Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas

One major unresolved question in the field of pancreas biology is whether ductal cells have the ability to generate insulin-producing β-cells. Conclusive examination of this question has been limited by the lack of appropriate tools to efficiently and specifically label ductal cells in vivo. We generated Sox9CreER(T2) mice, which, during adulthood, allow for labeling of an average of 70% of pancreatic ductal cells, including terminal duct/centroacinar cells. Fate-mapping studies of the Sox9(+) domain revealed endocrine and acinar cell neogenesis from Sox9(+) cells throughout embryogenesis. Very small numbers of non-β endocrine cells continue to arise from Sox9(+) cells in early postnatal life, but no endocrine or acinar cell neogenesis from Sox9(+) cells occurs during adulthood. In the adult pancreas, pancreatic injury by partial duct ligation (PDL) has been suggested to induce β-cell regeneration from a transient Ngn3(+) endocrine progenitor cell population. Here, we identify ductal cells as a cell of origin for PDL-induced Ngn3(+) cells, but fail to observe β-cell neogenesis from duct-derived cells. Therefore, although PDL leads to activation of Ngn3 expression in ducts, PDL does not induce appropriate cues to allow for completion of the entire β-cell neogenesis program. In conclusion, although endocrine cells arise from the Sox9(+) ductal domain throughout embryogenesis and the early postnatal period, Sox9(+) ductal cells of the adult pancreas no longer give rise to endocrine cells under both normal conditions and in response to PDL.     

Lectin as a marker for staining and purification of embryonic pancreatic epithelium

The embryonic pancreatic epithelium, and later the ductal epithelium, is known to give rise to the endocrine and exocrine cells of the developing pancreas, but no specific surface marker for these cells has been identified. Here, we utilized Dolichos Biflorus Agglutinin (DBA) as a specific marker of these epithelial cells in developing mouse pancreas. From the results of an immunofluorescence study using fluorescein-DBA and pancreatic specific cell markers, we found that DBA detects specifically epithelial, but neither differentiating endocrine cells nor acinar cells. We further applied this marker in an immunomagnetic separation system (Dynabead system) to purify these putative multi-potential cells from a mixed developing pancreatic cell population. This procedure could be applied to study differentiation and cell lineage selections in the developing pancreas, and also may be applicable to selecting pancreatic precursor cells for potential cellular engineering.     

Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing β-cells

The Notch-signaling pathway is known to be fundamental in controlling pancreas differentiation. We now report on using Cre-based fate mapping to indelibly label pancreatic Notch-responsive cells (PNCs) at larval stages and follow their fate in the adult pancreas. We show that the PNCs represent a population of progenitors that can differentiate to multiple lineages, including adult ductal cells, centroacinar cells (CACs) and endocrine cells. These endocrine cells include the insulin-producing β-cells. CACs are a functional component of the exocrine pancreas; however, our fate-mapping results indicate that CACs are more closely related to endocrine cells by lineage as they share a common progenitor. The majority of the exocrine pancreas consists of the secretory acinar cells; however, we only detect a very limited contribution of PNCs to acinar cells. To explain this observation we re-examined early events in pancreas formation. The pancreatic anlage that gives rise to the exocrine pancreas is located in the ventral gut endoderm (called the ventral bud). Ptf1a is a gene required for exocrine pancreas development and is first expressed as the ventral bud forms. We used transgenic marker lines to observe both the domain of cells expressing ptf1a and cells responding to Notch signaling. We do not detect any overlap in expression and demonstrate that the ventral bud consists of two cell populations: a ptf1-expressing domain and a Notch-responsive progenitor core. As pancreas organogenesis continues, the ventral bud derived PNCs align along the duct, remain multipotent and later in development differentiate to form secondary islets, ducts and CACs.     

Exocrine pancreas development in zebrafish

Although many of the genes that regulate development of the endocrine pancreas have been identified, comparatively little is known about how the exocrine pancreas forms. Previous studies have shown that exocrine pancreas development may be modeled in zebrafish. However, the timing and mechanism of acinar and ductal differentiation and morphogenesis have not been described. Here, we characterize zebrafish exocrine pancreas development in wild type and mutant larvae using histological, immunohistochemical and ultrastructural analyses. These data allow us to identify two stages of zebrafish exocrine development. During the first stage, the exocrine anlage forms from rostral endodermal cells. During the second stage, proto-differentiated progenitor cells undergo terminal differentiation followed by acinar gland and duct morphogenesis. Immunohistochemical analyses support a model in which the intrapancreatic ductal system develops from progenitors that join to form a contiguous network rather than by branching morphogenesis of the pancreatic epithelium, as described for mammals. Contemporaneous appearance of acinar glands and ducts in developing larvae and their disruption in pancreatic mutants suggest that common molecular pathways may regulate gland and duct morphogenesis and differentiation of their constituent cells. By contrast, analyses of mind bomb mutants and jagged morpholino-injected larvae suggest that Notch signaling principally regulates ductal differentiation of bipotential exocrine progenitors.     

