Effect of triphenyldioxane on phase I xenobiotic metabolism enzymes in the liver of rats and rabbits.

We studied the effect of triphenyldioxane on phase I xenobiotic metabolism enzymes in the liver of rats and rabbits. Total cytochrome P450 content, protein concentration, and catalytic activity of CYP2B, CYP3A, and CYP2C isoforms were measured. Triphenyldioxane significantly increases specific activity of CYP2B and CYP2C in the liver of rats and rabbits, respectively. Immunoblotting analysis of microsomal enzymes in the liver of animals showed that the increase in specific activity of CYP is related to high content of apoenzymes. We showed for the first time that rats and rabbits are characterized by interspecies differences in the induction of cytochrome P450 isoforms under the influence of triphenyldioxane.

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells.

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.

Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5.

Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the gamma2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2- cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2- cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.

Human cord blood progenitors sustain thymic T-cell development and a novel form of angiogenesis.

There is growing interest in using human umbilical cord blood (CB) for allogeneic bone marrow transplantation (BMT), particularly in children. Thus, CB has been identified as a rich source of hematopoietic progenitors of the erythroid, myeloid, and B-cell lineages. Whether CB blood cells engrafting in the BM space also comprise T-cell progenitors capable of trafficking to the thymus and reconstituting a functional thymopoiesis in young recipients is presently unknown. Here, we show that CB progenitors, engrafted in the BM of immunodeficient mice, sustain human thymopoiesis by generating circulating T-cell progenitors capable of homing to and developing within a human thymic graft. Surprisingly, development of CB stem cells in this in vivo model extended to elements of the endothelial cell lineage, which contributed to the revascularization of transplants and wound healing. These results demonstrate that human CB stem cell transplantation can reconstitute thymic-dependent T-cell lymphopoiesis and show a novel role of CB-derived hematopoietic stem cells in angiogenesis.

Sustained proliferation of PDX-1+ cells derived from human islets.

Ex vivo expansion of human beta-cells is an important step toward the development of cell-based insulin delivery systems in type 1 diabetes. Here, we report that human pancreatic endocrine cells can be expanded through 15 cell doublings in vitro for an estimated total 30,000-fold increase in cell number. We believe that the cells resulting from these cultures are of beta-cell origin, since they uniformly express the transcription factor PDX-1 (STF-1, IDX-1, IPF-1), which is initially seen only in cells positive for insulin and negative for the ductal cell marker cytokeratin (CK)-19. To rule out the possibility that PDX-1 expression might be induced by the culture conditions used here, cells from isolated human pancreatic ducts were cultured under the same conditions as the islet cells. Cells in these cultures expressed CK-19 but not PDX-1. Although the expanded beta-cells continued to express PDX-1, insulin expression was lost over time. Whether reexpression of islet-specific genes in vitro is essential for successful cell transplantation remains to be determined.

Gene transfer to human pancreatic endocrine cells using viral vectors.

We have studied the factors that influence the efficiency of infection of human fetal and adult pancreatic endocrine cells with adenovirus, murine retrovirus, and lentivirus vectors all expressing the green fluorescent protein (Ad-GFP, MLV-GFP, and Lenti-GFP, respectively). Adenoviral but not retroviral vectors efficiently infected intact pancreatic islets and fetal islet-like cell clusters (ICCs) in suspension. When islets and ICCs were plated in monolayer culture, infection efficiency with all three viral vectors increased. Ad-GFP infected 90-95% of the cells, whereas infection with MLV-GFP and Lenti-GFP increased only slightly. Both exposure to hepatocyte growth factor/scatter factor (HGF/SF) and dispersion of the cells by removal from the culture dish and replating had substantial positive effects on the efficiency of infection with retroviral vectors. Studies of virus entry and cell replication revealed that cell dispersion and stimulation by HGF/SF may be acting through both mechanisms to increase the efficiency of retrovirus-mediated gene transfer. Although HGF/SF and cell dispersion increased the efficiency of infection with MLV-GFP, only rare cells with weak staining for insulin were infected, whereas approximately 25% of beta-cells were infected with Lenti-GFP. We conclude that adenovirus is the most potent vector for ex vivo overexpression of foreign genes in adult endocrine pancreatic cells and is the best vector for applications where high-level but transient expression is desired. Under the optimal conditions of cell dispersion plus HGF/SF, infection with MLV and lentiviral vectors is reasonably efficient and stable, but only lentiviral vectors efficiently infect pancreatic beta-cells.

Vascularization of purified pancreatic islet-like cell aggregates (pseudoislets) after syngeneic transplantation.

