Pancreatic Cell Cultures Research Articles

INFLUENCE OF MICROSTRUCTURED COLLAGEN HYDROGEL ON PANCREATIC ISLET CELL CULTURES

Aim. A study of the in fl uence of microstructured collagen hydrogel (biomatrix) on survival and growth charac- teristics of islet cell cultures at their co-incubation. Materials and methods. As a biomatrix, the microstructured collagen hydrogel of linear series Sphero ® GEL was used. Islet cell cultures obtained from newborn rabbit pan- creas were inoculated onto the biomatrix surface, covered with growth medium, and placed in a CO 2 incubator. Changes occurring with biomatrix and cultures were observed by means of an inverted microscope and histolo- gical studies, including immunohistochemical analysis. Results. The presence of the microstructured collagen hydrogel matrix during the incubation of fl oating islet cell cultures promoted long-term preservation of the structural integrity and hormonal activity. Simultaneously the formation of cultures of pancreatic progenitor cells (islet cells precursors) was observed. Conclusion. Collagen hydrogel has a favorable effect on the formation and survival of islet cell cultures and can be used as a matrix of a tissue-engineered pancreas construct.

Lipid‐dependent proliferation of breast and pancreatic cancer cell lines (605.7)

Cancer cells rely heavily on anaerobic glycolysis for energy production, and have the capacity to synthesize their own sup¬ply of biologically active compounds of predominantly lipid origin. We hypothesize that any perturbation of this lipid pool, caused by drug treatment or changes in lipid metabolism, will result in significant alterations in the biological processes of these cells and result in cell death. In these studies, the lipid content of breast and pancreatic cell culture models has been assessed. Staining with Nile Red and Oil Red O indicate that primary cancer cell lines contain significantly higher lipid levels than metastatic lines and that the subcellular localization of these lipids also varies. Separation, identification, and quantitation of total lipid extracts by TLC and LCMS indicates that the proportions of phospholipids also vary in relation to the level of tumor cell differentiation. Treatment with drugs and transfection of cells with plasmids for specific UGT isoforms, which are known to conjugate various bioactive lipids and to be down regulated in several cancers, affects the lipid content and results in decreased cell proliferation and death. Nanodelivery of these drugs and plasmids significantly enhances these effects. Examining the role of lipids in cancer cell homeostasis is a novel approach to cancer research.Grant Funding Source: Supported By DoD‐W81XWH1110795 and UAMS TRI ABCRP

Characterization of Trypanosoma cruzi infectivity, proliferation, and cytokine patterns in gut and pancreatic epithelial cells maintained in vitro

Trypanosoma cruzi infects all nucleated cells in both humans and experimental animals. As a prelude to our studies of T. cruzi pathogenesis in the gastrointestinal system, we have initiated in vitro cultures of gut (Caco-2 and HT-29) and pancreatic (Panc-1) epithelial cells. We show that along with primary human fibroblasts, all three cell lines are susceptible to infection and support proliferation of T. cruzi. Infection with T. cruzi modified dramatically the cytokines elaborated by these cells. Substantially greater quantities of IL-5 and TGF-β1 were produced by fibroblasts and Caco-2 and Panc-1 cells, whereas secretion of IFN-γ and TNF-α was greatly reduced in all three cell types. Since these cells are not known to be the primary sources of IFN-γ, we examined IFN-γ mRNA expression in these cells. Both Caco-2 and Panc-1 cells were found to express IFN-γ mRNA, validating its secretion. These findings may provide insight into signaling pathways that mediate innate immunity to T. cruzi and pathogenesis of gastrointestinal and pancreatic alterations in Chagas disease.

Antidiabetic and Beta Cell-Protection Activities of Purple Corn Anthocyanins

Antidiabetic and beta cell-protection activities of purple corn anthocyanins (PCA) were examined in pancreatic beta cell culture and db/db mice. Only PCA among several plant anthocyanins and polyphenols showed insulin secretion activity in culture of HIT-T15 cells. PCA had excellent antihyperglycemic activity (in terms of blood glucose level and OGTT) and HbA1c-decreasing activity when compared with glimepiride, a sulfonylurea in db/db mice. In addition, PCA showed efficient protection activity of pancreatic beta cell from cell death in HIT-T15 cell culture and db/db mice. The result showed that PCA had antidiabetic and beta cell-protection activities in pancreatic beta cell culture and db/db mice.

