Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation

Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol.

[1]  H. Clevers,et al.  Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors , 2012, Nature.

[2]  K. Zaret.  Genetic programming of liver and pancreas progenitors: lessons for stem-cell differentiation , 2008, Nature Reviews Genetics.

[3]  Hans Clevers,et al.  Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis , 2013, The EMBO journal.

[4]  M. Dickie,et al.  Obese, a new mutation in the house mouse. , 1950, The Journal of heredity.

[5]  M. Grompe,et al.  Stem cells and liver regeneration. , 2009, Gastroenterology.

[6]  M. Yarmush,et al.  Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. , 2002, Biophysical chemistry.

[7]  H. Clevers,et al.  Controlled gene expression in primary Lgr5 organoid cultures , 2011, Nature Methods.

[8]  Michael Schumacher,et al.  Modeling human development and disease in pluripotent stem cell-derived gastric organoids , 2014, Nature.

[9]  T. Adachi,et al.  Self-organizing optic-cup morphogenesis in three-dimensional culture , 2011, Nature.

[10]  Takanori Takebe,et al.  Vascularized and functional human liver from an iPSC-derived organ bud transplant , 2013, Nature.

[11]  M. Grompe,et al.  Generation and Regeneration of Cells of the Liver and Pancreas , 2008, Science.

[12]  Toshiro Sato,et al.  Efficient genetic engineering of human intestinal organoids using electroporation , 2015, Nature Protocols.

[13]  K. Zaret,et al.  Hex homeobox gene controls the transition of the endoderm to a pseudostratified, cell emergent epithelium for liver bud development. , 2006, Developmental biology.

[14]  T. He,et al.  Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids , 2014, PloS one.

[15]  A. Zorn,et al.  Liver development , 2008 .

[16]  L. Donehower,et al.  Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours , 1992, Nature.

[17]  B. Trump,et al.  Mouse liver cell culture , 1981, In Vitro.

[18]  B. Koo,et al.  A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture , 2014, Journal of visualized experiments : JoVE.

[19]  Hans Clevers,et al.  In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration , 2013, Nature.

[20]  K. Kinzler,et al.  Constitutive Transcriptional Activation by a β-Catenin-Tcf Complex in APC−/− Colon Carcinoma , 1997, Science.

[21]  Hans Clevers,et al.  Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. , 2013, Cell stem cell.

[22]  Thomas Gridley,et al.  A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency. , 2002, Development.

[23]  Hans Clevers,et al.  Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. , 2010, Cell stem cell.

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

[25]  H. Edlund,et al.  The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. , 1996, Development.

[26]  E. Kroon,et al.  Production of pancreatic hormone–expressing endocrine cells from human embryonic stem cells , 2006, Nature Biotechnology.

[27]  D. Stolz,et al.  Histological organization in hepatocyte organoid cultures. , 2001, The American journal of pathology.

[28]  M. Spector,et al.  Organoid Models of Human and Mouse Ductal Pancreatic Cancer , 2015, Cell.

[29]  Madeline A. Lancaster,et al.  Cerebral organoids model human brain development and microcephaly , 2013, Nature.

[30]  H. Clevers,et al.  Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche , 2009, Nature.

[31]  Hans Clevers,et al.  Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling , 2011, Nature.

[32]  G. R. van den Brink,et al.  A protocol for lentiviral transduction and downstream analysis of intestinal organoids. , 2015, Journal of visualized experiments : JoVE.

[33]  S. Duncan,et al.  Embryonic development of the liver , 2005, Hepatology.

[34]  M. Sander,et al.  Pancreas organogenesis: from lineage determination to morphogenesis. , 2013, Annual Review of Cell and Developmental Biology.

[35]  S. Glaser,et al.  cAMP stimulates the secretory and proliferative capacity of the rat intrahepatic biliary epithelium through changes in the PKA/Src/MEK/ERK1/2 pathway. , 2004, Journal of hepatology.

[36]  A. Ranga,et al.  Artificial three-dimensional niches deconstruct pancreas development in vitro , 2013, Development.

[37]  J. Jonkers,et al.  Mouse models of BRCA1 and BRCA2 deficiency: past lessons, current understanding and future prospects , 2006, Oncogene.

[38]  D. Melton,et al.  Generation of Functional Human Pancreatic β Cells In Vitro , 2014, Cell.

[39]  B. Trump,et al.  Mouse liver cell culture , 1981, In Vitro.

[40]  D. Ledbetter,et al.  Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. , 1989, Science.

[41]  Bon-Kyoung Koo,et al.  Modeling mouse and human development using organoid cultures , 2015, Development.

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

[43]  Link,et al.  Mouse models of BRCA 1 and BRCA 2 deficiency : past lessons , current understanding and future prospects , 2022 .