Kohtaro Minami^1,2, Susumu Seino^{1, 2,3}

1Division of Cellular and Molecular Medicine, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan, ²Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, 54 Shogoinkawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan, ³Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

FIGURES

Figure 1. The stem cell system. Stem cells in adult tissues are thought to possess capacity for self-renewal and generation of committed progenitor cells. These progenitor cells proliferate and differentiate into several types of functionally differentiated cells.

Figure 2. Pancreatic acinar cell-derived spherical cell clusters. (A) Phase contrast photomicrograph of cultured pancreatic acinar cells. Isolated pancreatic acinar cells were cultured in suspension in the presence of EGF. The cells began to aggregate within a few days and formed smooth spheroids after 4 days of culture. (B) Double immunostaining for F-actin and E-cadherin. F-actin was stained by Alexa Fluor 488-conjugated phalloidin (Molecular Probes; green), and E-cadherin was detected with ECCD-2 anti-E-cadherin antibody (Takara; red). Both F-actin and E-cadherin localized to the plasma membrane region of each cell in the spheroids.

Figure 3. Generation of insulin-positive cells from pancreatic acinar cells. (A) Double immunostaining of insulin and C-peptide. The insulin-positive cells are also positive for C-peptide. Scale bar, 20 m. (B) Expression of pancreatic cell markers during culture. While amylase-positive cells were markedly decreased, CK-positive ductal structures became apparent by the culture. Insulin/amylase and amylase/CK double-positive cells were detected. Scale bars, 50 m. Reprinted with permission from Ref. 20 with modification.

Figure 4. Cell lineage tracing by Cre/loxP-based system. (A) The scheme of pancreatic acinar cell specific cell marking. In cells from the R26R-ECFP mouse, expression of the fluorescent protein (ECFP) is activated through the action of Cre recombinase to remove a transcriptional "stop" sequence. When amylase/elastase-expressing acinar cells are infected with adenovirus expressing Cre recombinase under control of either amylase or elastase promoter, the cells are labeled permanently with ECFP. (B) Lineage tracing of labeled acinar cells. Pancreatic acinar cells from R26R-ECFP were labeled by infection of Ad-pAmy-Cre at approximately 50% efficiency. Because fluorescence of ECFP is diminished after fixation, ECFP-expression was detected using anti-GFP antibody. Cells positive for insulin (arrow heads), ECFP (arrows), and both insulin and ECFP (asterisks) are observed. Photographs of higher magnification of insulin/ECFP double-positive cells are shown (lower panels). Scale bars, 20 m. Reprinted with permission from Ref. 20 with modification.

Figure 5. Insulin secretory properties of pancreatic acinar-derived cells. (A) Immunoelectron microscopic analysis for insulin. Insulin immunoreactivities were detected in the secretory granules. Scale bar, 200 nm. (B) Insulin secretion in pancreatic acinar-derived cells. Insulin secretion was stimulated by 30 mM KCl, 0.1 M glibenclamide (Glib), 0.1 mM carbachol (CCh), or increased concentrations of glucose (G3; 3 mM, G10; 10 mM, G20; 20 mM) for 60 min. Potentiation by GLP-1 (7-36 amide) (100 nM) is also shown. Data are means ± S.E. of three to seven independent experiments. Reprinted with permission from Ref. 20 with modification.

Figure 6. Model for pancreatic exocrine-to endocrine transdifferentiation. Enzymatic dissociation of exocrine pancreas disrupts epithelial structures of acini, which causes dedifferentiation of the acinar cells. Meanwhile, EGF receptors are activated, followed by activation of the PI3-kinase pathway. Within a few days of culture, spherical cell clusters are formed and genes characteristic of pancreatic beta-cells are induced (redifferentiation).