Induction of mouse pancreatic ductal differentiation, an in vitro assay

Despite recent technical advances for studying lineage tracing and gene functions, our knowledge of pancreatic duct progenitor cells and mechanisms involved in their differentiation remains a huge void in our understanding of pancreatic development. A deeper insight into ductal differentiation is needed because ductal cells may harbor pancreatic stem/progenitor cells that could give rise to new islets. Also, since the most common pancreatic tumors form structures expressing ductal cell-specific markers, studies of ductal development may provide better markers for pancreatic tumor classification. One major longstanding problem in the study of pancreatic ductal differentiation has been the lack of an effective in vitro model. We thus wished to develop an in vitro system for the study of pancreatic duct development. In doing so, we have developed a specific culture condition to promote ductal differentiation of E11.5 pancreatic rudiments. Normally, pancreatic explants cultured in vitro develop to form endocrine, acinar, as well as ductal cells. Here, we report that addition of a combination of EGF, fibroblast growth factor-10, and platelet-derived growth factor-AA to the explant cultures promotes ductal differentiation, while preventing endocrine and acinar differentiation. This culture system for differentiation and enrichment of pancreatic ductal cells may allow identification of gene(s) involved in ductal development.     

Preferential expression of cystein-rich secretory protein-3 (CRISP-3) in chronic pancreatitis

BACKGROUND: Chronic pancreatitis (CP) is a progressive inflammatory process resulting in exocrine and endocrine pancreatic insufficiency in advanced stages. Cysteine-rich secretory protein (CRISP-3) has been identified as a defense-associated molecule with predominant expression in the salivary gland, pancreas and prostate. AIMS: In this study, we investigated CRISP-3 expression in normal pancreatic tissues, chronic pancreatitis tissues, pancreatic cancer tissues and pancreatic cancer cell lines, as well as in other gastrointestinal organs. MATERIALS AND METHODS: 15 normal pancreatic tissues, 14 chronic pancreatitis tissues and 14 pancreatic cancer tissues as well as three pancreatic cancer cell lines were analyzed. Moreover, hepatocellular carcinoma and esophageal, stomach and colon cancers were also analyzed together with the corresponding normal controls. RESULTS: CRISP-3 was expressed at moderate to high levels in chronic pancreatitis tissues and at moderate levels in pancreatic cancer tissues but at low levels in normal pancreatic tissues, and was absent in three pancreatic cancer cell lines. CRISP-3 expression was below the level of detection in all cancerous gastrointestinal tissues and in all normal tissues except 2 of 16 colon tissue samples. CRISP-3 mRNA signals and immunoreactivity were strongly present in the cytoplasm of degenerating acinar cells and in small proliferating ductal cells in CP tissues and CP-like lesions in pancreatic cancer tissues. In contrast, CRISP-3 expression was weak to absent in the cytoplasm of cancer cells as well as in acinar cells and ductal cells in pancreatic cancer tissues and normal pancreatic tissues. CONCLUSION: These results reveal that the distribution of CRISP-3 in gastrointestinal tissues is predominantly in the pancreas. High levels of CRISP-3 in acinar cells dedifferentiating into small proliferating ductal cells in CP and CP-like lesions in pancreatic cancer suggests a role of this molecule in the pathophysiology of CP.     

Thyroid hormones promote endocrine differentiation at expenses of exocrine tissue

Diabetes is caused by loss or dysfunction of pancreatic beta cells. Generation of beta cells in vitro is a promising strategy to develop a full-scale cell therapy against diabetes, and the development of methods without gene transfer may provide safer protocols for human therapy. Here we show that thyroid hormone receptors are expressed in embryonic murine pancreas. Addition of the thyroid hormone T3 in an ex vivo culture model of embryonic (E12.5) dorsal pancreas, mimicking embryonic pancreatic development, promoted an increase of ductal cell number at expenses of the acinar compartment. Double labeled cells expressing specific markers for ductal and acinar cells were observed, suggesting cell reprogramming. Increased mRNA levels of the pro-endocrine gene Ngn3 and an increased number of beta cells were detected in cultures treated previously with T3 suggesting that ductal cells promoted by T3 can subsequently differentiate into endocrine cells. So, indirectly, T3 induced endocrine differentiation. Moreover, T3 induced the expression of the pro-endocrine gene Ngn3 in the acinar 266-6 cell line. The pro-endocrine effect of T3 in the pancreatic explants and in the acinar cell line, was abrogated by the Akt inhibitor Ly294002 indicating the involvement of Akt signaling in this process. Altogether we show numerous evidences that define T3 as a promising candidate to generate endocrine cells from exocrine tissue, using ectopically gene expression free protocols, for cell therapy against diabetes.     

