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-2HT-29SW480 Advantages: Easy to culture Ability to rapidly expand >21K papers in Pubmed Disadvantages: Aneuploid Limited ability to differentiate

Dark Ages Caco-2HT-29SW480 Advantages: Easy to culture Ability to rapidly expand >21K papers in Pubmed Disadvantages: Aneuploid Limited ability to differentiate

Renaissance (2008-current )

Renaissance ( ) 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, , 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, (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 EpigeneticsEnvironment Microbiome Other host cells Infection susceptibility Drug response Disease susceptibility ExposomeGenome

Exposome Genome 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

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

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 Acknowledgements