This document provides an overview of a presentation on organoids in veterinary research and precision medicine. The following key points are discussed: 1. Organoids are 3D mini-organs derived from adult stem cells that maintain tissue microarchitecture and physiology. They provide advantages over 2D cell lines for research. 2. The presenter's work focuses on standardizing canine organoid technology and developing canine liver organoids for translational research applications. 3. Bladder cancer organoids derived from urine samples show potential for precision medicine by enabling personalized drug testing and diagnosis.

1. IDEXX
7/17/2023
ORGANOIDS IN VETERINARY
RESEARCH AND PRECISION
MEDICINE
VOJTECH GABRIEL http://www.vojtechgabriel.com

2. Outline
1.About me (5 min)
2.Organoids Introduction (5-10 min)
3.Standardization of Canine Organoid Technology
and Canine Liver Organoid Development (20 min)
4. Bladder Cancer Organoids for Precision
(Personalized) Medicine (10 min)
5. Side Project Organoids (2 min)
6. Questions

3. University of Veterinary
Sciences Brno
2012 – 2018
DVM

4. Small
Animal
Clinic
• Veterinarian
• Lecturer
(Internal
Medicine)

5. Gastroenterolog
y
Endoscopy
2018 - 2019

6. Iowa State
University
• PhD
• 2020-2023
• Biomedical
Sciences
• Cell Biology
• Stem Cell
Research

7. CO-MAJOR PROFESSORS
Karin Allenspach
European College of Veterinary
Internal Medicine (ECVIM)
President
Jonathan P. Mochel
European Association for Veterinary
Pharmacology and Toxicology
(EAVPT)
President

8. SMART Lab
• Canine Organoids
• Originally intestine
• First project liver
• Rapid expansion to
different fields in
last few years

9. Organoids
• Mini-organs
• 3D
• Microarchitecture
• Physiology
• Adult Stem Cell (AdSCs)
• Extracellular Matrix
• Growth Factor

10. 2D Cell Lines
• Easy to Culture
• Low-cost Maintenance
• Standardization
• Changed gene expression
• Monocultures (Goblet cells)
• Lack of receptors (Caco2)

12. 6. Differentiation of
hepatocyte-like cells
Standardization of
Canine Organoid
Technology and Canine
Liver Organoid
Development

13. Reasoning
• Lack of standardization
• Interlaboratory variability
• Different results
• Intestinal and hepatic organoids
• Organoid culture techniques
• Isolation, maintenance, and harvest

16. ARTICLE
AND
VIDEO

17. LIVER
ORGANOID
PROBLEM
WE HAVE:
Bile Duct Cells (Cholangiocytes)
3-5% of cells
WE WANT:
Hepatocyte-like cells (some
properties of hepatocytes)

18. Partial functional
differentiation
“…these in vitro functions remained less pronounced than those of freshly
isolated hepatocytes.”
- Huch et al. (2013)
”The inability to induce full hepatocyte maturation in vitro is well known in
the field…”
- Nantasanti et al. (2015)
”…they (hepatic organoids) did not reach full maturation…”
- Kruitwagen et al. (2017)

20. Differentiation media components
Bone morphogenetic
protein 7 (BMP7)
Fibroblast Growth
Factor 19 (FGF19)
Dexamethasone
(Dex)
Gamma-Secretase
Inhibitor IX
(DAPT)

21. 20 media compositions
Encompass all combinations of 4 components
4 control groups

22. ORGANOID ALBUMIN PRODUCTION

23. RNASCOPE (RNA-ISH)

24. M01 Undifferentiated
LGR5 (stem cell marker)

25. M01 SOX9+ Undifferentiate
d
SOX9 (ductal cell marker)
M01 SOX9-

26. RNASCOPE: XENOBIOTIC METABOLISM

27. M01 Undifferentiated
CYP2B11 (metabolic marker) / CYP2B6

28. M01 Undifferentiated
CYP3B12 (metabolic marker)

29. P-glycoprotein Expression
• Primarily in cholangiocytes (control)
• Less in hepatocytes

31. Organ-on-a-chip

32. Canine Organ-on-a-chip
First Description and Preliminary
Characterization of a Microfluidic
Canine Liver-on-a-Chip
ECVIM 2021
Narrated presentation
• Homemade chip (<$10)
• Growth w/o Matrigel

33. 6. Differentiation of
hepatocyte-like cells
Bladder Cancer
Organoids for
Precision
(Personalized)
Medicine

34. First Isolation
of Canine
Healthy
Bladder
Organoids
ResearchGate

35. “THE POWER OF
A URINE SAMPLE”
• Veterinary oncology
expanding
• Chemotherapeutics
effectiveness
- highly subjective to the
animal
• The Targeted Disease:
Transitional Cell Carcinoma
https://www.neighborhoodvets.com/services/other/blog/small-sample-big-results-
understanding-your-senior-pets-urinalysis

36. How does it work?

37. Urinary-Derived 3D Carcinoma Organoids: A Promising Tool for the Diagnosis and Individualized Therapy of
Bladder Cancer
ISSCR 2022
POSTER

38. Mayo Clinic
Collaboration
• Muscle Invasive Bladder Cancer
• Derived from urine of human
patients
Using canine organoids to advance therapeutic drug development in
bladder cancer: A one health approach
ISSCR 2022

39. 6. Differentiation of
hepatocyte-like cells
Additional side
project
organoids

40. Turtle organoids
Supercooling
Anoxic/Hypoxic
Water contamination (metals)

41. And other organoids…
• Tenrec organoids
• Squirrel organoids
• Snake Venom Gland organoids
• Stony corral organoids
• Bovine organoids
• Porcine organoids
• Parasite organoids

42. PUBLICATIONS (NEWEST TO OLDEST) PT.1
 Synbiotic-IgY Therapy Modulates the Mucosal Microbiome and Inflammatory Indices in Dogs
with Chronic Inflammatory Enteropathy: A Randomized, Double-Blind, Placebo-Controlled
Study Sahoo, D.K., Allenspach, K., Mochel, J.P.,Parker, V., Rudinsky A.J., Winston, J.A.,
Bourgouis-Mochel, A., Ackermann, M., Heilmann, R.M., Koller, G., Yuan, L., Stewart, T.,
Morgan, S., Scheunemann, K.R., Iennarella-Servantez, C., Gabriel, V., Zdyrski, C., Pilla,
R., Suchodolski, J.S., Jergens, A.E., Vet. Sci. 2023, 10(1), 25;
doi:10.3390/vetsci10010025
 Canine Intestinal Organoids: A Novel In Vitro System to Model Intestinal Permeability of
Therapeutic Drugs in Preclinical Drug Discovery and Animal Health Sahoo, D.K., Martinez,
M.N., Dao, K., Gabriel, V., Zdyrski, C., Atherly, T., Iennarella-Servantez, C., Jergens,
A.E., Burns, L.E., Schrunk, D., Volpe, D.A., Allenspach, K., Mochel, J.P., Scientific
Reports in review
 Dose-response of benazepril on biomarkers of the classical and alternative pathways of
the renin-angiotensin-aldosterone system in dogs Sotillo, S., Ward, J.L., Guillot, E.,
Domenig, O., Yuan, L., Smith, J.S., Gabriel, V., Iennarella-Servantez, C.A., Mochel,
J.P., Scientific Reports in review
 Establishment and Characterization of Novel Canine Organoids with Organ Specific
Physiological Similarity Zdyrski*, C., Gabriel*, V., Ospina, O., Wickham, H., Sahoo,
D.K., Dao, K., Aguillar Meza, L.S., Bedos, L., Honold, S., Piñeyro, P., Mochel, J.P.,
Allenspach, K., eLife in review

