Human Pancreatic Ductal Cells Research Articles

Chronic Pancreatitis and Pancreatic Cancer: Prediction and Mechanism

We investigated the SPINK 1 mutations in 156 sporadic pancreatic cancer (PCa), and 8 pancreatic cancer with chronic pancreatitis (CPPCa) patients, and in 527 healthy subjects. The results demonstrated that 3 of 8 patients with CPPCa (37.5%) had the SPINK 1 gene N34S mutation. In addition, 3 of 156 sporadic PCa patients (1.9%) and 1 of them (0.6%) had the N34S and IVS3+2T>C mutation, respectively. The combined frequency of 2.5% was significantly higher than that of healthy subjects (0.38%), suggesting that the SPINK 1 mutation is an important risk factor for the development of pancreatic cancer. To investigate the genetic difference between sporadic PCa and CPPCa, we investigated several factors involved in the pathogenesis of PCa in 6 CPPCa and 15 sporadic PCa patients. The factors examined were genes including K-ras, p53, smad 4, p-smad 1, CXCL 14, NF-kB subunit p65 and Wnt 5a. No significant difference was found in the comparative examination of these factors, suggesting that the molecular disorders appeared to occur similarly in CPPCa as well as sporadic PCa. To assess the role of fibrosis in pancreatic carcinogenesis, we investigated the effects of pancreatic stellate cells (PSCs), which are largely responsible for pancreatic fibrogenesis, on duct cells, in vitro and in vivo. Activated PSCs were found surrounding precancerous duct cells in the tissues of a dimethylbenzanthracene mouse model and those of human PCa. Consistently, human pancreatic epithelial duct cells cultured with PSC conditioned media showed increased cell proliferation and colony formation, suggesting that PSCs may promote pancreatic ductal tumorigenesis.

Bicarbonate Transport by the Human Pancreatic Ductal Cell Line HPAF

The human pancreatic duct cell line, HPAF, has been shown previously to secrete Cl(-) in response to Ca(2+)-mobilizing stimuli. Our aim was to assess the capacity of HPAF cells to transport and secrete HCO3(-). HPAF cells were grown as confluent monolayers on permeable supports. Short-circuit current was measured by voltage clamp. Intracellular pH (pHi) was measured by microfluorometry in cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In HCO3(-)-free solutions, ATP-evoked changes in short-circuit current were inhibited by bumetanide, and the recovery of pHi from acid loading was abolished by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). In the presence of HCO3(-), ATP-evoked secretion was no longer inhibited by bumetanide, and there was a strong EIPA-insensitive recovery from acid loading, which was inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS). ATP, but not forskolin, stimulated HCO3(-) efflux from the cells. In the absence of HCO3(-), ATP-evoked Cl(-) secretion is driven by a basolateral Na(+)-K(+)-2Cl(-) cotransporter, and pH(i) is regulated by apical and basolateral Na(+)/H(+) exchangers. In the presence of HCO3(-), ATP-evoked secretion is sustained in the absence of Na(+)-K(+)-2Cl(-) cotransporter activity and is probably driven by basolateral Na(+)-HCO3(-) cotransport.

Islet neogenesis-associated protein-related pentadecapeptide enhances the differentiation of islet-like clusters from human pancreatic duct cells

The differentiation of pancreatic ductal epithelial cells into β-cells has been considered as an alternative method for increasing the number of islets for transplantation. Critical factors have been introduced into the in vitro differentiation protocol for pancreatic duct cells in order to enhance the production of β-cells. Islet neogenesis-associated protein (INGAP) is an initiator of islet neogenesis and the peptide sequence 104–118 of INGAP has been shown to stimulate an increase in β-cell mass in animals and also found in human pathological states involving islet neogenesis. To establish a novel method for the differentiation of β-cells from human pancreatic duct cells with INGAP-related pentadecapeptide (INGAP-PP), the pancreatic duct cells were isolated, purified and expanded in vitro and differentiated using a four-step protocol that included nicotinamide, exendin-4, transforming growth factor β 1 and INGAP-PP/Scrambled peptide (Scrambled-P). The production of islet-like clusters (ILCs) in the INGAP-PP group was significantly higher than that in the Scrambled-P control group after differentiation from an equal number of duct cells. The duct cells showed positive staining and expression for cytokeratin 19, pancreatic duodenal homeobox-1, nestin, and were negative for insulin and glucagon, as detected by both immunofluorescence and RT-PCR. Following differentiation the cells became insulin and glucagon positive. In addition, the ILCs from the INGAP-PP group secreted higher levels of insulin and C-peptide than the Scrambled-P group under a high glucose challenge. We conclude that INGAP peptide enhances the in vitro differentiation of pancreatic duct cells into islet-like clusters.

