Stimulation of cAMP signalling allows isolation of clonal pancreatic precursor cells from adult mouse pancreas
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- Yamamoto, T., Yamato, E., Taniguchi, H. et al. Diabetologia (2006) 49: 2359. doi:10.1007/s00125-006-0372-7
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Duct cells of the pancreas are thought to include latent progenitors of islet endocrine cells that can be induced to differentiate by appropriate morphogens. Here we developed a method for isolating pancreatic ductal epithelial cells from adult mice that overcomes the shortcomings of previous methods.
Materials and methods
Pancreatic ductal cells were grown in serum-free DMEM/F12 medium in the presence of cholera toxin or 8-bromo-cyclic adenosine monophosphate, which is known to be an intracellular cAMP generator. Single cell cloning was performed by limiting dilution in serum-free medium.
The isolated clonal cells expressed high levels of cytokeratin and Ipf1 (formerly known as Pdx-1). Adenovirus-mediated expression of ngn3 (also known as Neurog3) and Ptf1a in these cells induced expression of insulin and somatostatin, and of carboxypeptidase A, respectively. Furthermore, albumin production was induced by dexamethasone or by long-term culture in serum-containing medium.
Stimulation of the cAMP-dependent signalling allowed us to isolate clonal pancreatic ductal cells from adult mice. These cells are able to partially differentiate into endocrine cells, exocrine cells and hepatocyte-like cells and are therefore considered to have the characteristics of endodermal progenitor cells.
KeywordscAMP Cholera toxin Cytokeratin Insulin Ngn3 Pancreatic ductal cells Pdx-1 Serum-free
8-bromo-cyclic adenosine monophosphate
carbonic anhydrase II
fetal bovine serum
keratinocyte growth factor
pancreatic and duodenal homeobox 1 (encoded by the Ipf1 gene)
Pdx-1-positive pancreatic duct-derived
It was thought that islet endocrine cells were predominantly derived from the precursor cells that reside among pancreatic epithelial duct cells or duct-associated cells both during embryonic development and later in life; islet cell neogenesis from ducts has been observed in experimental injury models in rats [1, 2] and in transgenic mice over-expressing certain growth factors or cytokines [3, 4]. Furthermore, recent studies have demonstrated that isolated murine and human pancreatic ductal cells could be directed to differentiate into glucose-responsive insulin-producing cells in vitro [5, 6].
Cultures of pancreatic duct cells have been established using cells from several animals: mice [7, 8], rats , hamsters , cattle , guinea-pigs  and humans . Various basal media enriched with a wide variety of supplements have been used for these duct cell cultures. However, the experimental utility of pancreatic duct cell culture systems has been hampered by difficulties encountered in eliminating fibroblast contamination and by the limited survival in culture. Furthermore, these culture systems gave rise to heterogeneous cell populations and made it difficult to analyse the molecular mechanisms regulating ductal cell differentiation.
Here, to further investigate the mechanisms of the proliferation of pancreatic duct cells from adult mice and to evaluate the latent abilities of the duct cells to differentiate into other cells morphologically and biochemically, we developed a method for the isolation and cultivation of Pdx-1-positive pancreatic duct-derived (PPPD) cells from normal adult mice without genetic manipulation.
