Dr. Talita Resende - Organoids as an invitro model for enteric diseases
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1) The document discusses using intestinal organoids as an in vitro model to study enteric diseases in pigs. Organoids mimic intestinal tissue and present all organ-specific cell types. 2) The author's project uses mouse enteroids to study the pathogen Lawsonia intracellularis, which causes proliferative enteropathy in pigs. Preliminary results show the enteroids are permissive to L. intracellularis infection. 3) The goal is to further study the mechanisms of proliferation caused by L. intracellularis using the enteroid model to develop new treatment and control strategies for the disease. The author is also working to characterize and develop pig intestinal organoids for host-pathogen interaction studies.
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Dr. Talita Resende - Organoids as an invitro model for enteric diseases
- 1. Organoids as an in vitro model for enteric disease Talita Resende PhD candidate Advisors: Dr. Connie Gebhart & Dr. Fabio Vannucci Collaborator: Dr. Milena Saqui-Salces Allen D. Leman Swine Conference 2018
- 2. Enteric diseases – infectious agents Image adapted from: Holman, Devin B., et al MSystems 2.3 (2017): e00004-17.
- 3. Enteric diseases – infectious agents Image adapted from: Holman, Devin B., et al MSystems 2.3 (2017): e00004-17. PEDV TGEV PDCoV E. coli, enterotoxigenic Rotaviruses Brachyspira hyodysenteriae, B. hampsonii, B. pilosicoli Salmonella Typhimurium, Cholerasuis Lawsonia intracellularis Coccidiosis Trichuris Ascaris suis
- 4. Enteric diseases – infectious agents Images: Holman, Devin B., et al MSystems 2.3 (2017): e00004-17 Van den Bossche, L. (2017) (Doctoral dissertation, Ghent University)..
- 5. Host–pathogen interactions Intestine Dutta and Clevers, 2017 • Animal models Instrumental to host–microbe interactions • Well established • Uncontrollable microbial diversity Inter-species variances Expensive Ethical concerns yourgenome.org gdforum.freeforums.net zdmsociety.org https://www.diabeticool.com/
- 6. Host–pathogen interactions Intestine Dutta and Clevers, 2017 Pampaloni et al, 2007 • Single cell type cultures Closer to the ‘real situation’ • Well established • Relatively cheap • Lack the relationship between different cell types gdforum.freeforums.net zdmsociety.org Gebhart’s lab
- 7. • Organoids •Organotypic tridimensional (3D) structures •Matrix/scaffold based 3D in vitro culture systems –Supports the 3D growth •Originated from –Embryonic stem cells –Induced pluripotent cells –Primary tissues https://www.rndsystems.com cience.sciencemag.org Brain Intestine Liver Lung Host–pathogen interactions Dutta and Clevers, 2017 Pampaloni et al, 2007
- 8. • 3D in vitro/ex vivo models = Organoids • Present all organ-specific cell types on their surface Function as the tissue of origin • Self-renewed and self-organized • Reduce use of animals (1 animal = numerous organoids) Requires expertise to be established routinely Host–pathogen interactions Dutta and Clevers, 2017 Pampaloni et al, 2007
- 9. •Small intestine – enteroids •Colon - colonoids •Crypts and villi (small intestine) morphologically similar to the intestinal conformation •In vitro model with tissue-like morpho-physiology Intestinal organoids Saqui-Salces’ and Gebhart’s labs Fatehullah et al, 2016 Dutta and Clevers, 2017 https://www.rndsystems.com
- 11. • Some examples of applicability • Portal of entry • Shigella and M cells • Cellular modulation • Cryptosporidium parvum, Salmonella • Cytokine release and protein production • Clostridium difficile • Viral replication • Human Norovirus, Rotavirus • Non-infectious diseases: colorectal carcinoma Intestinal organoids - Host-pathogen interactions
- 12. • Cryptosporidium parvum (Zhang et al, 2016) • Inhibitory effect on the ex vivo propagation of enteroids from mice • Decreased expression level of intestinal stem cell markers Stem cells Intestinal organoids - Host-pathogen interactions
- 13. • Salmonella enterica Serovar Typhimurium • Cytokine release • Impact in production of α-defensin-5 (Crp5) Wilson et al, 2015 Forbester et al, 2015 Intestinal organoids - Host-pathogen interactions
- 14. • Rotavirus • Susceptibility to infection treatment efficacy tests Yin et al, 2017Yin et al, 2015 Mab “Antivirulence therapy” Intestinal organoids - Host-pathogen interactions
- 15. Our project
- 16. Our project albeitar.portalveterinaria.co m/ Vannucci & Gebhart • Proliferative enteropathy • Caused by Lawsonia intracellularis • Cellular proliferation – crypts • Unknown mechanism • Cell monocultures • Used over the years as in vitro models • Do not replicate cellular proliferation (Resende et al, 2018 – submitted for publication)
- 17. Supernatant (SN) Our project Media • Immunolabelling • RT-qPCR • Enteroid size (Area) L. intracellularis *Mouse enteroids
- 18. Apical side Lumen Basal DAPI L. intracellularis Nuclei L. intracellularis Our project Proliferation marker TAC marker Goblet cells marker
- 19. • Mouse enteroids are permissive to infection • Changes at 3dpi: area, Ki-67 expression, Muc2 expression • Check cellular proliferation, modulation of epithelial cells, apoptosis rate • Then what? • Initiate further research of the mechanisms of proliferation • New strategies for controlling the disease (antivirulence treatment, antibiotic alternatives) Our project
- 20. • Pig enteroids characterized (Gonzalez et al, 2013) • Nutrition investigation (Koltes and Gabler, 2015) • No host-pathogen interaction study to date • Where are we? • Successful development of pig enteroids • Currently: Characterization, evaluation of freezing- thawing effects • Collaboration with Dr. Milena Saqui-Salces Pig organoids? https://www.diabeticool.com/ HE PAS – globlet cells Ki-67 - proliferation
- 21. Final considerations • Intestinal organoids • The most promising “in vivo/ex vivo” model to investigate host-pathogen interactions • Closer to in vivo, but with controlled experimental conditions • Need to acquire expertise – new technique • Host-pathogen interactions of important enteropathogens in swine industry • Development of new strategies of treatment and control
- 22. • Dr. Milena Salqui-Salces – Ashley Manicor – Yue Guo – Lekha Medida • Dr. Gebhart’s lab – Lacey M. Lund – Erika Vasquez – Amanda Daniel • VDL Histo and IHC labs Acknowledgements Thank you! Questions? Talita Resende resen023@umn.edu Acknowledgements