Rat Tail Collagen Gel Research Articles

Engineered aligned endothelial cell structures in tethered collagen hydrogels promote peripheral nerve regeneration

Vascularisation is important in nerve tissue engineering to provide blood supply and nutrients for long-term survival of implanted cells. Furthermore, blood vessels in regenerating nerves have been shown to serve as tracks for Schwann cells to migrate along and thus form Bands of Büngner which promote axonal regeneration. In this study, we have developed tissue-engineered constructs containing aligned endothelial cells, or co-cultures of both endothelial cells and Schwann cells to test whether these structures could promote regeneration across peripheral nerve gaps. Type I rat tail collagen gels containing HUVECs (Human Umbilical Vein Endothelial Cells, 4 × 106 cells/ml) were cast in perforated tethering silicone conduits to facilitate cellular self-alignment and tube formation for 4 days of culture. For co-culture constructs, optimal tube formation and cellular alignment was achieved with a ratio of 4:0.5 × 106 cells/ml (HUVECs:Schwann cells). An in vivo test of the engineered constructs to bridge a 10 mm gap in rat sciatic nerves for 4 weeks revealed that constructs containing only HUVECs significantly promoted axonal regeneration and vascularisation across the gap, as compared to conventional aligned Schwann cell constructs and those containing co-cultured HUVECs and Schwann cells. Our results suggest that tissue-engineered constructs containing aligned endothelial cells within collagen matrix could be good candidates to treat peripheral nerve injury. Statement of significanceNerve tissue engineering provides a potential way to overcome the limitations associated with current clinical grafting techniques for the repair of severe peripheral nerve injuries. However, the therapeutic cells within engineered nerve tissue require effective vascularisation in order to survive. This work therefore aimed to develop engineered nerve constructs containing aligned tube-like structures made from endothelial cells. Not only did this provide a method to improve vascularisation, it demonstrated for the first time that aligned endothelial cells can outperform Schwann cells in promoting nerve regeneration in the rat sciatic nerve model. This has introduced the concept of developing pre-vascularised engineered nerve tissues, and indicated the potential usefulness of endothelial cell structures in tissue engineering for peripheral nerve repair.

Engineered Aligned Endothelial Cell Structures in Tethered Collagen Hydrogels Promote Peripheral Nerve Regeneration

Vascularisation is important in nerve tissue engineering to provide blood supply and nutrients for long-term survival of implanted cells. Furthermore, blood vessels in regenerating nerves have been shown to serve as tracks for Schwann cells to migrate along and thus form Bands of Bungner which promote axonal regeneration. In this study, we have developed tissue‑engineered constructs containing aligned endothelial cells, or co-cultures of both endothelial cells and Schwann cells to test whether these structures could promote regeneration across peripheral nerve gaps. Type I rat tail collagen gels containing HUVECs (Human Umbilical Vein Endothelial Cells, 4×106 cells/ml) were cast in perforated tethering silicone tubes to facilitate cellular self-alignment and tube formation for 4 days of culture. For co-culture constructs, optimal tube formation and cellular alignment was achieved with a ratio of 4:0.5×106 cells/ml (HUVECs:Schwann cells). An in vivo test of the engineered constructs to bridge a 10 mm gap in rat sciatic nerves for 4 weeks revealed that constructs containing only HUVECs significantly promoted axonal regeneration and vascularisation across the gap, as compared to conventional aligned Schwann cell constructs and those containing co-cultured HUVECs and Schwann cells. Our results suggest that tissue-engineered constructs containing aligned endothelial cells within collagen matrix could be good candidates to treat peripheral nerve injury.

Neural stem/progenitor cells on collagen with anchored basic fibroblast growth factor as potential natural nerve conduits for facial nerve regeneration.

Neural stem/progenitor cells (NS/PCs) were beneficial for the treatment of nervous system diseases. However, after transplantation, the beneficial was limited because the number of living NS/PCs decreased rapidly due to insufficient signaling molecules, such as growth factors, in the microenvironments surrounding transplanted cells. In the present study, we constructed collagen-based nerve conduit with anchored bFGF to achieve higher numbers of NS/PCs for repairing facial nerve injury. Compared with other methods involving neutral salt treatment or dialysis, the fabrication method of collagen scaffolds was simple, low-cost and safe, requiring a relatively short time to prepare. At 12weeks after transplantation, the functional and histological results of natural nerve conduits treated group showed significant similarities to the gold standard method of nerve autografting.

