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Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.

ABSTRACT: Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

SUBMITTER: Huch M

PROVIDER: S-EPMC3801438 | biostudies-other | 2013 Oct

REPOSITORIES: biostudies-other

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Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.

Huch Meritxell M Bonfanti Paola P Boj Sylvia F SF Sato Toshiro T Loomans Cindy J M CJ van de Wetering Marc M Sojoodi Mozhdeh M Li Vivian S W VS Schuijers Jurian J Gracanin Ana A Ringnalda Femke F Begthel Harry H Hamer Karien K Mulder Joyce J van Es Johan H JH de Koning Eelco E Vries Robert G J RG Heimberg Harry H Clevers Hans H

The EMBO journal 20130917 20

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearan ...[more]

PMID: 24045232

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Project description:BackgroundPancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human. Mouse pancreatic organoids exhibit unlimited expansion potential, while previously reported human pancreas organoid (hPO) cultures do not expand efficiently long-term in a chemically defined, serum-free medium. We sought to generate a 3D culture system for long-term expansion of human pancreas ductal cells as hPOs to serve as the basis for studies of human pancreas ductal epithelium, exocrine pancreatic diseases and the development of a genomically stable replacement cell therapy for diabetes mellitus.ResultsOur chemically defined, serum-free, human pancreas organoid culture medium supports the generation and expansion of hPOs with high efficiency from both fresh and cryopreserved primary tissue. hPOs can be expanded from a single cell, enabling their genetic manipulation and generation of clonal cultures. hPOs expanded for months in vitro maintain their ductal morphology, biomarker expression and chromosomal integrity. Xenografts of hPOs survive long-term in vivo when transplanted into the pancreas of immunodeficient mice. Notably, mouse orthotopic transplants show no signs of tumorigenicity. Crucially, our medium also supports the establishment and expansion of hPOs in a chemically defined, modifiable and scalable, biomimetic hydrogel.ConclusionshPOs can be expanded long-term, from both fresh and cryopreserved human pancreas tissue in a chemically defined, serum-free medium with no detectable tumorigenicity. hPOs can be clonally expanded, genetically manipulated and are amenable to culture in a chemically defined hydrogel. hPOs therefore represent an abundant source of pancreas ductal cells that retain the characteristics of the tissue-of-origin, which opens up avenues for modelling diseases of the ductal epithelium and increasing understanding of human pancreas exocrine biology as well as for potentially producing insulin-secreting cells for the treatment of diabetes.

Dataset Information

Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.

Publications

Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.

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