In Vitro Multilineage Differentiation and Self-Renewal of Single Pancreatic Colony-Forming Cells from Adult C57Bl/6 Mice

Abstract

In a previous study we established colony assays suitable for studying murine adult (2-4 months) pancreatic progenitor cells plated in semisolid media containing methylcellulose and extracellular matrix proteins. Using these assays, we found robust in vitro progenitor cell activities (multilineage differentiation and self-renewal) from pancreatic cells of adult mice in the CD-1 outbred background. However, it was not clear whether progenitor cell activities can be detected from inbred mice, a preferred mouse model for various genetic studies. It was also not clear whether a single cell is sufficient to self-renew. Here, we show that fluorescent activated cell sorting pancreatic CD133+ but not CD133 -cells from adult C57Bl/6 inbred mice are enriched for progenitor cells that self-renew and give rise to multilineage colonies in vitro. The number of cells in a colony is in proportion to its diameter. Around 60% of single handpicked 3-week-old colonies express trilineage markers, indicating most progenitors are tripotent for ductal, acinar, and endocrine lineage differentiation. Approximately 80% of primary (freshly sorted) colony-forming progenitor cells are capable of giving rise to secondary progenitors in vitro, indicating that a majority of the primary progenitors self-renew. A single cell is sufficient for self-renewal and a Wnt agonist, R-Spondin1, enhances the number of secondary progenitors from the primary progenitors. Together, our pancreatic colony assays allow quantitative analyses of progenitors at a single-cell level from inbred mice. These assays will be useful for elucidating in vitro mechanisms of pancreatic progenitor cell biology.

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

“…+ cells *5% were cystic colony-forming progenitor cells [45]. In this study, we subdivided the postnatal CD133 + population into CD133 high and CD133 low fractions, and we were able to enhance the colony-forming efficiency of CD133 high cells to *10% (Fig.…”

“…We have previously shown that single CD133 + cells isolated from adult (2-4 months old) pancreas can efficiently form colonies in the Matrigel-supplemented culture [45]. In the young postnatal pancreas, we further separated the CD133 + population into the CD133 high and CD133 low fractions, accounting for 8.1% -1.4% and 14.2% -0.8% (average -SD from seven independent experiments) of total live cells, respectively (Fig.…”

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.…”

<em>In Vitro</em> Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

“…To begin addressing the technical gap in the field of pancreatic progenitor cell biology, colony [7][8][9][10][11] or organoid [12][13][14][15] assays using 3D culture systems were devised. Two colony assays for pancreatic progenitors were developed in our laboratory: one contains a commercial preparation of murine extracellular matrix proteins (ECM) (see Methods and Equipment Table), and the other contains laminin hydrogel, a defined artificial ECM protein [7][8][9][10][11] .…”

“…Two colony assays for pancreatic progenitors were developed in our laboratory: one contains a commercial preparation of murine extracellular matrix proteins (ECM) (see Methods and Equipment Table), and the other contains laminin hydrogel, a defined artificial ECM protein [7][8][9][10][11] . Progenitor cells are mixed in semi-solid medium containing methylcellulose.…”