Enhanced tenogenic differentiation and tendon-like tissue formation by tenomodulin overexpression in murine mesenchymal stem cells.

As a highly specific marker of tenocytes, tenomodulin (Tnmd) functions remain largely unexplored. We investigated the effect of Tnmd overexpression on tenogenic differentiation of murine mesenchymal stem cells (mMSCs) via plasmid-mediated overexpression in the C3H10T1/2 cell line. The results showed that overexpressed Tnmd could significantly enhance cell proliferation (p<0.05) and the gene expressions of tenogenic-related molecules, including Tnmd, Scleraxis (Scx), collagens I, III and VI and decorin (p<0.05), and significantly inhibit mMSCs differentiation towards the adipogenic, chondrogenic and osteogenic lineages (p<0.05). Upon in vivo implantation with rat tail collagen gel subcutaneously in nude mice, Tnmd-overexpressed C3H10T1/2 cells formed neotendon-like tissue, which revealed a histological feature of wave-like dense collagen fibres and cells aligned in parallel. By contrast, a disorganized connective tissue structure with randomly distributed cells was observed in the control group. To further confirm this finding, a conditional Tnmd-overexpressing mouse model was established and the derived primary mMSCs could be induced to overexpress Tnmd with>two-fold upregulated gene expression (p<0.05) by the treatment of doxycycline (Dox). Similarly, conditional overexpression of Tnmd in primary mMSCs also led to faster proliferation (p<0.05), enhanced gene expression of tenogenic markers (p<0.05) and the inhibited expressions of adipogenic and osteogenic markers (p<0.05). The results of enhanced tenogenic differentiation and neotendon formation indicated that Tnmd may serve not only as a tenogenic marker but also as a positive regulator of MSCs tenogenic differentiation, which might be applied to MSCs-mediated tendon regeneration. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of cold preservation on the expression of GATA in intrahepatic bile duct

Objective To investigate the effects of cold preservation on the expression of GATA in intrahepatic bile duct. Methods The intrahepatic bile duct tissues of SD rats were obtained by collagenase perfusion combined with mechanical separation. After being cut into fragments, the intrahepatic bile duct tissues were cultured in rat tail collagen gel for 48 hours before experiment. All the rats were divided into the control group, cold preservation 1 hour (CP1 h) group and cold preservation 12 hours (CP12 h) group. There were 5 rats in each group. The mRNA and protein expressions of GATA were detected by Real-Time polymerase chain reaction and Western blot. Measurement data with normal distribution were presented as ±s. Comparison among 3 groups was done by ANOVA and pairwise comparisons were done by LSD test. Results The mRNA expressions of GATA3, GATA4, GATA6 were detected, while the mRNA expressions of GATA1, GATA2 and GATA5 were undetectable in intrahepatic bile duct tissue of the control group.The mRNA expressions of GATA4 in the CP1 h group, CP12 h group and the control group were 0.72 ± 0.08, 0.56 ± 0.07 and 0.96 ± 0.06, with significant difference among the 3 groups ( F =38.981, P 0.05) .The protein expressions of GATA4 in the CP1 h group, CP12 h group and the control group were 0.78 ±0.07, 0.64 ±0.06 and 0.99 ±0.10, with significant difference among the 3 groups ( F =24.211, P <0.05) .The protein expression of GATA4 in the CP12 h group was significantly lower than that in the CP1 h group and the control group, and the protein expression of GATA4 in the CP1 h group was significantly lower than that in the control group ( P <0.05) .The protein expressions of GATA6 in the CP1 h group, CP12 h group and the control group were 0.90 ±0.04, 0.75 ±0.06 and 0.98 ±0.11, with significant difference among the 3 groups ( F =11.651, P <0.05) .The protein expression of GATA6 in the CP12 h group was significantly lower than that in the CP1 h group and the control group ( P <0.05). Conclusion The expressions of GATA4 and GATA6 in the intrahepatic bile duct tissues are decreased significantly after cold preservation, which indicate that GATA4 and GATA6 might be involved in the pathophysiological process of the bile duct after cold preservation. Key words: Ischemic-type biliary lesions; Cold preservation; GATA; Intrahepatic bile duct

MP21-10 DEVELOPMENT OF AN EFFICIENT BLADDER TUMOR MODEL BY RECOMBINING WITH MESENCHYMAL CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2014MP21-10 DEVELOPMENT OF AN EFFICIENT BLADDER TUMOR MODEL BY RECOMBINING WITH MESENCHYMAL CELLS Kenichiro Ishii, Takeshi Sasaki, Manabu Kato, Kenjiro Ito, Motomu Sakuragi, Nobuyuki Oda, Hideki Kanda, Yasushi Yamada, Kiminobu Arima, Taizo Shiraishi, and Yoshiki Sugimura Kenichiro IshiiKenichiro Ishii More articles by this author , Takeshi SasakiTakeshi Sasaki More articles by this author , Manabu KatoManabu Kato More articles by this author , Kenjiro ItoKenjiro Ito More articles by this author , Motomu SakuragiMotomu Sakuragi More articles by this author , Nobuyuki OdaNobuyuki Oda More articles by this author , Hideki KandaHideki Kanda More articles by this author , Yasushi YamadaYasushi Yamada More articles by this author , Kiminobu ArimaKiminobu Arima More articles by this author , Taizo ShiraishiTaizo Shiraishi More articles by this author , and Yoshiki SugimuraYoshiki Sugimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.838AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Aberrant activation of tumor-stromal interactions is considered to play a critical role in the promotion of tumorigenesis. Many solid tumors are composed of carcinoma cells and surrounding stromal cells named as carcinoma-associated fibroblasts (CAFs). They secrete a number of growth factors such as EGF family, FGFs, HGF, and IGFs, which are also produced by rat mesenchymal cells (Ishii et al., Endocr Relate Cancer, 2009). Bladder cancer is the second most common genitourinary malignancy, with transitional cell carcinoma representing 90% of the cases. Unfortunately, bladder cancer has the high recurrence rate after treatment, even when superficial tumors are completely removed by a laser or electrical force. We hypothesize that the aberrant activation between stromal cells and bladder cancer cells is probably involved in the high recurrence rate of bladder cancer. The development of efficient tumor models that mimic human disease would benefit to investigate new cancer therapeutic agents. Here we have developed a new model of bladder tumors by recombining rat mesenchymal cells with T24 human bladder cancer cells. METHODS Rat mesenchymal cells were prepared from 18-day SD rat fetuses. Briefly, bladder, urogenital sinuses, and stomach were dissected from the fetuses and separated into epithelial and mesenchymal components. Recombinants were prepared by mixing 5 x 105 T24 cells and 1 x 105 rat mesenchymal cells in suspension. Pelleted cells were resuspended in neutralized type I rat tail collagen gels, and then grafted beneath the renal capsule of adult male nude mice. RESULTS As generally known, T24 tumors without rat mesenchymal cells were not well grown in vivo. In contrast, T24 tumors that formed with rat mesenchymal cells derived from urogenital sinus (UGM) and bladder (BLM) but not stomach (STM) showed accelerated cell proliferation and angiogenesis. In in vitro cell culture experiments, several growth factors such as EGF, TGF-alpha, and IL-6 stimulated cell proliferation of T24 cells. Only IL-6 treatment activated cellular PI3/Akt pathway in T24 cells, whereas IL-6 treatment reduced VEGF secretion from T24 cells. Cell motility of T24 cells was significantly increased by EGF and TGF-alpha treatment. CONCLUSIONS In this study, we demonstrate that our recombination model using mesenchymal cells may be useful to understand the tumor-stromal interactions in bladder cancer. Identifying mechanisms underlying the aberrant activation between stromal cells and bladder cancer cells is an initial step toward developing new cancer therapeutic agents in bladder cancer. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e229 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Kenichiro Ishii More articles by this author Takeshi Sasaki More articles by this author Manabu Kato More articles by this author Kenjiro Ito More articles by this author Motomu Sakuragi More articles by this author Nobuyuki Oda More articles by this author Hideki Kanda More articles by this author Yasushi Yamada More articles by this author Kiminobu Arima More articles by this author Taizo Shiraishi More articles by this author Yoshiki Sugimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cyclical strain modulates metalloprotease and matrix gene expression in human tenocytes via activation of TGFβ

Tendinopathies are a range of diseases characterised by degeneration and chronic tendon pain and represent a significant cause of morbidity. Relatively little is known about the underlying mechanisms; however onset is often associated with physical activity. A number of molecular changes have been documented in tendinopathy such as a decrease in overall collagen content, increased extracellular matrix turnover and protease activity. Metalloproteinases are involved in the homeostasis of the extracellular matrix and expression is regulated by mechanical strain. The aims of this study were to determine the effects of strain upon matrix turnover by measuring metalloproteinase and matrix gene expression and to elucidate the mechanism of action. Primary Human Achilles tenocytes were seeded in type I rat tail collagen gels in a Flexcell™ tissue train system and subjected to 5% cyclic uniaxial strain at 1Hz for 48h. TGFβ1 and TGFβRI inhibitor were added to selected cultures. RNA was measured using qRT-PCR and TGFβ protein levels were determined using a cell based luciferase assay. We observed that mechanical strain regulated the mRNA levels of multiple protease and matrix genes anabolically, and this regulation mirrored that seen with TGFβ stimulation alone. We have also demonstrated that the inhibition of the TGFβ signalling pathway abrogated the strain induced changes in mRNA and that TGFβ activation, rather than gene expression, was increased with mechanical strain. We concluded that TGFβ activation plays an important role in mechanotransduction. Targeting this pathway may have its place in the treatment of tendinopathy.

Breast epithelial tissue morphology is affected in 3D cultures by species‐specific collagen‐based extracellular matrix

Collagen-based gels have been widely used to determine the factors that regulate branching morphogenesis in the mammary gland. The patterns of biomechanical gradients and collagen reorganization influence the shape and orientation of epithelial structures in three-dimensional (3D) conditions. We explored in greater detail whether collagen type I fibers with distinct biomechanical and fiber-assembling properties, isolated from either bovine or rat tail tendon, differentially affected the epithelial phenotype in a tissue culture model of the human breast. Rat tail collagen fibers were densely packed into significantly longer and thicker bundles compared to those of the bovine type (average fascicle length 7.35 and 2.29 μm, respectively; p = 0.0001), indicating increased fiber alignment and biomechanical enablement in the former. MCF10A epithelial cells formed elaborated branched tubular structures in bovine but only nonbranched ducts and acini in rat tail collagen matrices. Ductal branching in bovine collagen was associated with interactions between neighboring structures mediated through packed collagen fibers; these fiber-mediated interactions were absent in rat tail collagen gels. Normal breast fibroblasts increased the final size and number of ducts only in rat tail collagen gels while not affecting branching. Our results suggest that the species of origin of collagen used in organotypic cultures may influence epithelial differentiation into alveolar or ductal structures and the patterns of epithelial branching. These observations underscore the importance of considering the species of origin and fiber alignment properties of collagen when engineering branching organs in 3D matrices and interpreting their role in the tissue phenotype.

Culture of newborn monkey liver epithelial progenitor cells in chemical defined serum-free medium

Studies with hepatic progenitor cells from non-human primates would allow better understanding of their human counterparts. In this study, rhesus monkey liver epithelial progenitor cells (mLEPCs) were derived from a small piece of newborn livers in chemical defined serum-free medium. Digested hepatic cells were treated in Ca(2+)-containing medium to form cell aggregates. Two types of cell aggregates were generated: elongated spindle cells and polygonal epithelial cells. Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 d in our cultures. The remaining type consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f, and E-cad, the markers of hepatic stem cells, but were negative for alpha-fetoprotein, albumin, and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as alpha-1-antitrypsin, intracytoplasmic glycogen storage, indocyanine green uptake, and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19-positive cholangiocytes with formation of doughnut-like structure. Thus, mLEPCs with bipotency were derived from newborn monkey liver and may serve as a preclinical model for assessment of cell therapy in humans.

Abstract 3640: Mitochondrial Uncoupling Protein 2 Facilitates Endothelial Cell Growth and Angiogenesis via Reduction of Mitochondrial Superoxide