Expression of neuropilin-1 in human pancreatic ductal carcinoma and MIA PaCa- II cell lines

Objective To investigate the expression of Neuropilin-1 in human pancreatic ductal adenocarcinoma (PDAC) specimens and cell lines. Methods Expression and distribution of Neuropilin-1 were detected in operation tissue specimens of human pancreatic ductal adenocarcinoma with HE, immunohistochemistry and RT-PCR. Results Immunohistochemical analysis revealed that Neuropilin-1 were expressed in human pancreatic adenocarcinoma specimens, precancerous tissue and MIA PaCa-Ⅱ cell lines, but was absent in normal pancreatic tissue, mRNA of Neuropilin-1 could also be detected the in human pancreatic adenocarcinoma specimens, precancerous tissue and MIA PaCaⅡ cell Lines, while could not be detected in normal pancreatic tissue. Neuropilin-1 expression in pancreatic adenocarcinoma were higher than that in normal pancreas, and the differences were significant( P < 0. 01 ). Conclusions Neuropilin-1 might play an potential role in carcinogenesis for pancreatic cancer. Neuropilin-1 may be relevant to perineural invasion of pancreatic carcinoma by some signal transduction. Key words: Neuropilin-l/Bl; Pancreatic neoplasms/ME

Up-regulation of L1CAM in Pancreatic Duct Cells Is Transforming Growth Factor β1– and Slug-Dependent: Role in Malignant Transformation of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is thought to originate from ductal structures, exhibiting strong desmoplastic reaction with stromal pancreatic myofibroblasts (PMF), which are supposed to drive PDAC tumorigenesis. Previously, we observed high expression of the adhesion molecule L1CAM (CD171) in PDAC cells accounting for chemoresistance. Thus, this study aimed to investigate whether PMFs are involved in the induction of tumoral L1CAM and whether this contributes to malignant transformation of pancreatic ductal cells and PDAC tumorigenesis. Immunohistochemistry of tissues from chronic pancreatitis specimens revealed considerable L1CAM expression in ductal structures surrounded by dense fibrotic tissue, whereas no L1CAM staining was seen in normal pancreatic tissues. Using the human pancreatic duct cell line H6c7, we show that coculture with PMFs led to a transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of L1CAM expression. Similarly, L1CAM expression increased in monocultured H6c7 cells after administration of exogenous TGF-beta1. Both TGF-beta1- and PMF-induced L1CAM expression were independent of Smad proteins but required c-Jun NH(2)-terminal kinase activation leading to the induction of the transcription factor Slug. Moreover, Slug interacted with the L1CAM promoter, and its knockdown abrogated the TGF-beta1- and PMF-induced L1CAM expression. As a result of L1CAM expression, H6c7 cells acquired a chemoresistant and migratory phenotype. This mechanism of TGF-beta1-induced L1CAM expression and the resulting phenotype could be verified in the TGF-beta1-responsive PDAC cell lines Colo357 and Panc1. Our data provide new insights into the mechanisms of tumoral L1CAM induction and how PMFs contribute to malignant transformation of pancreatic duct cells early in PDAC tumorigenesis.

Bestrophin expression and function in the human pancreatic duct cell line, CFPAC‐1

Pancreatic duct epithelial cells (PDECs) have been shown to express calcium activated chloride channels (CaCCs) and there is evidence for their involvement in fluid secretion from these cells. The molecular identity of the CaCC in PDECs remains unknown. Recently, the bestrophin family of proteins have been proposed as a potential molecular candidate for CaCCs. Expression of bestrophins is strongly correlated with the function of CaCCs in a variety of tissues. In the present study, the expression of bestrophins has been investigated in the cystic fibrosis pancreatic duct cell line, CFPAC-1. Iodide efflux analysis was used to characterise native CaCCs in CFPAC-1 cell monolayers. Efflux was induced with the addition of UTP (100 microM, 10.2 +/- 1.5 nmol min(-1)), which was blocked by the chloride channel blockers niflumic acid (81%) and DIDS (90%). The UTP-stimulated iodide efflux was shown to be Ca(2+) dependent and cAMP independent. RT-PCR analysis of RNA isolated from CFPAC-1 cells demonstrated positive identification of all four human bestrophin mRNAs. Western blot of CFPAC-1 cell protein isolates with antibodies specific to human bestrophin 1 (hBest1) showed that hBest1 protein was expressed in this cell line. HBest1 was present on the cell surface, demonstrated using biotinylation and confocal imaging, as well as in the cytoplasm. SiRNA-mediated silencing of hBest1 in CFPAC-1 cells reduced the UTP-stimulated iodide efflux by around 40%. This study provides evidence that the bestrophins are expressed in pancreatic duct cells and, more specifically, that hBest1 plays a role in the CaCCs found in these cells.