Pancreatic duct secretion: experimental methods, ion transport mechanisms and regulation

The pancreatic ductal tree conveys enzymatic acinar products to the duodenum and secretes the fluid and ionic components of pancreatic juice. The physiology of pancreatic duct cells has been widely studied, but many questions are still unanswered concerning their mechanisms of ionic transport. Differences in the transport mechanisms operating in the ductal epithelium has been described both among different species and in the different regions of the ductal tree. In this review we summarize the methods developed to study pancreatic duct secretion both in vivo and in vitro, the different mechanisms of ionic transport that have been reported to date in the basolateral and luminal membranes of pancreatic ductal cells and the regulation of pancreatic duct secretion by nervous endocrine and paracrine influences.     

Nestin expression in pancreatic exocrine cell lineages

Expression of nestin has been suggested to be a characteristic of pancreatic islet stem cells. To determine whether nestin is indeed expressed in such putative cells during embryonic development, or in the adult pancreas after injury, we performed a cell lineage analysis using two independent lines of transgenic mice encoding Cre recombinase under the control of rat nestin cis-regulatory sequences, each crossed with loxP-bearing R26R mice. F1 animals produced the reporter molecule beta-galactosidase only upon Cre-mediated recombination, thus solely in cells using (or having used) the transgenic nestin promoter. In early pancreatic primordia, beta-galactosidase was observed in mesenchymal and epithelial cells. At later developmental stages or in adults, vast clusters of acinar cells and few ductal cells were labeled, in addition to fibroblasts and vascular cells, but no endocrine cells were tagged by beta-galactosidase. This correlated with the transient expression, observed with an anti-nestin antibody, of endogenous nestin in about 5% of epithelial cells during development (whether in cord-forming arrangements or in nascent acini), and in vascular and mesenchymal structures. After partial pancreatectomy, there was a transient increase of the number of anti-nestin-labeled endothelial cells, but again, no endocrine cells bore beta-galactosidase. Together, these findings show that nestin is expressed in the pancreatic exocrine cell lineage, and suggest that consistent nestin expression is not a major feature of islet endocrine progenitor cells.     

Activated Notch1 prevents differentiation of pancreatic acinar cells and attenuate endocrine development

Mice carrying loss-of-function mutations in certain Notch pathway genes display increased and accelerated pancreatic endocrine development, leading to depletion of precursor cells followed by pancreatic hypoplasia. Here, we have investigated the effect of expressing a constitutively active form of the Notch1 receptor (Notch1(ICD)) in the developing pancreas using the pdx1 promoter. At e10.5 to e12.5, we observe a disorganized pancreatic epithelium with reduced numbers of endocrine cells, confirming a repressive activity of Notch1 upon the early differentiation program. Subsequent branching morphogenesis is impaired and the pancreatic epithelium forms cyst-like structures with ductal phenotype containing a few endocrine cells but completely devoid of acinar cells. The endocrine cells that do form show abnormal expression of cell type-specific markers. Our observations show that sustained Notch1 signaling not only significantly represses endocrine development, but also fully prevents pancreatic exocrine development, suggesting that a possible role of Notch1 is to maintain the undifferentiated state of common pancreatic precursor cells.     

TNF-Like Weak Inducer of Apoptosis (TWEAK) Promotes Beta Cell Neogenesis from Pancreatic Ductal Epithelium in Adult Mice

Aim/Hypothesis

The adult mammalian pancreas has limited ability to regenerate in order to restore adequate insulin production from multipotent progenitors, the identity and function of which remain poorly understood. Here we test whether the TNF family member TWEAK (TNF-like weak inducer of apoptosis) promotes β-cell neogenesis from proliferating pancreatic ductal epithelium in adult mice.

Methods

C57Bl/6J mice were treated with Fc-TWEAK and pancreas harvested at different time points for analysis by histology and immunohistochemistry. For lineage tracing, 4 week old double transgenic mice CAII-CreER^TM: R26R-eYFP were implanted with tamoxifen pellet, injected with Fc-TWEAK or control Ig twice weekly and analyzed at day 18 for TWEAK-induced duct cell progeny by costaining for insulin and YFP. The effect of TWEAK on pancreatic regeneration was determined by pancytokeratin immunostaining of paraffin embedded sections from wildtype and TWEAK receptor (Fn14) deficient mice after Px.

