will stem cells be a key resource in the future for regulating inflammation and treatment of ibd?...
TRANSCRIPT
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?
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
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 SW480
Advantages:Easy to cultureAbility to rapidly expand>21K papers in Pubmed
Disadvantages:AneuploidLimited ability to differentiate
Dark AgesCaco-2 HT-29 SW480
Advantages:Easy to cultureAbility to rapidly expand>21K papers in Pubmed
Disadvantages:AneuploidLimited ability to differentiate
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)
(Sato, T. et al. Nature 459, 262-266, 2009)
Organoids aka ‘miniguts’
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
Patient 1 Drug A = best response
Drug B = best response
Drug A & B = best response
Patient 2
Patient 3
Exhibit similar
symptoms or have same diagnosis
Development of in vitro Systems and Assays Is Critical for Individualized Medicine Approaches
Patient 1 Drug A = best
Drug B = best
Drug A & B = best
Patient 2
Patient 3
Collect sample
Cell-based assay
Inter-individual Variation in Intestinal Epithelial Cells
Epithelial Cell Function
Genetics
Epigenetics EnvironmentMicrobiome
Other host cells
Infection susceptibility
DrugresponseDisease
susceptibility
ExposomeGenome
ExposomeGenome
Hypothesis: Functional variation will be present in primary intestinal epithelial cell
lines from different individuals
Question: What is driving the inter-individual
variation observed for particular readouts?
Variations in epithelial cell functional readouts
Establishment of Human Intestinal Epithelial Cell Cultures
Proliferative Stem cells
Established primary cell line
1) Collagenase treatment
2) Filtration
Tissue culture plate well
Matrigel
Epithelial spheroid
lumen
Bar, 100 µm
Bar, 100 µm
Human Intestinal Epithelial Cell Cultures Can be Rapidly Expanded
50% L-WRN CMConditioned
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 dailyBar, 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
Stappenbeck labKelli VandussenHiroyuki MiyoshiClara MoonStacy RyuNaomi SonnekChristina HickeyHaerin JungGerard KaikoTa-Chiang LiuRC LaiChristine LuoNick ManieriNikki MalvinGreg RippbergerAshley SteedLulu SunMarta Wegorzewska
Matt CiorbaJeff Marinshaw
Phil TarrNurmohammad
Shaikh
Dave SibleyGeorgia Wilke
Skip VirginMegan BaldridgeDebbie Lenschow
Helen and David Piwnica-Worms
DDRCCRodney NewberryDarren Nix
Cedars-SinaiDermot McGovern
Harvard/Broad InstituteRamnik Xavier
GTACRich HeadMike HeinzChris Sawyer
Acknowledgements