Tetrapeptide stimulates functional activity of pancreatic cells in aging

In this study, the molecular mechanisms of pancreoprotective action of the tetrapeptide H-Lys-Glu-Asp-Trp-NH2 in aging human pancreatic cells have been investigated. It has been established that the tetrapeptide under study increases the expression of matrix metallopreteinases MMP2 and MMP9, serotonin, glycoprotein CD79α, antiapoptotic protein Mcl-1, and proliferation markers PCNA and Ki67, as well as decreases the expression of proapoptotic protein p53 in aged pancreatic cell cultures. Thus, the clinical effect of the tetrapeptide observed in elderly patients with type-2 diabetes mellitus and pancreatitis may be due to its ability to activate the expression of signaling molecules, i.e., markers of functional activity of pancreatic cells.

Enhanced penetration into 3D cell culture using two and three layered gold nanoparticles

Nano-scale particles sized 10–400 nm administered systemically preferentially extravasate from tumor vasculature due to the enhanced permeability and retention effect. Therapeutic success remains elusive, however, because of inhomogeneous particle distribution within tumor tissue. Insufficient tumor vascularization limits particle transport and also results in avascular hypoxic regions with non-proliferating cells, which can regenerate tissue after nanoparticle-delivered cytotoxicity or thermal ablation. Nanoparticle surface modifications provide for increasing tumor targeting and uptake while decreasing immunogenicity and toxicity. Herein, we created novel two layer gold-nanoshell particles coated with alkanethiol and phosphatidylcholine, and three layer nanoshells additionally coated with high-density-lipoprotein. We hypothesize that these particles have enhanced penetration into 3-dimensional cell cultures modeling avascular tissue when compared to standard poly(ethylene glycol) (PEG)-coated nanoshells. Particle uptake and distribution in liver, lung, and pancreatic tumor cell cultures were evaluated using silver-enhancement staining and hyperspectral imaging with dark field microscopy. Two layer nanoshells exhibited significantly higher uptake compared to PEGylated nanoshells. This multilayer formulation may help overcome transport barriers presented by tumor vasculature, and could be further investigated in vivo as a platform for targeted cancer therapies.

Peptides Tissue-Specifically Stimulate Cell Differentiation during Their Aging

We verified expression of CXCL12 and Hoxa3 transcription factors of differentiation in cultures of human embryonic pancreatic and bronchial cells and CXCL12 and WEGC1 factors in a culture of human prostatic fibroblasts. Reduced expression of these differentiation markers was detected in late-passage (aging) cultures. The expression of differentiation factors CXCL12, Hoxa3, and WEGC1 was tissue-specifically stimulated by short peptides: pancragen (Lys-Glu-Asp-Trp) in pancreatic cells, bronchogen (Ala-Glu-Asp-Leu) in bronchial epithelial cells, and vesugen (Lys-Glu-Asp) in fibroblasts. The inducing effect of peptides on the expression of differentiation factors was more pronounced in aged cultures, which can serve as a mechanism of their geroprotective effect.

Purified Human Pancreatic Duct Cell Culture Conditions Defined by Serum-Free High-Content Growth Factor Screening

The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells.

Age‐dependent reduction of the PI3K regulatory subunit p85α suppresses pancreatic acinar cell proliferation

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is important for tissue proliferation. Previously, we found that tissue regeneration after partial pancreatic resection was markedly attenuated in aged mice as compared to young mice and that this attenuation was because of an age-dependent reduction of PI3K/Akt signaling in the pancreatic acini; however, the mechanisms for the age-associated decline of pancreatic PI3K/Akt signaling remained unknown. To better delineate the mechanisms for the decreased PI3K/Akt activation with aging, age-associated changes in cell proliferation and PI3K/Akt signaling were investigated in the present study using in vitro primary pancreatic acinar cell cultures derived from young and aged mice. In response to treatment with insulin-like growth factor 1 (IGF-1), acinar cells from young but not aged mice showed increased activation of PI3K/Akt signaling and cell proliferation, indicating that intrinsic cellular mechanisms cause the age-associated changes in pancreatic acinar cells. We also found that the expression of PI3K p85α subunit, but not IGF-1 receptor or other PI3K subunits, was significantly reduced in pancreatic acinar cells from aged mice; this age-associated reduction of p85α was confirmed in both mouse and human pancreatic tissues. Finally, small interfering RNA (siRNA)-mediated knockdown of p85α expression in acinar cells from young mice resulted in markedly attenuated activation of PI3K/Akt downstream signaling in response to IGF-1. From these results, we conclude that exocrine pancreatic expression of PI3K p85α subunit is attenuated by aging, which is likely responsible for the age-associated decrease in activation of pancreatic PI3K signaling and acinar cell proliferation in response to growth-promoting stimuli.