Decreased Expression of Angiotensin-Converting Enzyme 2 in Pancreatic Ductal Adenocarcinoma Is Associated with Tumor Progression

Angiotensin II (ANG II), the biologically active peptide of the renin-angiotensin system (RAS), is generated by angiotensin-converting enzyme (ACE) and is a regulator of cardiovascular homeostasis. Recently, there has been increasing evidence that ANG II is involved in the regulation of cell proliferation and migration, as well as angiogenesis via the ANG II-type 1 receptor (AT1R). These findings suggest that the ACE-ANG II-AT1R pathway is related to cancer biology. Previous reports have shown that ACE is preferentially expressed in pancreatic ductal adenocarcinoma (PDAC) tissues. Recently a homologue of ACE, angiotensin-converting enzyme 2 (ACE2), was reported to counterbalance the function of ACE, but the expression and role of ACE2 in PDAC are still unclear. In the present study, we analyzed the expression of ACE2 in invasive human PDAC and surrounding non-malignant tissues by Western blot analysis and immunohistochemistry. The ANG II concentration in homogenates of pancreatic tissues was measured with ELISA, and ACE2 protein was detected by Western blot analysis in BxPC3 and SW1990 human pancreatic ductal cancer cells. We have shown for the first time that the expression of ACE2 is decreased in PDAC tissues, in which ANG II was accumulated. Treatment of BxPC3 and SW1990 cells with ANG II decreased the expression of ACE2. Therefore, ANG II may contribute to the down-regulation of ACE2. Moreover, reduction of ACE2 expression by RNA interference promoted the proliferation of cultured pancreatic cancer cells. These findings suggest that ACE2 may have clinical potential as a novel molecular target for the treatment of PDAC.

Chmp1A functions as a novel tumor suppressor gene in human embryonic kidney and ductal pancreatic tumor cells

Chmp1A (Chromatin modifying protein 1A/Charged multivesicular protein 1A) is a member of the ESCRT-III (Endosomal Sorting Complex Required for Transport) family that was shown to function in endosome-mediated trafficking via multivesicular body (MVB) formation and sorting. Recent reports suggest that ESCRT complexes are also involved in cell cycle progression and tumor development. Using in vitro and in vivo model systems, we provide evidence that Chmp1A is a novel tumor suppressor, especially in the pancreas. We demonstrated that short hairpin RNA (shRNA) mediated stable silencing of Chmp1A in HEK 293T cells resulted in an increase of anchorage-independent growth in soft agar assay and tumor formation in xenograft assay. To investigate the involvement of Chmp1A in human tumor development we screened human cancer arrays and pancreatic tissue arrays. We discovered that Chmp1A mRNA and protein was reduced and/or altered (protein) in various human pancreatic tumors. To investigate the biological implication of these data, we either over-expressed or silenced Chmp1A in human pancreatic ductal tumor cells (PanC-1) and studied the effect of these manipulations on cell and tumor growth respectively. Stable over-expression of Chmp1A in PanC-1 cells resulted in cell growth inhibition and tumor xenograft inhibition respectively. In contrast, silencing of Chmp1 in PanC-1 cells resulted in the elevation of cell growth in vitro. Mechanistically, over-expression of Chmp1A strongly increased the protein level of P53 and phospho-P53. Taken together, our data indicates that Chmp1A is a novel tumor suppressor, especially in pancreas and that Chmp1A regulates tumor growth potentially through P53 signaling pathway.

CD40 activation in human pancreatic islets and ductal cells

CD40 expression on non-haematopoietic cells is linked to inflammation. We previously reported that CD40 is expressed on isolated human and non-human primate islets and its activation results in secretion of IL-8, macrophage inflammatory protein 1-beta (MIP-1beta) and monocyte chemoattractant protein-1 (MCP-1) through nuclear factor-kappaB and extracellularly regulated kinases 1/2 pathways. The objective of this study was to identify the pattern of gene expression, and to study viability and functionality affected by CD40-CD40 ligand (CD40L) interaction in human islets. Furthermore, we have studied the CD40-mediated cytokine/chemokine profile in pancreatic ductal cells, as they are always present in human islet transplant preparations and express CD40 constitutively. CD40-CD40L gene expression modulation was studied by microarray on islet cells depleted of ductal cells. Selected genes were validated by quantitative RT-PCR. The cytokine profile in purified ductal cells was evaluated by Luminex technology, based on the use of fluorescent-coated beads, known as microspheres, and capable of multiplex detection of proteins from a single sample. Glucose-stimulated insulin secretion and islet viability were assessed by perifusion and 7-aminoactinomycin D membrane exclusion, respectively. Statistical analysis of microarrays identified 30 genes exhibiting at least a 2.5-fold increase across all replicate arrays. The majority of them were related to oxidative stress/inflammation. Prominently upregulated were chemokine C-X-C motif ligand 1 (CXCL1), CXCL2 and CXCL3 belonging to the CXC family of chemokines related to IL-8. CD40-mediated CXCL1 secretion was confirmed by ELISA. The viability or in vitro function was not affected by CD40 activation. In addition to previously reported IL-8, MIP-1beta and MCP-1, CD40 stimulation in ductal cells produced IL-1beta, IFN-gamma, TNF-alpha, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. CD40 activation in islets and ductal cells produces cytokines/chemokines with a broad-spectrum range of biological functions.

Hedgehog inhibition prolongs survival in a genetically engineered mouse model of pancreatic cancer

Background and aims:Pancreatic cancer is among the most dismal of human malignancies. Current therapeutic strategies are virtually ineffective in controlling advanced, metastatic disease. Recent evidence suggests that the Hedgehog signalling...

Pancreatic Duct Cells in Human Islet Cell Preparations Are a Source of Angiogenic Cytokines Interleukin-8 and Vascular Endothelial Growth Factor

OBJECTIVE—Engraftment and function of human islet cell implants is considered to be dependent on their rapid and adequate revascularization. Studies with rodent islet grafts have shown that vascular endothelial growth factor (VEGF) expression by β-cells can promote this process. The present work examines whether human islet preparations produce VEGF as well as interleukin (IL)-8, another angiogenic protein, and assesses the role of contaminating duct cells in VEGF and IL-8–mediated angiogenesis.RESEARCH DESIGN AND METHODS—Human islet and pancreatic duct cell preparations are compared for their respective expression and production of VEGF and IL-8 during culture as well as following transplantation in nonobese diabetic (NOD)/scid mice. The associated angiogenic effects are measured in an in vitro aortic ring assay and in an in vivo chick embryo chorioallantoic membrane assay.RESULTS—Cultured pancreatic duct cells expressed 3- and 10-fold more VEGF and IL-8, respectively, than cultured human islet endocrine cells and released both proteins at angiogenic levels. The angiogenic effect of purified duct cells was higher than that of purified endocrine islet cells and was completely blocked by a combination of IL-8 and VEGF antibodies. Human duct cell implants under the kidney capsule of NOD/scid mice expressed higher levels of IL-8 and VEGF than human islet cell implants and induced circulating IL-8 and VEGF levels during the first day posttransplantation.CONCLUSIONS—Human duct cell–released IL-8 and VEGF may help revascularization of currently used human islet cell grafts. Further work should examine whether and when this effect can prevail over other inflammatory and immune influences of this cell type.

The effect of bile acids on human pancreatic duct cells

We have previously shown that exposure of intact guinea pig pancreatic ducts to bile acids, which may occur e.g. in acute pancreatitis, induces intracellular acidification and a marked increase in intracellular Ca2+ concentration ([Ca2+]i). Moreover, a low dose of chenodeoxycholate (CDC) stimulates the luminal anion exchanger which suggests a possible role of cystic fibrosis transmembrane conductance regulator (CFTR) in this effect. There are no available data concerning the effect of bile acids on human pancreatic duct cells. The aim of our study was to examine the effects of CDC or glycochenodeoxycholate (GCDC) in human cystic fibrosis CFPAC-1 pancreatic duct cells which are homozygous for the most common delF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutation or in CFPAC-1 cells which are corrected for the defective CFTR. Methods: We transduced polarized CFPAC-1 cells (n=4–6) with recombinant Sendai virus (SeV) constructs (multiplicity of infection=3) containing the CFTR or β-galactosidase (LacZ) cDNA. 48–72h after infection, the intracellular pH (pHi) and [Ca2+]i of the cells were recorded by microfluorimetry. Results: Both apical and basolateral administration of CDC or GCDC caused dose-dependent (100–1000µM) reversible intracellular acidification. Our results did not show significant differences in the rate of intracellular acidification caused by bile acids in SeV-CFTR and SeV-LacZ transduced cells. The exposure of cell lines to bile acids induced a marked increase in [Ca2+]i and CDC caused the largest response. Similarly to that observed in primary ducts, the effect of CDC administration from the apical membrane was greater than from the basolateral membrane. Furthermore, glycine conjugated bile acids also resulted in Ca2+ signalling when administered from either membrane of the cells. Conclusions: Human pancreatic duct cells respond similarly to administration of bile acids compared to that observed in guinea pigs. CFTR expression is not necessary for pHi changes and Ca2+ signalling caused by bile acids.

N-Acetylcysteine Derivative Inhibits CD40-Dependent Proinflammatory Properties of Human Pancreatic Duct Cells

We recently observed that duct cells constitutively express CD40, a membrane molecule whose engagement results in duct cell activation and proinflammatory cytokine secretion. This observation suggests a potential role of this pathway in the pathogenesis of type 1 diabetes, islet graft rejection, or acute pancreatitis. In this article, we investigated whether a salt derivative of N-acetyl-L-cysteine, Nacystelyn, could modulate CD40 expression on duct cells and the response of activated duct cells to CD40 engagement. We assessed the effects of Nacystelyn on CD40 expression and function in human caucasian pancreatic adenocarcinoma, ATCC n degrees THB-80 (CAPAN-2) cells, a human pancreatic duct cell line. CD40 expression was analyzed by flow cytometry. To assess CAPAN-2 cell responses to CD40 engagement, we looked at nuclear factor-kappaB transcription factor activation using enzyme-linked immunosorbent assay and electrophoretic mobility shift assay and cytokine mRNA levels by quantitative real-time reverse transcriptase polymerase chain reaction. We observed that Nacystelyn dose-dependently inhibited CD40 expression on CAPAN-2 cells as well as CD40-induced nuclear factor kappaB activation and proinflammatory cytokines up-regulation. Our data suggest that Nacystelyn could be considered as a useful tool to prevent immune and inflammatory responses in pancreatic disorders by interfering with the CD40 pathway in pancreatic duct cells.

Interleukin‐8 increases vascular endothelial growth factor and neuropilin expression and stimulates ERK activation in human pancreatic cancer

Interleukin-8 (IL-8) is associated with tumorigenesis by promoting angiogenesis and metastasis. Although up-regulation of IL-8 is indicated in many cancers, its function in pancreatic cancer has not been well characterized. In this study we examined the expression of IL-8 on pancreatic cancer cells and clinical tissue specimens, and investigated the effect of exogenous IL-8 on gene expression, and signaling in human pancreatic cancer cells. We found that pancreatic cancer cells expressed higher amount of IL-8 mRNA than normal human pancreatic ductal epithelium cells. IL-8 mRNA was also substantially overexpressed in 11 of 14 (79%) clinical pancreatic-adenocarcinoma samples compared with that in their surrounding normal tissues. Exogenous IL-8 up-regulated the expression of vascular endothelial growth factor(165), and neuropilin (NRP)-2 in BxPC-3 cells, one of human pancreatic cancer cell lines. IL-8 expression was inducible by hypoxia mimicking reagent cobalt chloride. In addition, IL-8 activated extracellular signal-regulated kinase (ERK)1/2 signaling pathway in BxPC-3 cells. Our studies suggest that IL-8 might be a malignant factor in human pancreatic cancer by induction of vascular endothelial growth factor and NRP-2 expression and ERK activation. Targeting IL-8 along with other antiangiogenesis therapy could be an effective treatment for this malignancy.

In Vitro Induction of Endocrine Differentiation Markers in Human Pancreatic Ductal Cells by Islet Neogenesis Associated Protein (INGAP)

In Vitro Induction of Endocrine Differentiation Markers in Human Pancreatic Ductal Cells by Islet Neogenesis Associated Protein (INGAP)

Expression and Subcellular Localization of a 35-kDa Carbonic Anhydrase IV in a Human Pancreatic Ductal Cell Line (Capan-1)