Results

TWEAK stimulates proliferation of ductal epithelial cells through its receptor Fn14, while it has no mitogenic effect on pancreatic α- or β-cells or acinar cells. Importantly, TWEAK induces transient expression of endogenous Ngn3, a master regulator of endocrine cell development, and induces focal ductal structures with characteristics of regeneration foci. In addition, we identify by lineage tracing TWEAK-induced pancreatic β-cells derived from pancreatic duct epithelial cells. Conversely, we show that Fn14 deficiency delays formation of regenerating foci after Px and limits their expansion.

Conclusions/Interpretation

We conclude that TWEAK is a novel factor mediating pancreatic β-cell neogenesis from ductal epithelium in normal adult mice.     

Immunohistochemical localization of pancreatic spasmolytic polypeptide (PSP) in the pig.

Pancreatic spasmolytic polypeptide (PSP) is a peptide that is isolated from the porcine pancreas and that affects intestinal motility and growth of intestinal tumour cells in vitro. The peptide was recently demonstrated to be present in large amounts in pancreatic juice. The cellular origin of the peptide, however, is largely unclarified and the localization was therefore studied of PSP in pigs using immunohistochemistry. Positive immunoreactions were seen in the pancreas, the stomach, the duodenum, the jejunum and the ileum. In the pancreas, the PSP immunoreaction was seen in all acinar cells; no immunoreaction was seen in the endocrine islets. In the stomach, it was localized to the mucous cells of the glands in the cardiac gland region, the corpus and the pylorus. In the duodenum a strong immunoreaction was present in Brunner's glands and in the cells of their excretory ducts. In the jejunum and ileum, PSP immunoreactivity was seen in some of the cells in the epithelium of the crypts of Lieberkühn. A peptide chromatographically identical to highly purified PSP was identified in pancreas and stomach extracts. Thus epithelial cells in all parts of the stomach and small intestine contribute to the supply of PSP to the gut lumen.     

Notch/Rbp-j signaling prevents premature endocrine and ductal cell differentiation in the pancreas

To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas.     

Immunohistochemical localization of pancreatic spasmolytic polypeptide (PSP) in the pig

Summary Pancreatic spasmolytic polypeptide (PSP) is a peptide that is isolated from the porcine pancreas and that affects intestinal motility and growth of intestinal tumour cells in vitro. The peptide was recently demonstrated to be present in large amounts in pancreatic juice. The cellular origin of the peptide, however, is largely unclarified and the localization was therefore studied of PSP in pigs using immunohistochemistry. Positive immunoreactions were seen in the pancreas, the stomach, the duodenum, the jejunum and the ileum. In the pancreas, the PSP immunoreaction was seen in all acinar cells; no immunoreaction was seen in the endocrine islets. In the stomach, it was localized to the mucous cells of the glands in the cardiac gland region, the corpus and the pylorus. In the duodenum a strong immunoreaction was present in Brunner's glands and in the cells of their excretory ducts. In the jejunum and ileum, PSP immunoreactivity was seen in some of the cells in the epithelium of the crypts of Lieberkühn. A peptide chromatographically identical to highly purified PSP was identified in pancreas and stomach extracts. Thus epithelial cells in all parts of the stomach and small intestine contribute to the supply of PSP to the gut lumen.     

Impaired differentiation of endocrine and exocrine cells of the pancreas in transgenic mouse expressing the truncated type II activin receptor

Activin A is expressed in endocrine precursor cells of the fetal pancreatic anlage. To determine the physiological significance of activins in the pancreas, a transgenic mouse line expressing the truncated type II activin receptor under the control of beta-actin promoter was developed. Histological analyses of the pancreas revealed that the pancreatic islets of the transgenic mouse were small in size and were located mainly along the pancreatic ducts. Immunoreactive insulin was detected in islets, some acinar cells, and in some epithelial cells in the duct. In addition, there were abnormal endocrine cells outside the islets. The shape and the size of the endocrine cells varied and some of them were larger than islets. These cells expressed immunoreactive insulin and glucagon. In the exocrine portion, there were morphologically abnormal exocrine cells, which did not form a typical acinar structure. The cells lacked spatial polarity characteristics of acinar cells but expressed immunoreactive amylase, which was distributed diffusely in the cytoplasm. Plasma glucose concentration was normal in the transgenic mouse before and after the administration of glucose. The insulin content of the pancreas in transgenic and normal mice was nearly identical. These results suggest that activins or related ligands regulate the differentiation of the pancreatic endocrine and exocrine cells.