MicroRNA miR-17-5p is overexpressed in pancreatic cancer, associated with a poor prognosis, and involved in cancer cell proliferation and invasion

The microRNA-17-92 cluster is an oncogene in human B cell lymphomas and lung cancers. Previous microRNA microarray data revealed that miR-17-5p, a member of the miR-17-92 cluster, is upregulated in pancreatic cancer. However, the involvement of miR-17-5p expression in pancreatic carcinogenesis has not well been studied. In the present study, we measured the miR-17-5p expression levels in pancreatic cancer cell lines, primary cultures of normal human pancreatic ductal cells, formalin-fixed paraffin-embedded (FFPE) tissue samples derived from 80 patients who underwent pancreatectomy for pancreatic cancer and microdissected cells (including normal ductal epithelial, pancreatic intraepithelial neoplasia-1B and invasive ductal carcinoma cells) by qRT-PCR. Furthermore, we investigated the effects of upregulation of miR-17-5p expression on the proliferation and invasion of pancreatic cancer cells. We found that pancreatic cancer cells expressed higher levels of miR-17-5p than primary cultured normal ductal cells. miR-17-5p was also overexpressed in pancreatic cancer in FFPE and microdissected samples. Furthermore, analysis of macrodissected FFPE samples revealed that high miR-17-5p expression was associated with a poor prognosis (p = 0.03). In addition, in vitro experiments revealed that SUIT-2 and KP-2 pancreatic cancer cells transfected with the miR-17-5p precursor showed significantly higher cell growth ratios than the corresponding control cells (p < 0.001 and p = 0.012, respectively), as well as significantly higher numbers of invading cells (p < 0.0001 for both). The present findings suggest that miR-17-5p plays important roles in pancreatic carcinogenesis and cancer progression, and is associated with a poor prognosis in pancreatic cancer.

Potential role of CXCL10 in the induction of cell injury and mitochondrial dysfunction

Chemokines have been known to play a critical role in pathogenesis of chronic pancreatitis and acinar cell death. However, the role played by one of the CXC chemokines: CXCL10 in regulation of acinar cell death has remained unexplored. Hence, this study was designed to assess the role of CXCL10 promoting apoptosis in ex vivo cultured acinar cells. Primary human pancreatic acinar cell cultures were established and exposed to varying doses of CXCL10 for different time intervals. Apoptotic induction was evaluated by both qualitative as well as quantitative analyses. Various mediators of apoptosis were also studied by Western blotting, membrane potential (Psim) and ATP depletion in acinar cells. Analysis of apoptosis via DNA ladder and cell death detection - ELISA demonstrated that CXCL10 induced 3.9-fold apoptosis when administrated at an optimal dose of 0.1 mug of recombinant CXCL10 for 8 h. Quantitative analysis using FACS and dual staining by PI-annexin showed increased apoptosis (48.98 and 53.78% respectively). The involvement of upstream apoptotic regulators like pJNK, p38 and Bax was established on the basis of their increased expression of CXCL10. The change of Psim by 50% was observed in the presence of CXCL10 in treated acinar cells along with enhanced expression of Cytochrome C, apaf-1 and caspase 9/3 activation. In addition, ATP depletion was also noticed in CXCL10 stimulated acinar cells. CXCL10 induces cell death in human cultured pancreatic cells leading to apoptosis and DNA fragmentation via CXCR3 signalling. These signalling mechanisms may play an important role in parenchymal cell loss and injury in pancreatitis.

