The goal of this study was to

The goal of this study was to identify an additional cell-surface marker that, when combined with CD133, could further distinguish and enrich PCFUs. Cell surface markers that were previously known to enrich non-pancreas progenitor cells, including CD71 (transferrin receptor), were screened. CD71 transports iron from the extracellular space into cells, and higher levels of CD71 expression are detected in erythroid progenitor purchase 5-Azacytidine (Fraser et al., 2007) as well as in some cancer cells (Keysar & Jimeno, 2010). The adult pancreas expresses CD71 (Gatter et al., 1983); however, its role in the pancreas has not been characterized. Here, we report that CD71 expression status fractionates pancreatic CD133+ ductal cells in adult mice. Among the subpopulations, CD133highCD71low cells are the most enriched for PCFUs, and approximately one in three CD133highCD71low cells is a PCFU. This enriched population will enable further studies on putative pancreas stem and progenitor cells in vivo.
Results
Discussion We report that sorting for CD133highCD71low cells enriches PCFU progenitors that form Ring or 1° Endocrine/Acinar colonies in Matrigel or laminin hydrogel, respectively. The enrichment of these PCFUs is achieved using a strategy that has been successful in purifying adult hematopoietic stem cells (HSCs) (Spangrude et al., 1988). Murine HSCs are often designated as lineage negative (Lin), indicating that they do not express the cell-surface markers CD4, CD8, B220, Mac-1, Gr-1, or TER119 for differentiated blood cells (Spangrude et al., 1988). Additional markers, including stem cell antigen (Sca-1) (Spangrude et al., 1988), CD117 (c-Kit) (Okada et al., 1991), CD48, and CD150 (Kiel et al., 2005), have been identified to either positively or negatively select HSCs. About one in two Lin−Sca-1+c-kit+CD48−CD150+ bone marrow cells is a HSC (Kiel et al., 2005). CD133 has been shown to positively enrich progenitors from embryos and neonates of murine (Hori et al., 2008; Oshima et al., 2007; Sugiyama et al., 2007; Ghazalli et al., 2015) and human (Sugiyama et al., 2007) pancreas, as well as from adult human pancreas (Lee et al., 2013). We (Jin et al., 2014) and others (Dorrell et al., 2014) have previously shown that pancreatic progenitor cells from adult mice can be enriched by CD133 selection. However, the cell culture techniques reported by others were more difficult to enumerate colonies. Cells were mixed in high concentrations (>33% vol/vol) of Matrigel and placed only at the edge of the culture well. After solidification, the Matrigel was thicker at the periphery than towards the center of the culture dish. When viewed under a phase-contrast light microscope, the images of the resulting colonies were largely overlapping, preventing effective counting. Quantification of colony was achieved by sparse organoid plating or single cell deposition methods (Dorrell et al., 2014), which were labor-intensive and resource-consuming. In contrast, the methylcellulose-containing semi-solid media that we used allowed us to evenly spread Matrigel and colonies across the culture well. In addition, a grid was attached underneath the culture well to further facilitate accurate numeration by avoiding double counting (Winkler et al., 2011). Using our quantitative assays, we find that approximately one in three adult pancreatic CD133highCD71low cells is a PCFU-Ring (Fig. 2C), compared to ~1 in 100 pre-sorted pancreatic cells (Jin et al., 2013; Jin et al., 2014) (Fig. 2C), which translates to a 33-fold enrichment. The efficiency of formation of Ring colonies from CD133highCD71low cells has now reached ~30% (Fig. 2C), compared to ~5% from CD133+ selection alone in B6 mice (Jin et al., 2014) and ~15% from CD133+Sox9/EGFP+ cells from CD1 mice (Jin et al., 2013). Importantly, the use of commercially-available antibodies rather than transgenic mice will enable the characterization of adult PCFUs in many laboratories.