Background: Growing evidence suggests that mitochondrial function contributes to cell phenotype. One important component of mitochondrial function is the membrane potential that is controlled, in part, by uncoupling proteins (UCPs). Based on our previous data, the UCP2 is predominantly expressed in cultured endothelial cells. Therefore, we sought to examine the role of UCP2 in endothelial cell growth and angiogenesis. Methods and Results: Murine lung endothelial cells (MLECs) were isolated from UCP2-null and wild-type mice. UCP2-null cells were found less proliferative than wild-type cells (P&lt;0.02, UCP2-null cells vs. wild-type cells, n=4). This defect of UCP2-null cells was rescued by UCP2 adenovirus transfection (19% increase, p&lt;0.02 vs. LacZ adenovirus treated cells, n=3), and also rescued by transfection with manganese superoxide dismutase (MnSOD) adenovirus (53% increase, P&lt;0.002 vs. LacZ adenovirus treated cells, n=3). We found a reciprocal relation such as no UCP2 expression and higher mitochondrial superoxide level in the MLECs (P&lt;0.005, UCP2-null cells vs. wild-type cells, n=3), suggesting that mitochondrial superoxide may regulate endothelial cell growth. Then, we prepared murine aortic rings from UCP2-null and wild-type mice and embedded in rat tail collagen gel. The sprouting angiogenesis of UCP2-null explants was significantly less than wild-type explants (P&lt;0.02, UCP2-null explants vs. wild-type explants, n=3– 4). Furthermore, MLECs from MnSOD-heterozygous mice showed less proliferation with lower expression of UCP2 protein and higher mitochondrial superoxide level compared to the MLECs from wild-type littermates (P&lt;0.02, MnSOD-heterozygous cells vs. wild-type cells, n=4 – 8). We also observed less sprouting angiogenesis in MnSOD-heterozygous aortic explants than wild-type aortic explants (P&lt;0.05, MnSOD-heterozygous explants vs. wild-type explants, n=3– 6). Conclusions: These data indicate that mitochondrial superoxide controls endothelial cell proliferation and angiogenesis, suggesting that mitochondrial metabolism modulates the endothelial cell growth and angiogenesis.

IL-13 induces a bronchial epithelial phenotype that is profibrotic

BackgroundInflammatory cytokines (e.g. IL-13) and mechanical perturbations (e.g. scrape injury) to the epithelium release profibrotic factors such as TGF-β2, which may, in turn, stimulate subepithelial fibrosis in asthma. We hypothesized that prolonged IL-13 exposure creates a plastic epithelial phenotype that is profibrotic through continuous secretion of soluble mediators at levels that stimulate subepithelial fibrosis.MethodsNormal human bronchial epithelial cells (NHBE) were treated with IL-13 (0, 0.1, 1, or 10 ng/ml) for 14 days (day 7 to day 21 following seeding) at an air-liquid interface during differentiation, and then withdrawn for 1 or 7 days. Pre-treated and untreated NHBE were co-cultured for 3 days with normal human lung fibroblasts (NHLF) embedded in rat-tail collagen gels during days 22–25 or days 28–31.ResultsIL-13 induced increasing levels of MUC5AC protein, and TGF-β2, while decreasing β-Tubulin IV at day 22 and 28 in the NHBE. TGF-β2, soluble collagen in the media, salt soluble collagen in the matrix, and second harmonic generation (SHG) signal from fibrillar collagen in the matrix were elevated in the IL-13 pre-treated NHBE co-cultures at day 25, but not at day 31. A TGF-β2 neutralizing antibody reversed the increase in collagen content and SHG signal.ConclusionProlonged IL-13 exposure followed by withdrawal creates an epithelial phenotype, which continuously secretes TGF-β2 at levels that increase collagen secretion and alters the bulk optical properties of an underlying fibroblast-embedded collagen matrix. Extended withdrawal of IL-13 from the epithelium followed by co-culture does not stimulate fibrosis, indicating plasticity of the cultured airway epithelium and an ability to return to a baseline. Hence, IL-13 may contribute to subepithelial fibrosis in asthma by stimulating biologically significant TGF-β2 secretion from the airway epithelium.

Evaluation of coculture model composed of sinusoidal endothelial cells and small hepatocytes

Tendinopathies are a range of diseases characterised by degeneration and chronic tendon pain and represent a significant cause of morbidity. Relatively little is known about the underlying mechanisms; however onset is often associated with physical activity. A number of molecular changes have been documented in tendinopathy such as a decrease in overall collagen content, increased extracellular matrix turnover and protease activity. Metalloproteinases are involved in the homeostasis of the extracellular matrix and expression is regulated by mechanical strain. The aims of this study were to determine the effects of strain upon matrix turnover by measuring metalloproteinase and matrix gene expression and to elucidate the mechanism of action. Primary Human Achilles tenocytes were seeded in type I rat tail collagen gels in a Flexcell™ tissue train system and subjected to 5% cyclic uniaxial strain at 1 Hz for 48 h. TGFβ1 and TGFβRI inhibitor were added to selected cultures. RNA was measured using qRT-PCR and TGFβ protein levels were determined using a cell based luciferase assay. We observed that mechanical strain regulated the mRNA levels of multiple protease and matrix genes anabolically, and this regulation mirrored that seen with TGFβ stimulation alone. We have also demonstrated that the inhibition of the TGFβ signalling pathway abrogated the strain induced changes in mRNA and that TGFβ activation, rather than gene expression, was increased with mechanical strain. We concluded that TGFβ activation plays an important role in mechanotransduction. Targeting this pathway may have its place in the treatment of tendinopathy.