Materials and methods
Isolation of pancreatic ductal cells and single-cell cloning
Pancreas tissue was obtained from 20-week-old normal adult C57BL/6J mice (Clea Japan, Tokyo, Japan) and digested with collagenase type XI (Sigma-Aldrich, Saint Louis, MO, USA). After repeated washes with cold Hanks’ balanced salt solution (Invitrogen, Carlsbad, CA, USA) containing 2-mercaptoethanol (Wako, Osaka, Japan), fractions enriched with pancreatic ductal fragments were passed through a stainless-steel mesh sieve with 850-μm pores. The pancreatic ductal fraction trapped on the sieve was cultured on a tissue culture plate in CMRL-1066 medium (Sigma-Aldrich) containing 10% fetal bovine serum (FBS; Cansera International, ON, Canada), 100 ng/ml cholera toxin (CTx; Sigma-Aldrich), and Antibiotic-Antimycotic (100 U/ml of penicillin, 100 μg/ml of streptomycin and 250 ng/ml of amphotericin B; Invitrogen). To reduce the fibroblast contamination in the primary culture, the medium containing ductal fragments was transferred to another plate 2 h after the initial plating. After 4 days, the culture medium was changed to serum-free medium consisting of 1:1 mixture of DMEM (Sigma-Aldrich) and Ham’s F-12 (Invitrogen) medium supplemented with 100 ng/ml CTx, 5 μg/ml insulin (Sigma-Aldrich), 5 μg/ml transferrin (Sigma-Aldrich), 5 ng/ml sodium selenite (Sigma-Aldrich), 0.2% bovine serum albumin (Sigma-Aldrich), 25 ng/ml keratinocyte growth factor (KGF; Sigma-Aldrich), and Antibiotic–Antimycotic. The culture was incubated at 37°C in a humidified 5% CO2 and 95% air atmosphere, and the medium was changed every 2 or 3 days. After more than 40 days in serum-free medium, first passage was performed. The resulting cells showed stable growth in serum-free medium for more than 1 year. To experimentally induce hepatocyte lineage, 10% FBS, CTx and Antibiotic-Antimycotic were included. Subcloning of the ductal cells was performed by limiting dilution in serum-free DMEM/F12 medium. Dissociated cells were placed at 1–10 cells/0.1 ml per well in a 96-well plate.
Ultrastructural studies were performed on cultured cells attached as a monolayer in tissue culture dishes after four passages. For electron microscopy, cells were fixed for 3 h with 2.5% glutaraldehyde, and then post-fixed with 1% osmium tetroxide. After overnight dehydration in graded ethanol solutions and propylene oxide, the cells were embedded in a mixture of Epon-Araldite. Thin sections (approximately 80 nm) were cut using a Reichert Ultracut OM-U4 microtome (Microm, Walldorf, Germany) and were stained with uranyl acetate and lead citrate. Transmission electron microscopy was carried out using a Hitachi H-7100 electron microscope (Hitachi Hi-technologies, Tokyo, Japan).
Cell proliferation analysis
Cell proliferation was assessed by counting the number of cells per colony. 8-Bromo-cyclic adenosine monophosphate (8-Br-cAMP; Biomol, Plymouth Meeting, PA, USA), PD98059 (Sigma-Aldrich), and KT5720 (Sigma-Aldrich) were respectively added to serum-free DMEM/F12 medium at an optimal concentration.
Total RNA was extracted from cultured cells by the acid guanidinium-phenol-chloroform method. The cDNA was prepared from total RNA using a ReverTra Ace-α kit (Toyobo, Tokyo, Japan) with oligo-dT primers according to the manufacturer’s instructions. The primer sequences and PCR conditions used for RT-PCR were previously described . PCR was performed with Taq DNA polymerase (Promega, Madison, WI, USA) and within the log phase of the reaction (25-35 cycles). For quantitative estimation of Ins2 and GAPDH mRNA, real-time RT-PCR was performed on the ABI PRISM 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq (TaKaRa Bio, Otsu, Japan). The relative amount of Ins2 mRNA was determined using GAPDH mRNA as a standard. Amplification conditions comprised initial denaturation at 95°C for 10 s, followed by 40 cycles of denaturation at 95°C for 5 s and annealing and extension at 60°C for 30 s. The primer sequences for Ins2 were: forward, 5′-GATCCGCTACAATCAAAAACCAT-3′; reverse, 5′-ATCCACAGGGCCATGTTGAA-3′. Those for GAPDH were: forward, 5′-TGTGTCCGTCGTGGATCTGA-3′; reverse, 5′-CCTGCTTCACCACCTTCTTGA-3′.
