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.
Highlights
As first demonstrated for intestinal crypts (Korinek et al, 1998), Wnt signalling plays a crucial role in the regulation of multiple types of adult stem cells and progenitors (Clevers and Nusse, 2012)
Wnt signalling and Lgr5 expression are upregulated during pancreas regeneration following partial duct ligation (PDL) We first sought to document Wnt pathway activation in normal adult pancreas and following acute damage
To assess whether the organoid cells are capable of differentiating towards fully mature endocrine lineages we developed a whole-organ morphogenetic assay based on the re-aggregation of dissociated cells from embryonic pancreas on one hand and organoids generated from adult pancreas on the other hand (Figure 6A; Supplementary Figure S7B)
Summary
As first demonstrated for intestinal crypts (Korinek et al, 1998), Wnt signalling plays a crucial role in the regulation of multiple types of adult stem cells and progenitors (Clevers and Nusse, 2012). It is fair to say that no robust, long-term culture system exists today that is capable of maintaining potent, clonal expansion of adult non-transformed pancreas progenitors over long periods of time under defined conditions. We have recently described a 3D culture system that allows long-term expansion of adult small intestine, stomach and liver cells without the need of a mesenchymal niche, while preserving the characteristics of the original adult epithelium (Sato et al, 2009; Barker et al, 2010; Huch et al, 2013). We exploit the Wnt-Lgr5-Rspo signalling axis to generate culture conditions that allow long-term expansion of adult pancreatic duct cells, which maintain the ability to differentiate towards both duct and endocrine lineages when provided the proper signals
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