To clarify whether avascular purified endocrine cell aggregates derived from islets of Langerhans (pseudoislets) revascularize similarly to what is known for intact pancreatic islet grafts, we studied the process of angiogenesis and revascularization of syngeneically transplanted pseudoislets using intravital fluorescence microscopy. Pseudoislets were composed of pure beta-cells (B) or non-beta-cells (NB), as well as of mixed beta- and non-beta-cells (B/NB; 70/30%) or nonsorted-cells (NC), and were transplanted into the dorsal skinfold of Syrian golden hamsters. Intact islet grafts served as controls. At day 6 after transplantation, microvascularization of all types of pseudoislets was found to be less than in controls, as indicated by a reduced number of transplants that contained newly formed microvessels (take-rate: B, 38.8; NB, 38.7; B/NB, 43.8; and NC, 40.3% vs. intact islet grafts, 71.9%; P < 0.01). Moreover, those pseudoislets that had developed a microvascular network revealed a significantly lower functional capillary density (145.8+/-49.5 to 241.0+/-47.5 cm(-1) vs. intact islet grafts: 459.8+/-65.6 cm(-1); P < 0.05). After 20 days, the take-rate of pseudoislets was still lower (B, 67.4; NB, 45.3; B/NB, 48.4; and NC, 64.2%) when compared with intact islet grafts (88%; P < 0.05); however, islet-like aggregates with vascularization now showed an islet-specific glomerulus-like network of capillaries with a functional capillary density (498.5+/-49.1 to 601.4+/-124.0 cm[-1]) similar to that of intact islet grafts (644.3+/-26.8 cm[-1]). We conclude that the dissociation of pancreatic islets, followed by reaggregation of the purified endocrine cells to islet-like clusters (pseudoislets), delays the process of angiogenesis and revascularization after free transplantation; however, this does not influence the capacity to form an intact islet-specific microvasculature (angio-architecture), which appears to be independent from the cellular composition of pseudoislets.

KSA antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells: morphoregulatory roles in pancreatic islet development.

Cell adhesion molecules (CAMs) are important mediators of cell-cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell-cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.

Migration of breast epithelial cells on Laminin-5: differential role of integrins in normal and transformed cell types.

We examined the role of Laminin-5 (Ln-5) an extracellular matrix component of breast gland basement membrane, in supporting migration of normal (HUMEC), immortalized (MCF-10A), and malignant breast epithelial cells that exhibit different degrees of metastatic potential (MDA-MB-435>MDA-MB-231>MCF-7). HUMEC, MCF-10A, and MCF-7 cells all adhered to purified Ln-5 through the alpha3beta1 integrin receptor in adhesion assays. However, HUMEC and MCF-10A cells remained statically adherent, while MCF-7 cells migrated on Ln-5 in Transwell and colloidal gold displacement assays. Anti-alpha3 integrin antibodies blocked migration of MCF-7 cells on Ln-5. MDA-MB-231 and MDA-MB-435 cells bound and migrated on Ln-5 through a beta1 integrin receptor that is insensitive to antibodies that block the function of alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, and alphaV integrin subunits. Migration of all cell types tested was blocked by CM6, a monoclonal antibody directed to a cell adhesion site on the alpha3 chain of Ln-5. Thus, Ln-5 may play an important role in regulating adhesion and migration in normal and transformed breast epithelium. Our results indicate that the type of integrin utilized by breast cells to interact with Ln-5, as well as its functional state, may determine whether cells will be statically adherent or migratory on Ln-5.

Phosphatidylinositol 3-kinase is a negative regulator of cellular differentiation.

Phosphatidylinositol 3-kinase (PI3K) has been shown to be an important mediator of intracellular signal transduction in mammalian cells. We show here, for the first time, that the blockade of PI3K activity in human fetal undifferentiated cells induced morphological and functional endocrine differentiation. This was associated with an increase in mRNA levels of insulin, glucagon, and somatostatin, as well as an increase in the insulin protein content and secretion in response to secretagogues. Blockade of PI3K also increased the proportion of pluripotent precursor cells coexpressing multiple hormones and the total number of terminally differentiated cells originating from these precursor cells. We examined whether any of the recently described modulators of endocrine differentiation could participate in regulating PI3K activity in fetal islet cells. The activity of PI3K was inversely correlated with the hepatocyte growth factor/scatter factor-induced downregulation or nicotinamideinduced upregulation of islet-specific gene expression, giving support to the role of PI3K, as a negative regulator of endocrine differentiation. In conclusion, our results provide a mechanism for the regulation of hormone-specific gene expression during human fetal neogenesis. They also suggest a novel function for PI3K, as a negative regulator of cellular differentiation.

Ex vivo expansion of human pancreatic endocrine cells.

