|Rev Diabet Stud,
Colony-Forming Progenitor Cells in the Postnatal Mouse Liver and Pancreas Give Rise to Morphologically Distinct Insulin-Expressing Colonies in 3D Cultures
Liang Jin1,2, Tao Feng1,2, Jing Chai1,2, Nadiah Ghazalli1,3, Dan Gao1, Ricardo Zerda4, Zhuo Li4, Jasper Hsu1, Alborz Mahdavi5, David A. Tirrell6, Arthur D. Riggs1, Hsun Teresa Ku1,3
1Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope, Duarte, California 91010, USA
2These authors contributed equally to this work
3Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute, City of Hope, Duarte, California 91010, USA
4Electron Microscopy Core, Beckman Research Institute, City of Hope, Duarte, California 91010, USA
5Department of Bioengineering, California Institute of Technology, Pasadena, California 91125, USA
6Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
Address correspondence to: Hsun Teresa Ku, e-mail: email@example.com
Manuscript submitted February 15, 2013; resubmitted June 20, 2013; accepted July 9, 2013.
Keywords: in vitro colony assays, insulin expression, methylcellulose, Matrigel, laminin hydrogel, progenitor cell
In our previous studies, colony-forming progenitor cells isolated from murine embryonic stem cell-derived cultures were differentiated into morphologically distinct insulin-expressing 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+CD49flowCD107blow 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.
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