Angiotensin II regulates vascular smooth muscle cell pH, contraction, and growth via tyrosine kinase-dependent signaling pathways.

Angiotensin II (Ang II), a potent vasoactive peptide with mitogenic potential, influences vascular smooth muscle cell contraction and growth through receptor-linked pathways that increase intracellular free Ca2+ concentration ([Ca2+]i) and pH (pHi). Activation of these second messengers by Ang II may involve tyrosine kinase-dependent signaling pathways. This study determined the role of tyrosine kinases in Ang II-stimulated pHi, and in simultaneously measured contractile and [Ca2+]i responses, as well as growth in cultured vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto rats. pHi was determined by fluorescent digital imaging using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). Vascular smooth muscle cell [Ca2+]i and contractile responses were assessed simultaneously by fura 2 methodology and by photomicroscopy in cells grown on rat tail collagen gels. Cell growth was determined by DNA and protein synthesis as measured by [3H]thymidine and [3H]leucine incorporation, respectively. The Ang II receptor subtypes (AT1 or AT2) through which Ang II mediates effects were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD 123319 (a selective AT2 antagonist). To determine whether tyrosine kinases influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (a specific tyrosine kinase inhibitor). Ang II increased pHi in a dose-dependent manner (pD2, 9.2+/-0.2) and significantly increased vascular smooth muscle cell contraction (30%) and [Ca2+]i (pD2, 7.4+/-0.1). Ang II (10(-7) mol/L) increased DNA ([3H]thymidine incorporation) and protein synthesis ([3H]leucine incorporation). [Sar1,Ile8]Ang II and losartan but not PD 123319 abolished Ang II-elicited responses. Tyrphostin A-23 significantly attenuated Ang II-stimulated pHi responses; it also inhibited [Ca2+]i and contractile responses and cell growth. The inactive analogue tyrphostin A-1 did not alter Ang II-stimulated actions. These results provide novel evidence for a role of tyrosine kinases in Ang II-mediated pHi responses in vascular smooth muscle cells and indicate that tyrosine kinases participate in the regulation of signal transduction associated with AT1 receptor subtype-mediated contraction and growth.

Transdifferentiation of human islets to pancreatic ductal cells in collagen matrix culture

Transdifferentiation is a change from one differentiated phenotype to another, involving morphological and functional phenotypic markers. Stability of the cellular phenotype is probably related to the extracellular milieu, as well as cytoplasmic and nuclear components that interact to control gene expression, and the conversion of cell phenotype is likely to be accomplished by selective enhancement of gene expression, which controls the terminal developmental commitment of cells. In this paper, we show the induction of cultured human islets cells to alter their usual phenotypic expression and attain morphological and functional characteristics of duct cells. Islets were isolated by collagenase digestion of pancreata that were removed from cadaveric organ donors. The islets were purified on a two-step density gradient of bovine serum albumin and were then placed into a three-dimensional rat-tail collagen gel matrix supplemented with NuSerum epithelial growth factor and cholera toxin. During the initial 96 h of culture, the islets underwent a cystic transformation that was associated with (1) the maintenance of immunoreactivity for neuron-specific enolase, an endocrine cell marker, but a progressive loss of insulin gene expression, (2) a loss of immunoreactivity for insulin protein, and (3) the appearance of CK-19, a marker for ductal cells. After the transformation was complete, the cells had the ultrastructural appearance of primitive duct-like cells. Cyst enlargement after the initial 96 h was associated, at least in part, with cell replication, as reflected in the 1500% increase in the incorporation of tritiated thymidine. These experiments are consistent with the transdifferentiation of an islet cell to a ductal cell. The exact mechanisms involved still need to be fully elucidated.