Monolayer cultures of epithelial cells were washed four times with PBS, fixed for 15 min in 4% paraformaldehyde (PFA), and then incubated with blocking reagent (Roche, Mannheim, Germany). Immunofluorescence staining was performed using the following primary antibodies: mouse anti-human pancytokeratin monoclonal antibody (dilution 1:200, Sigma-Aldrich), rabbit anti-rat Pdx-1 polyclonal antibody (1:200, gift from Y. Kajimoto, Osaka University, Osaka, Japan), rabbit anti-mouse albumin polyclonal antibody (Biogenesis, Poole, UK), and goat anti-mouse HNF3β polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies were Alexa 568-conjugated goat anti-mouse IgG antibody, Alexa 568-conjugated donkey anti-goat IgG antibody and Alexa 488-conjugated goat anti-rabbit IgG antibody (Molecular Probes, Eugene, OR, USA). Alexa 568 was excited at 543 nm and Alexa 488 at 488 nm, using the Leica confocal laser scanning microscope system TCS SP2 (Leica Microsystems, Wetzlar, Germany).
Western blot analysis
Duct-derived cells and MIN6 cells were lysed in detergent lysis buffer. A total of 20-μg protein extracts was separated by SDS-PAGE, then electrotransferred onto an Immobilon P membrane (Millipore, Bedford, MA, USA). The membrane was incubated with rabbit anti-rat Pdx-1 polyclonal antibody (dilution 1:1,000). After incubation with horseradish-peroxidase-conjugated goat anti-rabbit IgG second antibody (New England Biolabs, Beverly, MA, USA), the membrane was subjected to an ECL detection system (Amersham Biosciences, Piscataway, NJ, USA).
Construction of adenoviral vector expressing mouse ngn3 and Ptf1a
A full-length cDNA of mouse ngn3 (also known as Neurog3) was cloned from mouse brain cDNA, and that of mouse Ptf1a was cloned from mouse pancreas cDNA. Adenovirus vectors (AdVs) expressing these cDNAs were generated as described previously  (Fig. 3a). Duct-derived cells were infected with control adenovirus vector (AdV-EGFP), AdV-ngn3, or AdV-Ptf1a at a multiplicity of infection (MOI) of 50 for 2 h at 37°C.
Induction of hepatocyte genes by dexamethasone
Duct-derived cells were seeded onto a six-well tissue culture plate in serum-free DMEM/F12 medium and cultured for 2 weeks with different concentrations of dexamethasone (Sigma-Aldrich).
We carried out descriptive statistics and analysis of variance (ANOVA) followed by two-tailed Student’s t-test using Statsview software (Abacus Concepts, Berkeley, CA, USA).
Culture of pancreatic cells and analysis of cell proliferation
The duct fraction was cultured in CMRL-1066 medium containing FBS for 4 days. After the first passage, the cells were cultured in serum-free DMEM/F12 containing CTx, KGF, insulin, transferrin and sodium selenite. Over the subsequent 40 days, only a fraction of the adherent cells expanded, while the spindle-shaped fibroblasts stopped proliferating and gradually disappeared. After second passage, KGF was removed because it was no longer required for cell proliferation. The resulting adherent cells were maintained for more than a year under serum-free conditions.
Morphology of pancreatic ductal epithelial cells
Ultrastructural studies of the pancreatic precursor cells demonstrated typical features of the epithelium (Fig. 2bi). The plasma membrane possessed specialisations, including numerous surface microvilli, and exhibited close interdigitations with tight junctions (Fig. 2bii and biii). One or two nuclei were eccentrically located and irregularly shaped, displaying clumping of the chromatin in the region of the nuclear membrane and some nucleoli (Fig. 2biv). None of the cells exhibited the well-developed secretory granules that are characteristic of acinar or islet cells.
