1. Volume 132, Issue 2, Pages 720-732 (February 2007)
Isolation of Mouse Pancreatic Ductal Progenitor Cells Expressing CD133 and c-Met by Flow Cytometric Cell Sorting 
Yuji Oshima, Atsushi Suzuki, Kaneaki Kawashimo, Momotarou Ishikawa, Nobuhiro Ohkohchi, Hideki Taniguchi 
Gastroenterology 
Volume 132, Issue 2, Pages (February 2007)
DOI: /j.gastro
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2. Figure 1
CD133 expression in neonatal, adult, and embryonic (E15.5) pancreata. CD133 (green) is expressed at the apical surfaces of pancreatic ductal epithelium, which express ductal cell marker CK7 (red) in (A, C, E, and G) neonatal and (B, D, F, and H) adult mice. Both CD133 and CK7 are expressed throughout the pancreatic ducts, (A and B) main ducts, (C and D) interlobular ducts, (E and F) intralobular ducts, and (G and H, arrowheads) intercalated ducts. CD133 and CK7 staining colocalizes at the apical surface of ductal epithelium (A and B insets, yellow). Double immunostaining of CD133 (green) and E-cadherin (red) also confirm these observations in (I) neonatal and (J) adult pancreas. Panels in I and J show the following: left upper panels, main ducts; right upper panels, interlobular ducts; left lower panels, intralobular ducts; right lower panels, intercalated ducts and acinar cells. CD133 (green) is not expressed in islets (insulin, red) in (K, left panel) neonatal and (K, right panel) adult pancreas. CD133 (red) is not expressed in both small (L, left panel) and large (L, right panel) vessels, which are stained with endothelial marker, CD34 (green). In embryonic pancreas, CD133 expressions (green) are evident in the central portion of pancreas and acinar-like structures, and (M) CK7 (red) is diffuse. (N) Insulin-producing clusters (green) are present around main pancreatic ducts. Immunofluorescent images were captured by confocal microscopy. Nuclei are visualized by DAPI (indicated by the blue color). (A and B) Right lower panels (C–H, M, and N) and right panels are merged with differential interference contrast images. Scale bars = 20 μm.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Flow cytometric isolation of neonatal pancreatic ductal cells. (A) Total pancreatic cells were stained with monoclonal antibodies and analyzed by FACS. Dead cells were excluded with propidium iodide, and hematopoietic cells were excluded with a leukocyte marker CD45 and the erythroid cell marker Ter119. Nonhematopoietic (CD45−Ter119−) cells were fractionated with antibodies directed against CD133 and the endothelial marker CD34. The percentage of the gated cells is included in each panel. (B) Sorted cells were plated onto glass slides and stained with an anti-CK7 antibody (red) and then counterstained with DAPI (blue; upper panel, no gated cells; lower panel, CD133+CD34−CD45−Ter119− cells; scale bar = 20 μm). CK7-positive cells were counted with the aid of a fluorescence microscope. (C) The percentage of CK7-positive cells in cells from each fraction is shown. Mean ± SD. (D) CD133+CD34−CD45−Ter119− cells were characterized with CD49f, CD29, c-Kit, Thy-1, and Sca-1 (shaded areas, isotype controls; open areas, specific markers).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Cells derived from FACS of neonatal pancreas show colonies. There are 3 types of colonies: (A) ECs, (B) endothelial colonies, and (C) fibroblastic colonies. Immunocytochemistry of CK7 (middle panels, green) and CD34 (right panels, red). Phase-contrast views are provided in the left panels in A–C. (D–H) A single sorted ductal cell forms an EC in clonal density culture (200 cells/cm2, arrowhead in D–H). Single cell attached to the dish at (D) day 1, begins to divide and form an EC at (E) day 2, (F) day 3, (G) day 4, and (H) day 5 after sorting. The markings in the upper areas of D to H are scratches on the back of the cell culture dishes to identify the same field. Scale bars = 200 μm. (I) Two thousand cells were sorted into 6-well plates at a density of 200 cells/cm2 for each fraction. The number of ECs in each well was counted on day 7 after sorting (n = 9; independent experiment = 3; mean ± SD; *P < .01).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
In vitro differentiation and expression of multilineage markers in a clonal EC formed from a sorted CD133+CD34−CD45−Ter119− cell derived from neonatal pancreas. Clonal ECs were cultured for 14 days. Total RNA of each clonal colony was extracted separately at days 3 and 14. (A) Nine clonal colonies were analyzed by RT-PCR. All 9 clonal ECs did not express endocrine marker at day 3. After 14 days of cultivation, almost all clonal ECs expressed 2 or more endocrine markers (gapdh). (B–E) By immunocytochemistry, clonal ECs expressed several endocrine markers, including (B) insulin (left panel, green) and C-peptide (middle panel, red). (B) A merged composite image is provided (right panel, yellow; DAPI, blue). (C) Some insulin-positive cells (left panel, green) expressed glucagon (middle panel, red; right panel, merged view [yellow] with DAPI [blue]). (D) Pancreatic polypeptide-positive cells also were detected (green). (E) Pancreatic polypeptide (red) and ductal cell marker, DBA lectin (green), were segregated in the same colony. (F) All ECs expressed the ductal cell marker CK7 (left panel, phase; right panel, CK7 [red]) by immunocytochemistry. Scale bars: (B–E) 50 μm, (F) 200 μm.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Flow cytometric analysis and characterization of ductal cells from adult mouse pancreas. (A) Adult pancreas was dissociated to a single-cell suspension and stained with the same monoclonal antibodies used to study the neonatal pancreas. Nonhematopoietic cells were also separated into 4 fractions. (B) One thousand cells were sorted into cell culture plates at a density of 100 cells/cm2 from each fraction. The number of clonal ECs was counted on day 7 (mean ± SD, *P < .01). (C) mRNA expression of various markers was examined by RT-PCR as described in Figure 4. All 6 clonal ECs expressed the ductal cell marker, CK7, and carbonic anhydrase 2. However, endocrine and acinar cell markers were not detected by RT-PCR (hypoxanthine phosphoribosyltransferase [hprt]). (D and E) CK7 expression but not endocrine nor acinar cell markers were detected by immunocytochemistry (D, phase; E, CK7 (red); scale bar = 200 μm). (F) DBA staining (green) often is detected in ECs (CK7, red).
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Further enrichment of CD133+CD34−CD45−Ter119− cells for c-Met expression. (A) CD133+CD34−CD45−Ter119− cells in neonatal pancreas were fractionated based on c-Met expression. (B) 500 cells from fractions of total CD133+CD34−CD45−Ter119− cells, c-Met–negative (c-Met− CD133+CD34−CD45−Ter119−) cells and c-Met–positive (c-Met+ CD133+CD34−CD45−Ter119−) cells were sorted into cell culture plates. The number of colonies derived from these cells was counted after 7 days in culture (n = 6; mean ± SD; *P < .01). (C) One thousand c-Met+ CD133+CD34−CD45−Ter119− cells and 2000 c-Met− CD133+CD34−CD45−Ter119− cells were sorted into 6-well plates. After 12 days in culture, mRNA expression of various markers was analyzed by real-time PCR. Gapdh was used as an internal control. The relative amount of mRNA expressions of c-Met–positive cells was normalized to the expression level in c-Met–negative cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions