title: In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas.
abstract: Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133(+)Sox9/EGFP(+) ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.
authors.affiliation: Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope; Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope.
authors.forename: Jacob R
authors.initials: JR
authors.lastname: Tremblay
authors.affiliation: Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope.
authors.forename: Jeanne M
authors.initials: JM
authors.lastname: LeBon
authors.affiliation: Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope.
authors.forename: Angela
authors.initials: A
authors.lastname: Luo
authors.affiliation: Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope.
authors.forename: Janine C
authors.initials: JC
authors.lastname: Quijano
authors.affiliation: Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope.
authors.forename: Lena
authors.initials: L
authors.lastname: Wedeken
authors.affiliation: Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope.
authors.forename: Kevin
authors.initials: K
authors.lastname: Jou
authors.affiliation: Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope.
authors.forename: Arthur D
authors.initials: AD
authors.lastname: Riggs
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology.
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
authors.affiliation: Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope; hku@coh.org.
authors.forename: H Teresa
authors.initials: HT
authors.lastname: Ku
mesh_terms: D000375:Aging; D000818:Animals; D002454:Cell Differentiation; D050417:Insulin-Secreting Cells; D051379:Mice; D010179:Pancreas; D013234:Stem Cells