Physalia physalis Poison Depolarizes Beta Cell Membrane and Increases Insulin Secretion

Peptide toxins isolated from marine cnidarians are considered as useful tools for studying ionic channels and promising drugs for therapeutics. Hemolytic and cardiotoxic activities have been described in Physalia physalis venom, and two toxins with anticholinergic and antiglutamatergic effects have been isolated in its high molecular weight fraction.In this work, we explored some crude extract of P. physalis and its low molecular weight fractions on ionic currents and insulin secretion of pancreatic rat beta-cells. P. physalis specimens were collected at the north littoral of La Habana, Cuba. Crude extract were purified by a gel filtration (Superdex 200). Mass spectrometry MALDI-TOF and RP-HPLC (C18 column) were performed on each fraction collected from gel filtration. The biological activity on insulin secretion was tested in a reverse hemolytic plaque assay on primary cultures of pancreatic beta cells from male Wistar rats.In basal glucose (5.6 mM), the crude extract (25 protein ug/mL) increased by 77% the average immunoplaque area, which is directly proportional to insulin secreted by isolated cells, without disrupting of beta cells or erythrocytes integrity. Low molecular fractions from gel filtration did not exceed of 20 kDa, and most of them eluted before the 20 % of acetonitrile on RP-HPLC. These fractions increased both, the percentage of insulin-secreting cells and the average immunoplaque area at a basal glucose level, suggesting a direct effect on TRP-type channels that are responsible for beta cell depolarization.Physalia physalis poison contains polar bioactive compounds capable of enhance the secretory behavior of pancreatic beta cells from male Wistar rats by depolarizing the membrane, in non stimulant glucose conditions.Supported by Instituto de Ciencia y Tecnologia del DF, ICYT (PICSD 08-72).

In vitro effects of oestrogens, antioestrogens and SERMs on pancreatic solid pseudopapillary neoplasm-derived primary cell culture.

Background: Solid-pseudopapillary neoplasms of the pancreas (SPNs) are uncommon tumours usually frequent in young women. Although the pathogenesis of SPNs is uncertain a potential influence of the sex hormone milieu on the biology of these tumours has been suggested. The controversial expression of oestrogen receptors (ERs) in SPNs, provide a rationale for studying the effects of oestrogenic molecules on SPN development. Methods: The expression of a large series of hormonal ligands and receptors was evaluated in tissue specimens and in a primary cell culture (SPNC), obtained from a SPN in young female patient. The effects of 17β-oestradiol (17βE2), ICI 182,780 and tamoxifen (Tam) on cell replication and growth were examined. Results: We have established SPNC primary line. Immunocytochemical analysis was positive for vimentin, cyclin D1 and β-catenin and negative for cytokeratin, CD10 and neuroendocrine markers, in line with the immunostaining features of the tumoral tissue. Expression of ERα, ERβ and progesterone mRNAs was demonstrated in SPNC and tumor tissue. A proliferative and antiproliferative action of 17βE2 and Tam respectively were proved in SPNC. Conclusions: In conclusion, we provide the first direct evidence that oestrogenic molecules can influence proliferation of SPNC, offering future strategies in the control of this neoplasia via selective ER modulators.

In Vitro Effects of Oestrogens, Antioestrogens and SERMs on Pancreatic Solid Pseudopapillary Neoplasm-Derived Primary Cell Culture

Background: Solid-pseudopapillary neoplasms of the pancreas (SPNs) are uncommon tumours usually frequent in young women. Although the pathogenesis of SPNs is uncertain a potential influence of the sex hormone milieu on the biology of these tumours has been suggested. The controversial expression of oestrogen receptors (ERs) in SPNs, provide a rationale for studying the effects of oestrogenic molecules on SPN development.Methods: The expression of a large series of hormonal ligands and receptors was evaluated in tissue specimens and in a primary cell culture (SPNC), obtained from a SPN in young female patient. The effects of 17β-oestradiol (17βE2), ICI 182,780 and tamoxifen (Tam) on cell replication and growth were examined.Results: We have established SPNC primary line. Immunocytochemical analysis was positive for vimentin, cyclin D1 and β-catenin and negative for cytokeratin, CD10 and neuroendocrine markers, in line with the immunostaining features of the tumoral tissue. Expression of ERα, ERβ and progesterone mRNAs was demonstrated in SPNC and tumor tissue. A proliferative and antiproliferative action of 17βE2 and Tam respectively were proved in SPNC.Conclusions: In conclusion, we provide the first direct evidence that oestrogenic molecules can influence proliferation of SPNC, offering future strategies in the control of this neoplasia via selective ER modulators.

Immobilization of primary cultures of insulin-releasing human pancreatic cells

Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type1 Diabetes, however, it is severely limited by the shortage of organ donors. Ex-vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 Diabetes. It has recently been shown that, even in the absence of fibrotic overgrowth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells. Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing. Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.

Expression of vascular endothelial growth factor C in pancreatic cancer and its effect upon lymph node metastasis

To investigate the expression of vascular endothelial growth factor C (VEGF-C) and the effect of antisense oligonucleotide (ASODN) upon lymph node metastasis of pancreatic cancer cell. We selected 15 cases of human pancreatic cancer and detected the expression of VEGF-C in primary tumor and lymph node metastasis tissues with immunohistochemistry. Meanwhile, the spontaneous lymphatic metastasis model in nude mice was established with orthotopic implantation for the human pancreatic cancer cell line PANC-1, isolation and culture of primary tumor and spontaneous lymphatic metastasis. The effect of VEGF-C special ASODN upon the apoptosis of pancreatic cancer cell derived from primary tumor and spontaneous lymphatic metastasis were detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme linked immunosorbent assay (ELISA), flow cytometer and terminal deoxynucleotidyl transferase mediated-dUTP nick end labeling (TUNEL). In tissues of human pancreatic cancer, the values of VEGF-C on lymph nodes metastasis were more higher than primary tumor (P < 0.05). About the expression of VEGF-C on pancreatic cancer cell derived from spontaneous lymphatic metastasis and primary tumor in nude mice model, the mRNA levels of VEGF-C were 0.87 +/- 0.11 and 0.61 +/- 0.15 respectively, the VEGF-C levels in culture supernatants were (1682 +/- 157) pg/ml and (1404 +/- 128) pg/ml. The expression of VEGF-C on pancreatic cancer cells derived from lymphatic metastasis were also more higher than primary tumor (P < 0.05). In vitro and vivo, transfection of VEGF-C ASODN decreased the expression of VEGF-C in pancreatic cancer cell. In control group, scramble-sense oligonucleotide (SODN) group and ASODN group, the apoptosis rates of pancreatic cancer cells derived from lymph node metastasis were (2.8 +/- 1.0)%, (5.0 +/- 2.1)%, (13.2 +/- 2.2)% respectively in vitro, and were (1.8 +/- 0.5)%, (2.0 +/- 0.7)%, (4.4 +/- 1.0)% respectively in vivo, the apoptosis was increased significantly after transfection of VEGF-C ASODN (all P < 0.01). But pancreatic cancer cells derived from primary tumor were not effected (all P > 0.05). In human pancreatic cancer and nude mice model, the expression of VEGF-C on lymphatic metastasis was higher than primary tumor. The apoptosis of pancreatic cancer cells derived from spontaneous lymphatic metastasis were promoted by transfection of VEGF-C ASODN specially.

The miR-30 family microRNAs confer epithelial phenotype to human pancreatic cells

Epithelial-to-mesenchymal transition is a phenomenon necessary for embryonic development and also seen during certain pathological conditions. We show here for the first time that reduction in miR-30 family microRNAs, is responsible for mesenchymal transition of primary cultures of human pancreatic epithelial cells. We found that miR-30 family microRNAs target mesenchymal gene transcripts and maintain them in a translationally inactive state. Forced depletion using miR-30 family specific anti-miRs leads to mesenchymal transition while ectopic overexpression maintains the epithelial phenotype. We also show that miR-30 family microRNAs increase in abundance during differentiation of pancreatic islet-derived mesenchymal cells into hormone-producing islet-like cell aggregates. Our studies in human adult diseased pancreas also demonstrate that miR-30 family microRNAs are expressed at lower abundance in fibrotic lesions during pancreatitis. Together, our data confirm that miR-30 family microRNAs form a part of the regulatory signaling events involved in cellular response of pancreatic epithelial cells during mesenchymal transition.

