Identification of a novel putative pancreatic stem/progenitor cell marker DCAMKL-1 in normal mouse pancreas

Abstract

Stem cells are critical in maintaining adult homeostasis and have been proposed to be the origin of many solid tumors, including pancreatic cancer. Here we demonstrate the expression patterns of the putative intestinal stem cell marker DCAMKL-1 in the pancreas of uninjured C57BL/6 mice compared with other pancreatic stem/progenitor cell markers. We then determined the viability of isolated pancreatic stem/progenitor cells in isotransplantation assays following DCAMKL-1 antibody-based cell sorting. Sorted cells were grown in suspension culture and injected into the flanks of athymic nude mice. Here we report that DCAMKL-1 is expressed in the main pancreatic duct epithelia and islets, but not within acinar cells. Coexpression was observed with somatostatin, NGN3, and nestin, but not glucagon or insulin. Isolated DCAMKL-1+ cells formed spheroids in suspension culture and induced nodule formation in isotransplantation assays. Analysis of nodules demonstrated markers of early pancreatic development (PDX-1), glandular epithelium (cytokeratin-14 and Ep-CAM), and isletlike structures (somatostatin and secretin). These data taken together suggest that DCAMKL-1 is a novel putative stem/progenitor marker, can be used to isolate normal pancreatic stem/progenitors, and potentially regenerates pancreatic tissues. This may represent a novel tool for regenerative medicine and a target for anti-stem cell-based therapeutics in pancreatic cancer.

Fig. 1.

Pancreatic DCAMKL-1 expression in adult mice. A: DCAMKL-1 expression (brown) in the main pancreatic duct (left) (×200) and in the periphery of pancreatic islets (middle) (×400). No DCAMKL-1 expression is observed in acinar cells or accessory ducts (right) (×400). B: immunofluorescence demonstrating DCAMKL-1 (red) and somatostatin (green) staining of pancreatic islets. Colocalization is demonstrated in merged image. C: DCAMKL-1 (red) and glucagon (green) immunofluorescence staining of pancreatic islets. No colocalization is demonstrated in merged image. D: immunofluorescence demonstrating DCAMKL-1 (red) and insulin (green) staining of pancreatic islets. No colocalization is observed in merged image. Images on the far right in B–D are the magnified portion of the corresponding merged images. In the immunofluorescence staining, nuclei are stained blue with Hoechst dye.

Fig. 2.

DCAMKL-1 and other putative pancreatic stem/progenitor cell markers. Newborn mice pancreas demonstrates DCAMKL-1 staining (A; arrows) and neurogenin 3 (NGN3; B; arrows) (×600). Immunofluorescence staining for DCAMKL-1 (C; red) and NGN3 (D; green) in the newborn mice pancreas. E–F: colocalization demonstrated in merged image with nuclei stained blue with Hoechst dye (×400). Adult mouse pancreatic tissue serial sections stained with DCAMKL-1 (G), NGN3 (H), and nestin (I) (×200). Immunofluorescence staining of newborn mouse pancreas demonstrated the presence of DCAMKL-1 (J) and nestin (K). L–M: colocalization demonstrated in merged image with nuclei stained blue with Hoechst dye (×400). Insets in J–M are magnified images.

Fig. 3.

FACS-based isolation of DCAMKL-1 cells from mouse pancreas. FACS plots of sorted cells. A: side scatter oval gate R1. B: polygon gate R2 represents sorted fluorescent cells (red) from gate R1 (0.36% of total cells). The graphs represent the mRNA expression levels of DCAMKL-1 (C), NGN3 (D), nestin (E), somatostatin (F), insulin (G), and glucagon (H) in DCAMKL-1+ and DCAMKL-1− sorted cells. *P < 0.01.

Fig. 4.

DCAMKL-1 sorted cells demonstrate growth in vitro and in vivo. FACS isolated DCAMKL-1+ cells in suspension culture at day 1 (A) and demonstrates spheroid formation at day 21 (B) (×400). C: magnified portion of image B. Athymic nude mice 4 wk after subcutaneous injection with either Matrigel alone (D) or spheroid with Matrigel (E); arrow indicates nodular growth. F: image demonstrates a tan-gray soft tissue outgrowth with blood vessel formation under the skin of the DCAMKL-1 spheroid-injected mouse.

Fig. 5.

Evaluation of nodular growth from spheroid injection. Hematoxylin and eosin staining of cells that appeared to be epithelial in nature and formed early isletlike structures (A) and cells that lined up around central spaces, which appeared to be poorly formed glands (B), as indicated by arrows. Cells around the central spaces were positive for glandular epithelial marker cytokeratin-14 (C) and PDX-1 (D), a marker of early pancreatic development. The early islet formations expressed endocrine markers somatostatin (E) and secretin (F). Additionally, cells within these nodules expressed the epithelial marker Ep-CAM (G), proliferation marker Ki67 (H) and DCAMKL-1 (I–J). Positive staining demonstrated by brown coloration (diaminobenzidine); all images shown at ×200 magnification.