43. PUBLICATIONS (NEWEST TO OLDEST) PT.2
 Culture and Characterization of Canine and Feline Corneal Epithelial Organoids: A New
Tool for the Study and Treatment of Corneal Diseases Bedos, L., Wickham, H., Gabriel,
V., Zdyrski, C., Allbaugh R.A., Sahoo D.K., Sebbag, L., Mochel, J.P., Allenspach K.,
Front. Vet. Sci. - Veterinary Pharmacology and Toxicology
 Ketogenic diets in healthy dogs induce gut and serum metabolome changes suggestive of
anti-tumourigenic effects: A model for human ketotherapy trials Allenspach, K.,
Borcherding, D.C., Iennarella-Servantez, C.A., Mosichuk, A.P., Atherly, T., Sahoo, D.,
Kathrani, A., Suchodolski, J.S., Bourgois-Mochel, A., Serao, M.R., Serao, N.V., Willette
A., Perez, B.A., Gabriel, V., Mao, S., Kilburn, L., Dang, V., Borts, D., Almada, L.L.,
Fernandez-Zapico, M.E., Philips, G.J., Jergens, A.E., Mochel, J.P. Clinical and
Translation Medicine, 2022, v. 12, i. 9, doi:10.1002/ctm2.1047
 Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures
for Use in Biomedical Research Gabriel, V., Zdyrski, C., Sahoo, D. K., Dao, K.,
Bourgois-Mochel, A., Kopper, J., Zeng, X. L., Estes, M. K., Mochel, J. P., Allenspach,
K. J. Vis. Exp. (179), e63515, doi:10.3791/63515 (2022).
 Canine Intestinal Organoids in a Dual-Chamber Permeable Support System. Gabriel, V.,
Zdyrski, C., Sahoo, D. K., Dao, K., Bourgois-Mochel, A., Atherly, T., Martinez, M. N.,
Volpe, D. A., Kopper, J., Allenspach, K., Mochel, J. P. J. Vis. Exp. (181), e63612,
doi:10.3791/63612 (2022).

44.  The role of Campylobacter spp. in chronic enteropathy in dogs. Vavra M., Borilova G.,
Fusek M., Gabriel V., Ceplecha V., Skoric M., Crha M. Acta Vet. Brno 2019, 88: 341-347,
doi:10.2754/avb201988030341
 Causes of lower urinary tract disease in Czech cat population. Hribova B., Ceplecha V.,
Rehakova K., Proks P., Gabriel V., Kohoutova L., Crha M. Acta Vet. Brno 2019, 88: 433-
441, doi:10.2754/avb201988040433
PUBLICATIONS (NEWEST TO OLDEST) PT.3

45. ABSTRACTS (NEWEST TO OLDEST) PT.1
 ACVIM 2022, 6/24/2022, Allenspach K., Mochel J.P., Bourgois-Mochel A., Sahoo D.,
Zdyrski C., Gabriel V., Mao S., Iennarella-Servantez C., Lingnan Y., Liu Y., Rudinsky
A., Winston J., Parker V., Jergens A. “Efficacy of a synbiotic-IgY supplement in
treatment of canine inflammatory bowel disease – clinical observations”
 ACVIM 2022, 6/24/2022, Gabriel V., Livania V., Diaz-Reganon D., Liu D., Meyerholtz
D.K., Mochel J.P., Allenspach K. “Decreased Duodenal Enterocyte Height and Width is a
Hallmark of Chronic Enteropathy in Dogs”
 ISSCR 2022, 6/16/2022 Mochel J.P., Sahoo D.K., Zdyrski C., Wickham H.M., Gabriel V.,
Bourgois-Mochel A., Lucien F., Frank I., Cheville J., Allenspach K. “Urinary-Derived 3D
Carcinoma Organoids: A Promising Tool for the Diagnosis and Individualized Therapy of
Bladder Cancer”
 ISSCR 2022, 6/15/2022 Mochel J.P., Mosichuk A., Sahoo D.K., Wickham H.M., Gabriel V.G.,
Zdyrski C., Bourgois-Mochel A., Trepanier L., Thomson C., Lucien-Matteoni F., Frank I.,
Cheville J., Allenspach K. “Using Canine Organoids to Advance Therapeutic Drug
Development in Bladder Cancer: A One Health Approach”
 ISSCR 2021, 6/21/2021 Gabriel V., Zdyrski C.H., Dao K., Sahoo D.K., Iennarella-
Servantez C., Bourgois-Mochel A., Jergens A.E., Mochel J.P., Allenspach K. “Preliminary
Characterization of Canine Hepatic Spheroids: A Potential Model for Translational
Research in Gastroenterology”