Cell transplantation as a therapy for type 1 diabetes is facilitated by ex vivo cell expansion of pancreatic beta-cells without loss of differentiative characteristics. The aim of this study was to determine the optimal conditions for in vitro growth of functional human pancreatic endocrine tissue. We examined the mitogenicity of matrixes from a variety of cell lines; proliferation was greater in cells growing on matrixes from bladder carcinoma cell lines, especially in monolayers grown on matrix from the human cell line HTB-9. After 14-day culture, there was a more than 100-fold proliferative increase, which was augmented to a more than 200-fold when hepatocyte growth factor/scatter factor was added; however, hepatocyte growth factor/scatter factor induced a rapid decrease in insulin content. Without the growth factor, fetal cell monolayers expanded 4-fold with no insulin loss; however, after 12-fold expansion, the insulin levels decreased to 40% of those in unexpanded cells. Adult islet cells expanded 3-fold without insulin loss. After 5-fold expansion, insulin levels decreased by 25% compared to those in free floating islets while retaining a normal response to secretagogues. Together, these results indicate that HTB-9 matrix provides the best stimulatory effect on replication of human endocrine cells, with little loss of in vitro function.

Trehalose: a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage.

The scarcity of available tissue for transplantation in diabetes and the need for multiple donors make it mandatory to use an optimal cryopreservation method that allows maximal recovery and preservation of beta-cell function. We have developed a method to cryopreserve islets with excellent survival of endocrine cells. Current methods use DMSO as cryoprotectant. Our method involves introducing both DMSO and the disaccharide trehalose into the cells during cooling. Uptake and release of trehalose occurred during the thermotropic lipid-phase transition measured in pancreatic endocrine cells between 5 degrees and 9 degrees C, using [14C]trehalose. Recovery of adult islets after cryopreservation with 300 mmol/l trehalose was 92 vs. 58% using DMSO alone. In vitro function, in terms of insulin content and release in response to secretagogues, was indistinguishable from fresh islets. Grafts from islets cryopreserved with trehalose contained 14-fold more insulin than grafts from islets cryopreserved without trehalose. Results with human fetal islet-like cell clusters (ICCs) were more pronounced: recovery from cryopreservation was 94%, compared with 42% without trehalose. Complete functionality of fetal cells was also restored; tritiated thymidine incorporation and insulin content and release were similar to fresh tissue. After transplantation in nude mice, there was a 15-fold increase in insulin content of grafts from ICCs cryopreserved with trehalose compared with ICCs cryopreserved without trehalose. Thus, the addition of trehalose to cryopreservation protocols leads to previously unobtainable survival rates of human pancreatic endocrine tissue.

Isolation and characterization of a cell line from the epithelial cells of the human fetal pancreas.

Pancreatic cell lines are useful for basic studies of pancreatic biology and for possible application to cell transplantation therapies for diabetes. A retroviral vector expressing simian virus 40 (SV40) T antigen and H-rasval12 was used to infect a monolayer culture of epithelial cells from an 18-wk human fetal pancreas. Infected cells gave rise to a clonal epithelial cell line, designated TRM-1. This cell line expresses epithelial markers as well as gult2 and small amounts of insulin and glucagon. TRM-1 is the first cell line to be generated from the human fetal pancreas and also the first cell line derived directly from the fetal pancreas of any species. The approach that we have used to develop TRM-1 should be applicable to isolating cell lines from other stages of human pancreatic development.

A role for hepatocyte growth factor/scatter factor in fetal mesenchyme-induced pancreatic beta-cell growth.

We have investigated the role of hepatocyte growth factor/scatter factor (HGF/SF) in the growth and/or differentiation of pancreatic islet beta-cells. We found that in the human fetal pancreas immunoreactive HGF/SF receptor (c-met proto-oncogene product) is preferentially associated with the developing beta-cells. In the adult pancreas, c-met messenger RNA is highly enriched in the islets and the immunoreactive protein is also restricted to the islet beta-cells. HGF/SF messenger RNA content of fetal pancreas-derived fibroblasts is more than 10-fold higher than that of adult fibroblasts. Culture of human fetal pancreatic epithelial cells in conditioned medium from the fetal pancreatic fibroblasts caused a 2.4-fold stimulation of the formation of islet-like cell clusters that was due to both mitogenic and morphogenic effects. Beta-cell proliferation in the cell clusters was stimulated 3.5-fold by the conditioned medium, and this was associated with a marked decrease in insulin content. All of the effects of the conditioned medium were blocked by anti-HGF/SF antibody. Specificity was confirmed by overriding the blocking effect of the antibody with excess recombinant HGF/SF. Conditioned medium from adult pancreatic fibroblasts stimulated islet-like cell cluster formation only slightly, and did not affect beta-cell replication. These results suggest that HGF/SF secreted by fetal fibroblasts is mitogenic to beta-cells. Taken together, our findings indicate an important role for HGF/SF in fetal mesenchyme-induced pancreatic beta-cell growth.

