaşık 1 cm² doku kullanılması tavsiye edilir. İnsan dokusu gastrik rezeksiyonu veya biyopsiler elde edilebilir. Malzemenin Hazırlanması Not: Kullanılan bodrum matris Matrigel olduğunu. Her zaman buz üzerinde bodrum matrisi tutun. -20 ° C 'de taban matrisi saklayın ve kullanımdan önce buz üzerinde eritin. Bazal ortam HE…

Representative Results

Bu protokol, gastrik bezleri (Şekil 2) ayrılmasına izin verir. Bezler organoids içine bezleri büyümeye izin laminin ve kolajen bakımından zengin bir 3 boyutlu bir çerçeve (Şekil 3) sağlanması, bir kuyu içinde damla olarak katılaşan temel matris içine ekilir. Organoids tipik haliyle küçük kistler başlar ve 12-16 gün içinde, bunlar 50-300 um (Şekil 4) bir çapa sahip olan küreler genişletmek. Bazı organoids bazı küçük buddings geliştirecek,…

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…

Disclosures

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

  1. 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).
  2. Penkert, R. R., Kalejta, R. F. Human Embryonic Stem Cell Lines Model Experimental Human Cytomegalovirus Latency. mBio. 4 (3), e00298-13-e00298-13 (2013).
  3. Roelandt, P., et al. Human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis C virus. J Hepatol. 57 (2), 246-251 (2012).
  4. 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).
  5. 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).
  6. 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).
  7. 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).
  8. Ng, S., et al. Human iPSC-Derived Hepatocyte-like Cells Support Plasmodium Liver-Stage Infection In Vitro. Stem Cell Report. 4 (2), (2015).
  9. 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).
  10. Engevik, M. A., et al. Loss of NHE3 alters gut microbiota composition and influences Bacteroides thetaiotaomicron growth. AJP: GI. 305 (10), G697-G711 (2013).
  11. 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).
  12. McCracken, K. W., et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature. 516 (7531), 400-404 (2014).
  13. Bartfeld, S., et al. In Vitro Expansion of Human Gastric Epithelial Stem Cells and Their Responses to Bacterial Infection. Gastroenterology. 148 (1), (2014).
  14. Schlaermann, P., Toelle, B., et al. A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro. Gut. , (2014).
  15. Bertaux-Skeirik, N., et al. CD44 Plays a Functional Role in Helicobacter pylori-induced Epithelial Cell Proliferation. PLOS Pathogens. 11 (2), e1004663 (2015).
  16. Sato, T., et al. Single Lgr5 stem cells build crypt villus structures in vitro without a mesenchymal niche. Nature. 459 (7244), 262-265 (2009).
  17. 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).
  18. 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).
  19. Huch, M., et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature. 494 (7436), 247-250 (2013).
  20. Huch, M., et al. Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver. Cell. 160 (1-2), 299-312 (2015).
  21. Boj, S. F., et al. Organoid Models of Human and Mouse Ductal Pancreatic Cancer. Cell. 160 (1-2), 324-338 (2015).
  22. Karthaus, W. R., et al. Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell. 159 (1), 163-175 (2014).
  23. Bartfeld, S., et al. High-throughput and single-cell imaging of NF-kappaB oscillations using monoclonal cell lines. BMC cell. 11, 21 (2010).
  24. Blanchard, T. G., Nedrud, J. G. Laboratory Maintenance of Helicobacter Species. Curr Protoc Microbiol. , (2006).
  25. 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).
  26. 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).
  27. 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).
  28. 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).
  29. 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).
  30. Schwank, G., Andersson-Rolf, A., Koo, B. -. K., Sasaki, N., Clevers, H. Generation of BAC Transgenic Epithelial Organoids. PLoS ONE. 8 (10), e76871 (2013).
  31. Koo, B. -. K., et al. Controlled gene expression in primary Lgr5 organoid cultures. Nat Meth. 9 (1), 81-83 (2012).
  32. 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).
  33. 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).
  34. Van de Wetering, M., et al. Prospective derivation of a ‘Living Organoid Biobank’ of colorectal cancer patients. Cell. 161 (4), 933-945 (2015).

Play Video

Cite This Article
Bartfeld, S., Clevers, H. Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori. J. Vis. Exp. (105), e53359, doi:10.3791/53359 (2015).

View Video