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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