Synthetic Hydrogels for Human Intestinal Organoid Generation and Colonic Wound Repair

In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multicellular three-dimensional structures capable of giving rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumour-derived extracellular matrix (ECM), which severely limits the use of organoid technologies for regenerative and translational medicine. Here, we developed a fully defined, synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer that supports robust and highly reproducible in vitro growth and expansion of HIOs, such that three-dimensional structures are never embedded in tumour-derived ECM. We also demonstrate that the hydrogel serves as an injection vehicle that can be delivered into injured intestinal mucosa resulting in HIO engraftment and improved colonic wound repair. Together, these studies show proof-of-concept that HIOs may be used therapeutically to treat intestinal injury.

[1]  Gail H Deutsch,et al.  In vitro generation of human pluripotent stem cell derived lung organoids , 2015, eLife.

[2]  R. Fässler,et al.  Genetic analyses of integrin signaling. , 2011, Cold Spring Harbor perspectives in biology.

[3]  Philippe Jay,et al.  DCAMKL-1 expression identifies Tuft cells rather than stem cells in the adult mouse intestinal epithelium. , 2009, Gastroenterology.

[4]  Nick Barker,et al.  Organoids as an in vitro model of human development and disease , 2016, Nature Cell Biology.

[5]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[6]  Hans Clevers,et al.  Designer matrices for intestinal stem cell and organoid culture , 2016, Nature.

[7]  H. Kleinman,et al.  Laminin-1 and Laminin-2 G-domain Synthetic Peptides Bind Syndecan-1 and Are Involved in Acinar Formation of a Human Submandibular Gland Cell Line* , 1998, The Journal of Biological Chemistry.

[8]  Jason R Spence,et al.  How to make an intestine , 2014, Development.

[9]  O. Farokhzad,et al.  Annexin A1-containing extracellular vesicles and polymeric nanoparticles promote epithelial wound repair. , 2015, The Journal of clinical investigation.

[10]  G. Kang,et al.  Distribution of intestinal stem cell markers in colorectal precancerous lesions , 2016, Histopathology.

[11]  Shuh Narumiya,et al.  Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension , 1997, Nature.

[12]  G. Lajoie,et al.  Matrigel: A complex protein mixture required for optimal growth of cell culture , 2010, Proteomics.

[13]  Christopher T. Johnson,et al.  Synthetic matrices reveal contributions of ECM biophysical and biochemical properties to epithelial morphogenesis , 2016, The Journal of cell biology.

[14]  Hans Clevers,et al.  A functional CFTR assay using primary cystic fibrosis intestinal organoids , 2013, Nature Medicine.

[15]  V. Young,et al.  Persistence and Toxin Production by Clostridium difficile within Human Intestinal Organoids Result in Disruption of Epithelial Paracellular Barrier Function , 2014, Infection and Immunity.

[16]  S. Ichinose,et al.  Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell , 2012, Nature Medicine.

[17]  W. R. Taylor,et al.  Vasculogenic bio-synthetic hydrogel for enhancement of pancreatic islet engraftment and function in type 1 diabetes. , 2013, Biomaterials.

[18]  Elizabeth E. Hoskins,et al.  Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro , 2010, Nature.

[19]  Richard W. Farndale,et al.  Structure of the Integrin α2β1-binding Collagen Peptide , 2004 .

[20]  E. White,et al.  Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids , 2015, Biology Open.

[21]  B. Thiers.  Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors , 2008 .

[22]  H. Clevers,et al.  Identification of stem cells in small intestine and colon by marker gene Lgr5 , 2007, Nature.

[23]  Hans Clevers,et al.  Modeling Development and Disease with Organoids , 2016, Cell.

[24]  O. Klein,et al.  Transcriptome-wide Analysis Reveals Hallmarks of Human Intestine Development and Maturation In Vitro and In Vivo , 2015, Stem cell reports.

[25]  J. Wells,et al.  Generating human intestinal tissue from pluripotent stem cells in vitro , 2011, Nature Protocols.

[26]  Adrian Ranga,et al.  Defined three-dimensional microenvironments boost induction of pluripotency. , 2016, Nature materials.

[27]  Hans Clevers,et al.  Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver , 2015, Cell.

[28]  H. Clevers,et al.  Characterizing responses to CFTR-modulating drugs using rectal organoids derived from subjects with cystic fibrosis , 2016, Science Translational Medicine.

[29]  Hans Clevers,et al.  OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells. , 2009, Gastroenterology.

[30]  Richard W Farndale,et al.  Structure of the integrin alpha2beta1-binding collagen peptide. , 2004, Journal of molecular biology.

[31]  G. Daley,et al.  Use of differentiated pluripotent stem cells in replacement therapy for treating disease , 2014, Science.

[32]  A. McCulloch,et al.  Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes , 2009, PloS one.

[33]  S. Yamanaka,et al.  A developmental framework for induced pluripotency , 2015, Development.

[34]  Alyssa J. Miller,et al.  A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids , 2016, eLife.

[35]  H. Kleinman,et al.  Laminin-111-derived peptides and cancer , 2013, Cell adhesion & migration.

[36]  M. Campone,et al.  A Quantitative Proteomic Approach of the Different Stages of Colorectal Cancer Establishes OLFM4 as a New Nonmetastatic Tumor Marker* , 2011, Molecular & Cellular Proteomics.

[37]  G. Daley,et al.  The promise of induced pluripotent stem cells in research and therapy , 2012, Nature.

[38]  Timothy J Mitchison,et al.  Dissecting Temporal and Spatial Control of Cytokinesis with a Myosin II Inhibitor , 2003, Science.

[39]  Jay C. Sy,et al.  Maleimide Cross‐Linked Bioactive PEG Hydrogel Exhibits Improved Reaction Kinetics and Cross‐Linking for Cell Encapsulation and In Situ Delivery , 2012, Advanced materials.