The high intraluminal concentrations of HCO(3)(-) in the human pancreatic ducts have suggested the existence of a membrane protein supplying the Cl(-)/HCO(3)(-) exchanger. Membrane-bound carbonic anhydrase IV (CA IV) is one of the potential candidates for this protein. The difficulties in isolating human pancreatic ducts have led the authors to study the molecular mechanisms of HCO(3)(-) secretion in cancerous cell lines. In this work, we have characterized the CA IV expressed in Capan-1 cells. A 35-kDa CA IV was detected in cell homogenates and purified plasma membranes. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase-C indicated that this CA IV was not anchored by a glycosylphosphatidylinositol (GPI). In contrast, its detection on purified plasma membranes by an antibody specifically directed against the carboxyl terminus of human immature GPI-anchored CA IV indicated that it was anchored by a C-terminal hydrophobic segment. Immunoelectron microscopy and double-labeling immunofluorescence revealed that this CA IV was present on apical plasma membranes, and in the rough endoplasmic reticulum, the endoplasmic reticulum-Golgi intermediate compartment, the Golgi complex, and secretory granules, suggesting its transport via the classical biosynthesis/secretory pathway. The expression in Capan-1 cells of a 35-kDa CA IV anchored in the apical plasma membrane through a hydrophobic segment, as is the case in the healthy human pancreas, should make the study of its role in pancreatic HCO(3)(-) secretion easier.

CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a Sendai virus vector

Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF transmembrane conductance regulator (CFTR) Cl- channel. About 70% of CF patients are exocrine pancreatic insufficient due to failure of the pancreatic ducts to secrete a HCO3- -rich fluid. Our aim in this study was to investigate the potential of a recombinant Sendai virus (SeV) vector to introduce normal CFTR into human CF pancreatic duct (CFPAC-1) cells, and to assess the effect of CFTR gene transfer on the key transporters involved in HCO3- transport. Using polarized cultures of homozygous F508del CFPAC-1 cells as a model for the human CF pancreatic ductal epithelium we showed that SeV was an efficient gene transfer agent when applied to the apical membrane. The presence of functional CFTR was confirmed using iodide efflux assay. CFTR expression had no effect on cell growth, monolayer integrity, and mRNA levels for key transporters in the duct cell (pNBC, AE2, NHE2, NHE3, DRA, and PAT-1), but did upregulate the activity of apical Cl-/HCO3- and Na+/H+ exchangers (NHEs). In CFTR-corrected cells, apical Cl-/HCO3- exchange activity was further enhanced by cAMP, a key feature exhibited by normal pancreatic duct cells. The cAMP stimulated Cl-/HCO3- exchange was inhibited by dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2-DIDS), but not by a specific CFTR inhibitor, CFTR(inh)-172. Our data show that SeV vector is a potential CFTR gene transfer agent for human pancreatic duct cells and that expression of CFTR in CF cells is associated with a restoration of Cl- and HCO3- transport at the apical membrane.

CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a Sendai virus vector

CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a Sendai virus vector

Differentiation of Affinity-Purified Human Pancreatic Duct Cells to β-Cells

To test whether pancreatic duct cells are in vitro progenitors, they were purified from dispersed islet-depleted human pancreatic tissue using CA19-9 antibody. The purified fraction was almost entirely CK19+ with no insulin+ cells, whereas the unpurified cells (crude duct) were 56% CK19+ and 0.4% insulin+ of total cells (0.7% of CK19+ cells). These cells were expanded as monolayers, aggregated under serum-free conditions, and transplanted into normoglycemic NOD/SCID mice. In crude duct grafts, insulin+ cells increased to 6.1% of CK19+ cells. Purified duct cells had slow expansion and poor aggregation, as well as engraftment. The addition of 0.1% cultured stromal cells improved these parameters. These stromal cells contained no CK19+ cells and no insulin by either quantitative RT-PCR or immunohistochemistry; stromal cell aggregates and grafts contained no insulin+ cells. Aggregation of purified duct plus stromal preparations induced insulin+ cells (0.1% of CK19+ cells), with further increase to 1.1% in grafts. Insulin mRNA mirrored these changes. In these grafts, all insulin+ cells were in duct-like structures, while in crude duct grafts, 85% were. Some insulin+ cells coexpressed duct markers (CK19 and CA19-9) and heat shock protein (HSP)27, a marker of nonislet cells, suggesting the transition from duct. Thus, purified duct cells from adult human pancreas can differentiate to insulin-producing cells.

Differences in HCO3-/CO2 permeabilities at the apical and basolateral membranes of cystic fibrosis human pancreatic duct cells

Differences in HCO3-/CO2 permeabilities at the apical and basolateral membranes of cystic fibrosis human pancreatic duct cells