46. ABSTRACTS (NEWEST TO OLDEST) PT.2
 ECVIM 2021, 9/1/2021 Gabriel V., Mochel J.P., Mao S., Zdyrski C.H., Sahoo D.K.,
Iennarella-Servantez C., Bourgois-Mochel A., Thenuwara S., Dao K., Bedos L., Que L.,
Jergens A.E., Allenspach K. “First Description and Preliminary Characterization of a
Microfluidic Canine Liver-on-a-Chip”
 DDW 2021, 5/21/2021 Zdyrski C., Iennarella-Servantez C., Sahoo D.K., Ward J., Long E.,
Gabriel V., Minkler S., Mao S., Bourgois-Mochel A., Jergens A., Allenspach K., Mochel
J.P. “Homology Directed Repair in Canine Duodenal Enteroids to Mimic the Wild-Type P-
Glycoprotein Mutation” Gastroenterology Volume 160, Issue 6, Supplement, May 2021,
Pages S-625-S-626 doi: 10.1016/S0016-5085(21)02200-9
 ECVIM 2020, 9/2/2020 Gabriel V., Iennarella-Servantez C.A., Atherly T., Minkler S., Mao
S., Colosimo M., Kurr L., Borcherding D., Thenuwara S., Bourgois- Mochel A., Jergens
A.E., Mochel J.P., Allenspach K. “Culture and Maintenance of Canine Urinary Bladder
organoids and Hepatic Organoids” doi:.1111/jvim.15924
 ECVIM 2020, 9/2/2020 Allenspach K., Suchodolski J., Gabriel V., Iennarella-Servantez
C., Atherly T., Borcherding D., Ambrosini Y.M., Jergens A.E., Kilburn L.R., Bourgois-
Mochel A., Rossoni-Serrao M., Mochel J. “Short-term feeding with high-fat diet induces
dysbiosis-associated changes of fecal metabolites consistent with changes in serum
metabolomics in dogs” doi: 10.1111/jvim.15924
 ECVIM 2020, 9/2/2020 Iennarella-Servantez C., Gabriel V., Atherly T., Minkler S.,
Thenuwara S., Mao S., Colosimo M., Kurr L., Borcherding D., Bourgois-Mochel A., Jergens
A.E., Allenspach K., Mochel J.P. “Collection, Culture, and Characterization of Canine
Urothelial Carcinoma Organoids: Reverse Translational Clinical Research in the

47.  DDW 2020, 5/2/2020 Borcherding D.C., Atherly T., Bourgois-Mochel A., Searo M.R., Kilburn L.,
Ambrosini Y.M. Perez B.A., Gabriel V., Iennarella-Servantez C.A, Jergens A., Mochel J.P.,
Allenspach K. “Polyphenols Reverse the Pathologic Effects of Palmitic Acid and High Fat Diet
in Canine Enteroids” Gastroenterology May 2020 Volume 158 Issue 6 Supplement 1S-1-S-1603 doi:
10.1016/S0016-5085(20)31893-X
 ASAS Annual Meeting 2021, 7/14/2022 Iennarella-Servantez C., Kathrani A., Sahoo D.K., Long E.,
Zdyrski C., Gabriel V., Mao S., Bourgois-Mochel A., Resop M., Rund L., Rossoni-Searao M.C.,
Jergens A., Mochel J.P., Allenspach K., “Diet-induced Clinical Responsiveness of Translational
Dog Model for Human Western Diet (WD)-related Disease Research” Journal of Animal Science,
Volume 99, Issue Supplement_3, November 2021, Pages 58–59, doi: 10.1093/jas/skab235.104
ABSTRACTS (NEWEST TO OLDEST) PT.3

48. THANK YOU
ANY QUESTIONS?