Organotypic Model Systems for Examining

Host-Microbe Interactions

Vincent B. Young, MD/PhD

Department of Internal Medicine/Infectious Diseases
Department Microbiology & Immunology
University of Michigan Medical School, Ann Arbor

Clostridium difficile
Anaerobic, gram-positive,
spore-forming, toxin
producing bacterium
Spores: environmentally
stable reservoir of the
organism
Can be detected in a
small fraction of the
healthy population
hastane.harran.edu.tr/enfek/gastroente.htm

Britton and Young (2014)

Gastroenterology 146:1547-1553

Murine models of
C. difficile infection

Gut Microbes (2011) 2:145

Infect Immun (2012) 80:3786

Nat Comms (2014) DOI: 10.1038

Infect Immun (2015) 83:3838

How to isolate the host side of the

symbiosis: organoids

Leslie et al. (2015) Infect Immun

2015 83(1):138-45

Jhansi Leslie

Denovo
differentiation

iPSreprogramming
YamanakaFactors

HumanIntestinalOrganoid(HIO)
Mesenchyme+Epithelium
Simplegrowthconditions
(ENR)

biopsy

Enteroid

Epitheliumonly
Complexgrowthconditions
(ENRW,p38i,TGFi,etc)
Satoetal.,Gastroenterology,2011

James M. Wells and Jason R. Spence; Development 2014;141:752-760

HIOs contain major cell types of

the small intestine

Spence, JR et al.; Nature. 2011; 470:105-9

Organoids as an alternative?
Replacement, Reduction, Refinement

Do HIOs have a functional epithelial

barrier?
Nuclei/DPP4/ZO1/Ecad

Fluorescent 4kDa dextran (FD4)

Barrier permeability
assay

2h

10h

12h

14h

+EGTA

Control

+EGTA
Leslie et al. (2015) Infect Immun
2015 Jan;83(1):138-45

16h

18h

Pruitt & Lacy (2012)

Front Cell Infect Microbiol 2:28

Luminal TcdA and TcdB disrupt barrier function

2h

10h

12h

+EGTA

Control

+TcdB

+TcdA

+EGTA

Fluorescent 4kDa dextran (FD4)

+
purified C. difficile TcdA or TcdB

14h

16h

18h

Control

TcdA

TcdB

Toxigenic C.difficile strains disrupt

the HIO epithelial barrier!
Fluorescent 4kDa dextran (FD4)
+
vegetative C. difficile

C. difficile bacilli and spores within

the lumen of infected organoids

But wait a minute: Didnt you tell

me that C. difficile is an anaerobe?
Interesting observation: in one experiment, there actually
appeared to be growth (multiplication) of C. difficile within
organoids
Upon further investigation, appears that the microinjection
system was contaminated with E. coli
Could E. coli alter the environment in a way to permit C.
difficile growth?

E. coli and modulation of

luminal oxygen tension
David Hill

Hill et al. (unpublished data)

Finkbeiner, Hill et al. (2015) Stem Cell Rep doi: 10.1016/j.stemcr.2015.04.010

HIO epithelial gene expression

resembles the fetal intestine

Finkbeiner, Hill et al. (2015) Stem Cell Rep doi: 10.1016/j.stemcr.2015.04.010

HIO implantation in kidney capsule of

NOD-scid IL2Rgammanull (NSG) mice

Finkbeiner, Hill et al. (2015) Stem Cell Rep doi: 10.1016/j.stemcr.2015.04.010

E. coli and modulation of

luminal oxygen tension

Hill et al. (unpublished data)

E. coli induces enhances mucus

expression in HIOs

E. coli alters HIO transcription

Challenges & The Future

of Organoid Research
Understand how oxygen can be
monitored/manipulated (anaerobes)
Need to focus on both beneficial
(mutualistic) and pathogenic interactions
Engineering organoids: luminal flow, 2D
vs 3D, mechanical stress
Increasing complexity: immune
components, enteric nervous system,
complex microbial communities (how far
do we go?)

Acknowledgments
NAMSED investigators: Jason Spence,
Sha Huang, David Hill, Stephanie Spohn,
Jhansi Leslie, Mary ORiordan, Christiane
Wobus, Shuichi Takayama, Robert
Britton, Eric Martens
The Young Lab: Jhansi Leslie, Angela
Hopkinson, Casey Theriot, Judy Opp,
Katherine Wozniak, Mark Koenigsknecht,
David Hill, Anna Seekatz

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