Ductal Cyst Formation in Collagen-Embedded Adult Human Islet Preparations: A Means to the Reproduction of Nesidioblastosis In Vitro

Neogenesis of endocrine islets from ductal epithelium termed nesidioblastosis has been described in vivo after various experimental conditions (90% pancreatectomy or pancreas wrapping in the rodent) and in clinical pathologies. In the adult regenerating pancreas, a proliferation and organization of ductal epithelium into tubular structures precedes its differentiation into endocrine cells. Reproduction of nesidioblastosis in vitro may provide a novel approach to human islet propagation in vitro. With this aim, adult human islet preparations were cultured in diverse three-dimensional (3D) gels in the presence of serum. After 3-5 days in rat tail collagen gels, proliferating (bromodeoxyuridine-positive) cystic structures appeared associated with islets and as isolated spheres. Percentage labeling indexes of the cysts were 4.1, 18.7, 15.4, and 13.3% after 3, 5, 7, and 10 days of culture, respectively. Immunohistochemistry confirmed the ductal (carbohydrate antigen 19-9) and epithelial (keratin-1) nature of the cysts. No cysts were formed in agarose gels or Vitrogen 100, whereas the cyst number was increased by the quantity of serum (20% > 10%) and gels rich in extracellular matrix components and growth factors (Matrigel). The latter lead to tubular networks. Single endocrine islet cells were observed in the ductal cysts after 7 (2.8%) to 10 (5.6%) days in rat tail collagen. Our observations paralleled the changes characteristic of the regenerating pancreas in vivo. 3D culture may permit the identification of matrix and media constituents promoting the neogenesis of islets and may be the means to increase the mass of endocrine tissue obtained from adult cadaveric pancreases for transplantation.

Differential regulation of integrin-mediated proplatelet formation and megakaryocyte spreading.

Guinea pig bone marrow megakaryocytes were cultured on a type I rat tail collagen gel which stimulated proplatelet formation. Proplatelet formation was inhibited by monoclonal antibody LM609 to the alpha v beta 3 integrin (VnR), but not by monoclonal antibodies to the alpha 5, alpha 6, beta 1, or IIb beta 3(GPIIb-IIIa) integrin proteins. Megakaryocytes cultured on a plastic surface and stimulated with thrombin undergo a spreading and an adhesion reaction. This reaction is blocked in a dose-dependent manner by the tetrapeptide RGDS and by the monoclonal antibody PG2 to the GPIIb-IIIa integrin, but not by the monoclonal antibody LM609 to the VnR. Immunoprecipitation and affinity chromatography experiments demonstrate that guinea pig megakaryocytes have distinct GPIIb-IIIa and VnR integrins with similar electrophoretic mobility. Spreading was significantly inhibited in a dose-dependent fashion by drugs which elevate cellular cyclic AMP, including forskolin, dibutyryl cAMP, and isobutylmethylxanthine. In contrast to spreading, megakaryocyte proplatelet formation was stimulated by these agents in a dose-dependent manner. Megakaryocyte spreading was stimulated by the protein kinase C (PKC) activator phorbol myristate acetate (PMA) and inhibited by the PKC inhibitors Calphostin C and K5720 in a dose-dependent manner. PKC inhibitors did not inhibit megakaryocyte proplatelet formation. These results demonstrate that the closely related VnR and GPIIb-IIIa integrins regulate different aspects of megakaryocyte morphological change and appear to be associated with different second messenger systems.

Morphological characteristics of human endometrial epithelial cells cultured on rat-tail collagen matrix.

Epithelial cells were isolated from normal human endometrium and cultured on reconstituted matrices of rat-tail collagen gels. Cells attached within 24 h after plating. Initially, epithelial cells were not structurally polarized, had projections from both apical and basal domains and were generally flat to cuboidal in shape. When the gels were made free floating 36-48 h after initiation of the cultures, the epithelial cells became columnar in shape as the gels underwent contraction. Although a maintained growth profile was observed during the 10 days of culture, there was a linear decrease in gel matrix diameter along with increasing loss of transparency. Electron microscopy revealed the presence of apical microvilli, junctional complexes, lipid droplets and endoplasmic reticulum in cultured epithelial cells. The basolateral dilatations and lateral membrane plications were seen in these cells by 6-8 days in culture. With gel contraction, rearrangement of matrix material was observed. Occasionally there were basal lamina-like structures adjacent to the flattened basal surface and the formation of gland-like structures within the matrix. The three-dimensional primary culture of human epithelial cells on collagenous biomatrix appears to be a potential experimental tool for the study of cell-cell and cell-matrix interactions, and of the study of the effects of endocrine and paracrine factors on these cells in vitro.

Isolation and culture of rhesus monkey pancreatic ductules and ductule-like epithelium.

