Colony-Forming Progenitor Cells in the Postnatal Mouse Liver and Pancreas Give Rise to Morphologically Distinct Insulin-Expressing Colonies in 3D Cultures

Abstract

■ AbstractIn our previous studies, colony-forming progenitor cells isolated from murine embryonic stem cell-derived cultures were differentiated into morphologically distinct insulinexpressing colonies. These colonies were small and not light-reflective when observed by phase-contrast microscopy (therefore termed "Dark" colonies). A single progenitor cell capable of giving rise to a Dark colony was termed a Dark colony-forming unit (CFU-Dark). The goal of the current study was to test whether endogenous pancreas, and its developmentally related liver, harbored CFU-Dark. Here we show that dissociated single cells from liver and pancreas of one-week-old mice give rise to Dark colonies in methylcellulose-based semisolid culture media containing either Matrigel or laminin hydrogel (an artificial extracellular matrix protein). CFU-Dark comprise approximately 0.1% and 0.03% of the postnatal hepatic and pancreatic cells, respectively. Adult liver also contains CFU-Dark, but at a much lower frequency (~0.003%). Microfluidic qRT-PCR, immunostaining, and electron microscopy analyses of individually handpicked colonies reveal the expression of insulin in many, but not all, Dark colonies. Most pancreatic insulin-positive Dark colonies also express glucagon, whereas liver colonies do not. Liver CFU-Dark require Matrigel, but not laminin hydrogel, to become insulin-positive. In contrast, laminin hydrogel is sufficient to support the development of pancreatic Dark colonies that express insulin. Postnatal liver CFU-Dark display a cell surface marker CD133 + CD49f low CD107b low phenotype, while pancreatic CFU-Dark are CD133 -. Together, these results demonstrate that specific progenitor cells in the postnatal liver and pancreas are capable of developing into insulin-expressing colonies, but they differ in frequency, marker expression, and matrix protein requirements for growth.

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

“…The frequency of CFU-Dark is 0.03% of the total postnatal pancreatic cells and 0.1% of liver cells [24]. In comparison, the PCFUs described in this study comprised at least 1.0% of pancreatic cells (Figs.…”

“…Our colony assays provide quantitative means to characterize self-renewal and differentiation of PCFUs [20]. In a recent publication, we demonstrated that postnatal (1-week-old) liver and pancreas contained CFUDark, a class of rare progenitors that give rise to morphologically distinct insulin-expressing colonies [24]. In addition to Dark colonies, other types of postnatal colonies with cystic or compact structures from pancreas were observed but had not been characterized [24].…”

“…In a recent publication, we demonstrated that postnatal (1-week-old) liver and pancreas contained CFUDark, a class of rare progenitors that give rise to morphologically distinct insulin-expressing colonies [24]. In addition to Dark colonies, other types of postnatal colonies with cystic or compact structures from pancreas were observed but had not been characterized [24]. In this study, we sought to determine the lineage potentials of these young postnatal progenitors.…”

<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.…”