Characterisation of isolated pancreatic cells
Isolated pancreatic cells at passage 3 were analysed for their gene expression pattern. A high expression of Krtl-19 (which encodes cytokeratin 19) and Car2 (which encodes carbonic anhydrase II), markers of epithelial cells, was specifically detected. No gene expression of endocrine and exocrine pancreas marker genes was detected. These results, taken together with ultrastructural studies, suggest that these pancreatic cells were derived from pancreatic ductal epithelial cells and did not contain any islet or acinar cells. These pancreatic duct-derived cells also expressed Ipf1 (formerly known as Pdx-1), Isl1, Neurod1, Pax6 and Nkx6-1, whereas no expression of ngn3, Pax4, Nkx2-2, or Ptf1a was observed (Fig. 2c). Pdx-1 was detected in the nuclei of the most of the cells by immunocytochemistry (Fig. 2d). Western blot analysis detected the Pdx-1 protein of 46 kDa in these cells and also in MIN6 cells, a mouse insulinoma-derived beta cell line (Fig. 2e), although the amount of Pdx-1 protein was lower in the pancreatic duct-derived cells than in the MIN6 cells. These results suggested that the cells we isolated were probably derived from pancreatic ductal epithelial cells, but were different from normal pancreatic duct epithelial cells in their gene expression pattern. Thus we designated these pancreatic cells as Pdx-1-positive pancreatic duct-derived (PPPD) cells.
Differentiation into the pancreatic endocrine and exocrine lineage
Differentiation into the hepatocyte-like cell lineage
PPPD cells could be maintained by repeated passages for more than 1 year without growth inhibition, not only in serum-free conditions but also in serum-containing conditions. The presence of FBS in the medium supported better cell proliferation. The addition of CTx was also essential for cell proliferation in serum-containing medium.
Dexamethasone is reported to promote the conversion of AR42J cells, which were derived from a rat pancreatic acinar cell tumour, into hepatocytes . To analyse the effect of dexamethasone, PPPD cells were incubated for 2 weeks with several concentrations of dexamethasone (Fig. 4c). RT-PCR analysis revealed that the expression levels of the Alb1 gene increased with dexamethasone in a dose-dependent manner. Thus, PPPD cells were shown to have the ability to differentiate into the hepatocyte-like cell lineage.
In the present study, we established a feasible method for the long-term culture of pancreatic ductal epithelial cells from normal adult mice. Cell proliferation was maintained under serum-free conditions for more than 1 year after the isolation. The expansion from single cells in serum-free medium enabled us to establish several clonal cell lines. The establishment of serum-free culture conditions is considered to be very important for studies of cell differentiation, because the factors that affect cell growth or differentiation can be identified without the effect of unknown factors contained in the FBS.
The presence of cAMP-elevating factors, like CTx, a bacterial toxin secreted by Vibrio cholerae, was crucial for the proliferation and maintenance of PPPD cells. Cyclic AMP reportedly promotes the growth of various kinds of isolated epithelial cells [18, 19, 20, 21, 22, 23, 24, 25]. Recently, CTx was also used for the isolation of immortalised human breast epithelial cell lines with stem cell properties . Our experiments using pharmacological agents that specifically target different aspects of cAMP-dependent signalling, 8-Br-cAMP, CTx, PKA inhibitor and MAPK inhibitor, further supported the idea that the proliferation of pancreatic ductal epithelial cells is promoted by the cAMP signalling pathway.
Pdx-1 is detected in all embryonic protodifferentiated epithelial cells during pancreatic development . Several studies have demonstrated that adult pancreatic ductal cells that retain the capacity for proliferation re-express Pdx-1 in the course of islet differentiation and regeneration [27, 28]. In the present study, Pdx-1 protein was detected in the nuclei of most of the PPPD cells by immunocytochemistry and immunoblot analysis. These observations suggest that PPPD cells derived from the adult mouse pancreas have the potential to differentiate into islet endocrine cells.