MicroRNA-21 modulates biological functions of pancreatic cancer cells including their proliferation, invasion, and chemoresistance

Due to the poor prognosis of pancreatic cancer, novel diagnostic modalities for early diagnosis and new therapeutic strategy are urgently needed. Recently, microRNA-21 (miR-21) was reported to be strongly overexpressed in pancreatic cancer as well as in other solid cancers. We investigated the functional roles of miR-21, which have not been fully elucidated in pancreatic cancer. miR-21 expression was assessed in pancreatic cancer cell lines (14 cancer cell lines, primary cultures of normal pancreatic epithelial cells and fibroblasts, and a human normal pancreatic ductal epithelial cell line) and pancreatic tissue samples (25 cancer tissues and 25 normal tissues) by quantitative real-time reverse transcription-PCR amplification. Moreover, we investigated the proliferation, invasion, and chemoresistance of pancreatic cancer cells transfected with miR-21 precursor or inhibitor. miR-21 was markedly overexpressed in pancreatic cancer cells compared with nonmalignant cells, and miR-21 in cancer tissues was much higher than in nonmalignant tissues. The cancer cells transfected with the miR-21 precursor showed significantly increased proliferation, Matrigel invasion, and chemoresistance for gemcitabine compared with the control cells. In contrast, inhibition of miR-21 decreased proliferation, Matrigel invasion, and chemoresistance for gemcitabine. Moreover, miR-21 positively correlated with the mRNA expression of invasion-related genes, matrix metalloproteinase-2 and -9, and vascular endothelial growth factor. These data suggest that miR-21 expression is increased in pancreatic cancer cells and that miR-21 contributes to the cell proliferation, invasion, and chemoresistance of pancreatic cancer.

Three-dimensional Cultures of Rat Pancreatic RIN-5F Cells in Porous PLGA-collagen Hybrid Scaffolds

Three-dimensional cultures of pancreatic islet cells in porous scaffolds or hydrogels have been constructed as a biohybrid artificial pancreas. A thin mesh of a PLGA-collagen hybrid was used to culture rat RIN-5F cells. The hybrid mesh was coated with laminin, fibronectin, vitronectin, type IV collagen, and poly(L-lysine) were evaluated and mesh without coating was used as a control. Cell adhered and proliferated on all of the coated and uncoated meshes. The cells formed spheroids in the uncoated, poly(L-lysine)-, fibronectin-, vitronectin-, and type IV collagen-coated hybrid meshes, while forming a layered structure in the laminin-coated hybrid mesh. Cell adhesion on the coated PLGA-collagen hybrid meshes was higher than that for the uncoated hybrid mesh. The laminin-coated hybrid mesh showed the greatest level of adhesion. The insulin secretion capacity of RIN-5F cells was at the same level for all coated and uncoated PLGA-collagen hybrid meshes and higher than that of cells cultured on cell culture plates. The 3D cultured PLGA-collagen hybrid meshes promoted insulin production capacity. Gene expression analysis showed that genes encoding insulin I, insulin II, and the pancreatic transcription factor PDX-1 (pancreas/duodenum homeobox 1) was expressed. These results indicate that the PLGA-collagen hybrid meshes support adhesion, proliferation, and differentiation of RIN-5F cells that allows culturing pancreatic islet cells on 3D constructs.

In vitro cultures of human pancreatic stem cells: Gene and protein expression of designated markers varies with passage

Adult stem cells may possess great plasticity, but the cellular mechanisms regulating their fate are not fully understood. Prior to application of stem cell populations in regenerative medicine, major challenges remain to be overcome. Fundamental questions about in vitro growth and spontaneous differentiation of adult stem cell populations must be resolved. In this study, we comprehensively characterized a stem cell population derived from human pancreatic tissue by analyzing mRNA and protein expression in consecutive passages. We examined transcription and protein expression levels of markers related to stem cells or differentiated cells, respectively, as well as the growth rate of a primary human pancreatic stem cell population. In particular, the course of spontaneous mRNA and protein expression of the genes for alpha-smooth muscle actin (alpha-SMA), neurofilaments (NF), cytokeratin 18 (CK18) and nestin was examined during 11 passages by means of RT-PCR and immunocytochemistry. The cell population showed exponential growth over 10 of the 11 examined passages. Both the spontaneous expression of stem cell-related mRNA and protein as well as the characteristics of spontaneous differentiation were variable. Changes in mRNA and protein expression showed no direct correlation. These results demonstrate the unpredictable behaviour of spontaneously differentiating stem cells, being influenced by numerous, barely traceable extrinsic factors. Characterization studies of stem cell populations therefore require improved analysis techniques together with strictly controlled cell cultivation conditions to improve global gene and protein expression analyses.