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Will stem cells be a key resource in
the future for regulating
inflammation and treatment of IBD?
Thaddeus Stappenbeck MD, PhD
Will stem cells graduate and move
down the hall to the ‘big’ room?
Thaddeus Stappenbeck MD, PhD
Will stem cells graduate and move
down the hall to the ‘big’ room?
1) Cellular therapy – addition of
stem cells or their progeny
2) Use of cells for patient
stratification
Will stem cells graduate and move
down the hall to the ‘big’ room?
1) Cellular therapy – addition of
stem cells or their progeny –
HSCs, MSCs, epithelial stem
cells, iPSCs
2) Use of cells for patient
stratification
Direct incorporation of epithelial
stem cells
Mesenchymal stem cells
Direct incorporation of
hematopoietic stem cells
iPS cells
Will stem cells graduate and move
down the hall to the ‘big’ room?
1) Cellular therapy – addition of
stem cells or their progeny
2) Use of cells for patient
stratification – epithelial stem
cells from patients
Dark Ages (1950s-2008)
Caco-2
HT-29
Advantages:
Easy to culture
Ability to rapidly
expand
>21K papers in
Pubmed
SW480
Disadvantages:
Aneuploid
Limited ability to
differentiate
Dark Ages
Caco-2
HT-29
Advantages:
Easy to culture
Ability to rapidly
expand
>21K papers in
Pubmed
SW480
Disadvantages:
Aneuploid
Limited ability to
differentiate
Renaissance (2008-current)
Renaissance (2008-2013)
Critical factors for in vitro crypt culture
primary cells
Wnt ligands (e.g. Wnt3a)
R-spondin (e.g. Rspo1)
Noggin (BMP inhibitor)
Organoids aka ‘miniguts’
(Sato, T. et al. Nature 459, 262-266, 2009)
Expanded Platforms
• 3D - Organoid (stem cells and
multilineage)
• 3D - Spheroid (stem cell enriched)
• 2D – sheet of differentiated
• Co-culture pathogens
• Co-culture anaerobic commensals
• Co-culture with mesenchymal cells
Industrial Revolution
Individualized Medicine
Oootani et al., Nat Med 15, 701-6 (2009)
Miyoshi et al., Nat Prot (2013)
Wang et al., Gastroenterology (2013)
Yin et al., Nat Methods (2013)
The Goal of Understanding Inter-Individual
Variation: Personalized Medicine
Exhibit
similar
symptoms or
have same
diagnosis
Patient 1
Drug A = best response
Patient 2
Drug B = best response
Patient 3
Drug A & B = best response
Development of in vitro Systems and Assays Is
Critical for Individualized Medicine Approaches
Patient 1
Collect
sample
Cell-based
assay
Drug A = best
Patient 2
Drug B = best
Patient 3
Drug A & B = best
Inter-individual Variation in Intestinal
Epithelial Cells
Microbiome
Epigenetics
Environment
Genetics
Genome
Exposome
Other host
cells
Epithelial Cell Function
Infection
susceptibility
Disease
susceptibility
Drug
response
Hypothesis: Functional variation will be
present in primary intestinal epithelial cell
lines from different individuals
Variations in epithelial cell
functional readouts
Question: What is driving
the inter-individual
variation observed for
particular readouts?
Genome
Exposome
Establishment of Human Intestinal
Epithelial Cell Cultures
1) Collagenase
treatment
2) Filtration
Proliferative
Stem cells
Established
primary cell line
Tissue culture
plate well
Bar, 100 µm
Matrigel
Epithelial
spheroid
lumen
Bar, 100 µm
Human Intestinal Epithelial Cell
Cultures Can be Rapidly Expanded
50% L-WRN CM
Conditioned
medium
containing stem
cell growth
factors
lumen
Spheroids are enriched
for stem cells in
50% L-WRN CM
Intestinal stem cell marker
Same spheroid imaged daily
Bar, 100 µm
Generation of a Biobank of Human
Intestinal Epithelial Cell Lines
• 153 cell lines generated to date
• Includes 45 lines from other
IBD centers (Cedars-Sinai,
Harvard)
• 105 individuals with different
genetic landscapes
• No disease and disease
Human Epithelial Cells Form a Differentiated,
Polarized Monolayer on Transwell Membranes
Monolayers contain the major
differentiated cell types of the
intestinal epithelium
• Lines can be established within 2-3 weeks
• Epithelial cells can be rapidly expanded as
stem cells or differentiated
• Epithelial cells can form a polarized monolayer
covered by a mucus layer
• Allows for repeated experiments
Acknowledgements
Stappenbeck lab
Kelli Vandussen
Hiroyuki Miyoshi
Clara Moon
Stacy Ryu
Naomi Sonnek
Christina Hickey
Haerin Jung
Gerard Kaiko
Ta-Chiang Liu
RC Lai
Christine Luo
Nick Manieri
Nikki Malvin
Greg Rippberger
Ashley Steed
Lulu Sun
Marta Wegorzewska
Matt Ciorba
Jeff Marinshaw
Phil Tarr
Nurmohammad
Shaikh
Dave Sibley
Georgia Wilke
Skip Virgin
Megan Baldridge
Debbie Lenschow
Helen and David
Piwnica-Worms
DDRCC
Rodney Newberry
Darren Nix
Cedars-Sinai
Dermot McGovern
Harvard/Broad Institute
Ramnik Xavier
GTAC
Rich Head
Mike Heinz
Chris Sawyer