In the present study, PPPD cells were converted into cells expressing Ins1, Ins2 and Sst (somatostatin) genes by the adenovirus-mediated expression of ngn3, consistent with the report that neuroendocrine differentiation markers including insulin were induced in adult human pancreatic duct cells by adenovirus-mediated overexpression of ngn3 . These results suggest that ngn3 expression is critical for regeneration of pancreatic endocrine cells. Infection with Ptf1a-expressing adenovirus vector induced the expression of genes for Amy2, Cpa1 and PPy in PPPD cells. Thus, Ptf1a is essential for the induction of pancreatic exocrine enzymes. Although the molecular mechanisms that underlie the induction of PP are uncertain, this result is intriguing. Only a few cells were positively stained for insulin, somatostatin, or CXPA by immunocytochemistry. One of the possible reasons for this is that the infection efficiency of adenovirus vector was not good in PPPD cells. Another possibility is that a single transcription factor gene might not be enough for the induction of pancreatic endocrine hormones or exocrine enzymes. A study is in progress to improve the efficiency of differentiation into insulin-positive cells.
Adenovirus-mediated transduction experiments were repeatedly conducted in all five clones. Interestingly, Ins1 and Ins2 mRNA induction in clone-12 by the treatment with AdV-ngn3, and both Amy2 and Cpa1 mRNA induction in clone-2 by the treatment with AdV-Ptf1a were most prominent (data not shown). These results suggest that PPPD cells were composed of a heterogeneous population. In fact, there were some differences among five clones in the expression pattern of several pancreatic transcription factors, Isl1, Neurod1, Pax-6 and Nkx6-1, which parental PPPD cells moderately expressed (data not shown). We propose that pancreatic ductal epithelium contains heterogeneous precursor cells that have distinct characteristics in terms of transcriptional regulation.
In embryonic development, it appears that there is a common precursor for the hepatic and pancreatic lineages . The conversion of pancreas to liver has also been described in experimental models both in vivo and in vitro [7, 17]. After long-term culture of PPPD cells in serum-containing medium, hepatocyte markers were upregulated, while pancreatic transcription factor genes were downregulated in PPPD cells. Alb1 mRNA expression was also dose-dependently induced by treatment with dexamethasone. However, other mature liver markers, such as Pck1 (phosphoenolpyruvate carboxykinase), G6pc (glucose-6-phosphatase), Tat (tyrosine aminotransferase) and Otc (ornithine transcarbamylase) were undetectable by RT-PCR analysis (data not shown). Therefore, the albumin-producing PPPD cells are not mature hepatocytes. Our culture conditions may not be optimal for obtaining complete hepatocyte differentiation.
The next important point of this report is whether PPPD cells were truly derived from pancreatic duct cells or not. Recent studies used a lineage tracing system to demonstrate that explant cultures of pancreas developed the acinar-to-ductal transdifferentiation of mature exocrine cells in response to EGF receptor signalling . Baeyens et al. demonstrated by in vitro culture experiment that insulin-producing cells were generated from pancreatic exocrine cells via the acinar-to-ductal transdifferentiation . This conclusion was based on the existence of the cells that were binucleated and coexpressed amylase with cytokeratin 20. In our experiment, only some of the cultured cells co-expressed amylase and cytokeratin after the first 24 h in serum-free conditions (data not shown) and exhibited binuclearity, a characteristic of part of acinar cells by ultrastructural study. These amylase-positive cells were lost within a few weeks, and gene expression of pancreatic endocrine markers as well as exocrine markers became undetectable by RT-PCR analysis after first passage in serum-free conditions. Therefore, it does not seem likely that PPPD cells were derived from cell-types other than pancreatic ductal cells.
In conclusion, we established a reliable method for isolating pancreatic epithelial ductal cells from adult normal mouse pancreas. The presence of intracellular cAMP elevating factors was crucial to the proliferation and maintenance of pancreatic epithelial cells under both serum-free and serum-containing culture conditions. We also demonstrated that the cells designated as PPPD cells give rise not only to pancreatic endocrine and exocrine cells but also to albumin-producing cells. PPPD cells should allow us to investigate the precise molecular mechanisms involved in the differentiation of pancreatic ductal cells into insulin-producing cells and offer promise for a non-invasive therapy for type 1 diabetes and liver cirrhosis.
This work was supported by grants from the Japanese Ministry of Education, Science, Sports and Culture and from the Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, Japan Science and Technology Corporation. The authors declare that they have no duality of interest.