The objective of this work was to devise methods for the isolation and culture of duct epithelium from rhesus monkey pancreas with the expectation that such methods would be applicable to the human pancreas. This objective is important because of the role duct epithelium appears to play in human diseases such as pancreatic cancer and cystic fibrosis. Pieces of freshly procured pancreas were minced and enzymatically dissociated, resulting in a digest that contained a few isolated ductules (intralobular ducts) as well as numerous small tissue fragments consisting of roughly equal proportions of ductular and acinar cells. These fragments were suspended in a rat tail collagen gel and cultured for up to 2 weeks in a medium supplemented with cholera toxin, epidermal growth factor, and other additives. A few cystic ductular fragments were initially observed among a large number of predominantly solid fragments. Later, most of the solid fragments also became cystic and eventually resembled the ductules except for being spherical. Autoradiographic analysis of DNA synthesis showed that the cysts possessed a proliferative potential. The cysts consisted almost entirely of ductule-like epithelium with no recognizable acinar cells, and exhibited greatly reduced concentrations of the acinar marker enzymes amylase, chymotrypsin, and gamma-glutamyl transferase. In contrast, the specific activity of the duct marker enzyme carbonic anhydrase was elevated in freshly isolated digests compared with the whole pancreas and this elevated activity was maintained for 4-5 days of culture, after which it declined. Other evidence for the ductular nature of the cysts was their low density relative to freshly isolated acinar tissue, their ability to distend (suggestive of fluid/electrolyte secretion), and the accumulation of mucins at the apical borders of the cells. The results show that fragments of rhesus monkey pancreas that are enriched in ductular epithelium assume some of the properties of ductular cells when cultured in a collagen gel. These epithelial preparations should facilitate biochemical and physiological studies of this important pancreatic cell type.

Mucinlike glycoproteins from cat tracheal gland cells in primary culture.

In a recent study (D. J. Culp, D. K. P. Lee, D. P. Penney, and M. G. Marin. Am. J. Physiol. 263: L264-275, 1992), we reported that primary cultures of cat tracheal gland cells expressed histological, ultrastructural, and immunological characteristics of mucous cells when cultured on floating gels of rat tail collagen (released-gel cultures) compared with cells cultured on glutaraldehyde-fixed collagen gels (fixed-gel cultures). We therefore collected culture medium from gland cells grown under both culture conditions for determination and comparison of glycoconjugates with characteristics of mucin glycoproteins. Cells were cultured in the presence of [3H]glucosamine, and material of high molecular weight and density (HMD material) was isolated. HMD material from both culture conditions were each resistant to heparitinase and heparinase, whereas 72 and 25% of the radiolabel in released-gel and fixed-gel HMD material, respectively, was resistant to chondroitinase ABC. Material resistant to chondroitinase ABC was analyzed further. Both samples contained a single broad glycoprotein band [relative molecular weight (M(r)) > 250,000] after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had amino acid profiles similar to airway mucin. The sample from fixed-gel cultures had nearly equal amounts of carbohydrate and protein, was highly enriched in N-acetylglucosamine, contained mannose, displayed little blood group A immunoreactivity, and had few O-linked oligosaccharides. Conversely, the sample from released-gel cultures contained 80% carbohydrate, was composed of monosaccharides characteristic of airway mucins, displayed blood group A immunoreactivity, and contained oligosaccharides O-linked via N-acetylgalactosamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term culture and characteristics of normal rat liver bile duct epithelial cells

Background: A method was established for isolation and long-term culture of bile duct epithelial cells (BDEC) of normal adult rat liver that does not require the preparation of highly purified BDEC. Methods: After dissociation of the liver parenchyma by collagenase perfusion, the liver remnant containing the intact biliary tree was minced into small fragments, embedded in a rat tail collagen gel, and cultured for 6 days in hormonally defined serum-free Dulbecco's Modified Eagle Medium/F12 medium (SFDM). BDEC cultures were subsequently subcultured and maintained on rat tail collagen gels in SFDM medium containing 5 μmol/L forskolin and 5%–10% Nu Serum IV (Collaborative Research, Bedford, MA). Results: Established BDEC lines continued to express ductal specific markers including γ-glutamyl transpeptidase, cytokeratins 7 and 19, and a number of monoclonal antibody-defined bile duct antigens, such as OC.2, OC.3, and OV6. Conclusions: The availability of a method to establish normal BDEC lines will allow further investigation of the function of bile duct cells and their role in normal liver differentiation and carcinogenesis.