title: Colony-forming progenitor cells in the postnatal mouse liver and pancreas give rise to morphologically distinct insulin-expressing colonies in 3D cultures.
abstract: 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⁺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.
authors.affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope, Duarte, California 91010, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Jin
authors.affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope, Duarte, California 91010, USA.
authors.forename: Tao
authors.initials: T
authors.lastname: Feng
authors.affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope, Duarte, California 91010, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Chai
authors.affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope, Duarte, California 91010, USA.
authors.forename: Nadiah
authors.initials: N
authors.lastname: Ghazalli
authors.affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope, Duarte, California 91010, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Gao
authors.affiliation: Electron Microscopy Core, Beckman Research Institute, City of Hope, Duarte, California 91010, USA.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Zerda
authors.affiliation: Electron Microscopy Core, Beckman Research Institute, City of Hope, Duarte, California 91010, USA.
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope, Duarte, California 91010, USA.
authors.forename: Jasper
authors.initials: J
authors.lastname: Hsu
authors.affiliation: Department of Bioengineering, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Alborz
authors.initials: A
authors.lastname: Mahdavi
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
authors.affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope, Duarte, California 91010, USA.
authors.forename: Arthur D
authors.initials: AD
authors.lastname: Riggs
authors.affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope, Duarte, California 91010, USA.
authors.forename: Hsun Teresa
authors.initials: HT
authors.lastname: Ku
mesh_terms: D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D021461:Animals, Outbred Strains; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D003114:Colony-Forming Units Assay; D004338:Drug Combinations; D018507:Gene Expression Regulation, Developmental; D020100:Hydrogels / Q000737:chemistry; D007328:Insulin / Q000096:biosynthesis*; D007797:Laminin / Q000737:chemistry; D008099:Liver / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D008422:Materials Testing; D051379:Mice; D008810:Mice, Inbred C57BL; D046529:Microscopy, Electron, Transmission; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D010179:Pancreas / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D061251:Primary Cell Culture / Q000379:methods; D011509:Proteoglycans / Q000737:chemistry
chemical_list: D004338:Drug Combinations; D020100:Hydrogels; D007328:Insulin; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen