Obtaining, growing, and analysis of pancreatic progenitor cells. Adult stem and progenitor cells have been successfully used for cell-based therapies such as transplantation of hematopoietic stem cells for various diseases. Whether stem and progenitor cells in the adult pancreas can be identified and used for replacement therapy has been a highly controversial topic. To address this controversy, our laboratory has developed in vitro colony assays to detect and characterize individual pancreatic stem and progenitor-like cells. We found that a subpopulation of ductal cells in the adult murine pancreas has the abilities to self-renew and differentiate into multiple pancreatic lineages in three-dimensional space in methylcellulose-containing semisolid media. This protocol details the techniques used for culturing and characterizing these pancreatic stem and progenitor-like cells, which we have named pancreatic colony-forming units (PCFUs), as well as their progenies (colonies). The techniques presented here include dissociation of pancreases, sorting antibody-stained cells with a fluorescence-activated cell sorter, viral transduction of dissociated pancreatic cells, growth of PCFUs in semi-solid media, whole-mount immunostaining and Western blot analysis for proteins expressed in colonies, and kidney capsule transplantation of colonies for in vivo functional analysis.
Keywords: Differentiation; Organoid; Pancreas; Pancreatic colony-forming units; Progenitor; Self-renewal; Stem cells.