Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel

Abstract

The study of hematopoietic colony-forming units using semisolid culture media has greatly advanced the knowledge of hematopoiesis. Here we report that similar methods can be used to study pancreatic colony-forming units. We have developed two pancreatic colony assays that enable quantitative and functional analyses of progenitor-like cells isolated from dissociated adult (2-4 mo old) murine pancreas. We find that a methylcellulose-based semisolid medium containing Matrigel allows growth of duct-like "Ring/ Dense" colonies from a rare (∼1%) population of total pancreatic single cells. With the addition of roof plate-specific spondin 1, a wingless-int agonist, Ring/Dense colony-forming cells can be expanded more than 100,000-fold when serially dissociated and replated in the presence of Matrigel. When cells grown in Matrigel are then transferred to a Matrigel-free semisolid medium with a unique laminin-based hydrogel, some cells grow and differentiate into another type of colony, which we name "Endocrine/Acinar." These Endocrine/Acinar colonies are comprised mostly of endocrine-and acinar-like cells, as ascertained by RNA expression analysis, immunohistochemistry, and electron microscopy. Most Endocrine/Acinar colonies contain beta-like cells that secrete insulin/C-peptide in response to D-glucose and theophylline. These results demonstrate robust self-renewal and differentiation of adult Ring/Dense colony-forming units in vitro and suggest an approach to producing beta-like cells for cell replacement of type 1 diabetes. The methods described, which include microfluidic expression analysis of single cells and colonies, should also advance study of pancreas development and pancreatic progenitor cells. extracellular matrix proteins | Sry-related HMG box (Sox) 9 | Promonin 1 (CD133) | neurogenin 3 | dickkopf1 (Dkk1)

Growth factors and medium hyperglycemia induce Sox9+ductal cell differentiation into β cells in mice with reversal of diabetes

“…One recent study, which used in vitro semisolid medium culture, showed that Sox9 + CD133 + pancreatic ductal cells from adult mice are able to give rise to three cell lineages, including insulin-producing β cells, ductal epithelial cells, and acinar cells (23,24). These results indicate that Sox9 + CD133 + pancreatic ductal cells from adult mice have the potential to differentiate into insulin-producing β cells in culture, if induced by specific conditions.…”

“…Medium hyperglycemia induced the presence of Sox9/EGFP + Ins + cells among ductal epithelial cells, and GE treatment increased the frequencies of the Sox9/EGFP + Ins + cells. A recent study reported that Sox9 + CD133 + cells contained β-cell progenitors (23), and multipotent progenitors in the pancreas of mice and human could be Insulin + (28). Whether or not these Sox9 + Ins + ductal cells are progenitors or "hybrid" cells mentioned above remains unclear, and it is under investigation.…”

Postnatal Pancreas of Mice Contains Tripotent Progenitors Capable of Giving Rise to Duct, Acinar, and Endocrine Cells In Vitro

“…On the other hand, tri-lineage potential was demonstrated for adult centroacinar cells (enriched by high aldehyde dehydrogenase 1 enzymatic activity) [19] and adult ductal cells (enriched by CD133 and Sox9 co-expression) [20]. These cells can be isolated, expanded, and differentiated in vitro into all three pancreatic lineages, which include glucose-responsive b-like cells [19,20].…”

MicroRNA-26a targets ten eleven translocation enzymes and is regulated during pancreatic cell differentiation

“…Although the existence of adult pancreatic progenitor cells in vivo remains debatable (26)(27)(28)(29), we recently identified rare progenitor cells in the adult pancreas that possess extensive selfrenewal and differentiation capacities in vitro (30). These adult progenitor cells, which are CD133 + Sox9 + , are enriched in the ductal cell fraction and can activate Pdx1 and Ngn3 expression before terminal differentiation into endocrine-like cells (30).…”

“…These adult progenitor cells, which are CD133 + Sox9 + , are enriched in the ductal cell fraction and can activate Pdx1 and Ngn3 expression before terminal differentiation into endocrine-like cells (30). Whether TETs, TDG, and miRNAs may affect differentiation of pancreatic cell lineages has not been tested.…”