Cell adhesion and migration are regulated at distinct stages of thymic T cell development: the roles of fibronectin, VLA4, and VLA5.

T cell development in the thymus requires the establishment of stable interactions with cell-selecting elements such as the cortical epithelium followed by a regulated movement of selected progenitors to the medulla. Cell adhesion and migration are mediated by integrins in a number of biological systems though little is known regarding their function in the thymus. We demonstrated previously that immature CD3loCD69lo double positive human thymocytes adhere avidly to FN via the integrin, VLA4. We now demonstrate that the interaction of mature CD3hiCD69hi thymic subsets with FN triggers migration rather than firm adhesion. Migration requires the engagement of VLA4 in cooperation with VLA5 and both receptors regulate the persistence and directionality of movement. While migration capability is linked to maturation state, ligand concentration determines the efficiency of migration. In fact, FN and the alternatively spliced CS1 site are predominant in the thymic medulla, suggesting an instructive role of this ECM protein in vivo. Our studies identify a novel VLA4 and VLA5/FN-mediated pathway likely to be involved in regulating cell traffic between the cortex and medulla of the thymus. Moreover, the data provides evidence that VLA4 exists in at least two functional states at distinct stages of T cell development. While different states of VLA4 activation have been described on cell lines, this represents the first evidence supporting a biological significance for this integrin property.

ICA69 is expressed equally in the human endocrine and exocrine pancreas.

Islet cell autoantigen 69 kDa (ICA69) has been reported as a polypeptide antigen expressed in pancreatic beta cells, and autoimmunity against this antigen has been associated with insulin-dependent diabetes mellitus. We have studied the cell type specificity and ontogeny of ICA69 gene expression in man. The ICA69 gene was expressed in all adult human tissues. The level of expression was three-to five-times higher in the pancreas than in the brain, liver, intestine, kidney, spleen, lung or adrenal glands. Pancreatic ICA69 expression increased with age, adult levels being five times higher than the levels present at 13 weeks of gestation. Total RNA from four separate preparations of isolated human islets revealed levels of ICA69 mRNA similar to those found in the pancreas as a whole, although another islet antigen, glutamic acid decarboxylase 65, was highly enriched in the islets. In situ hybridization and immunohistochemical staining of sections of the fetal and adult pancreas revealed expression of the ICA69 gene and protein throughout the acinar, ductal, and islet tissue, but not in the mesenchyme. Analysis of ICA69 mRNA levels in human cell lines indicated expression in neural, endothelial and epithelial cells, but not in fibroblasts. In conclusion, ICA69, although highest in the pancreas, is widely distributed in other human tissues, excluding connective tissue. Within the human pancreas, ICA69 is not enriched in the islets or in the beta cells.

Ontogeny and tissue distribution of human GAD expression.

One of the major beta-cell autoantigens associated with IDDM is GAD. Although GAD expression has been detected in adult islets, transcriptional expression of the GAD genes has not been reported during human pancreatic ontogeny. We therefore analyzed patterns of GAD gene transcription by quantitating the mRNAs encoding both the 65- and 67-kDa isoforms (GAD65 and GAD67, respectively) in human fetal, postnatal, and adult pancreases, as well as in isolated adult islets, and examined their tissue-specific expression. Significant levels of pancreatic GAD65 transcripts were already detected at 13 weeks of gestation and were expressed at higher levels in the fetal and infantile pancreas than in the adult pancreas. Isolated adult pancreatic islets were highly enriched in GAD65 mRNA. In contrast, GAD67 transcripts were not detectable in fetal and postnatal pancreases. In addition to the pancreas, marked GAD expression was detected in the brain, whereas other tissues examined contained either low or undetectable GAD transcripts. Triple immunofluorescent staining of fetal and adult pancreases revealed colocalization of GAD65 with alpha- and beta-cells. In the fetal pancreas, strong immunoreactivity for GAD65 was also evident in epithelial cells, which lacked expression of insulin or glucagon, some of which were present in the ductal epithelium, suggesting that GAD65 expression might correlate with endocrine determination. In summary, 1) this is the first demonstration of GAD65 expression in the human fetal pancreas, implicating a potential role during islet development, and 2) GAD65 may be a useful marker for the identification of primitive islet cells.

Growth factor/matrix-induced proliferation of human adult beta-cells.

Proliferation of human beta-cells in vitro is desirable for both transplantation and biological studies. In this study, human pancreatic islets obtained from cadavers were kept in tissue culture plates that favored cell attachment. When the cells attached to the matrix produced by the rat-bladder carcinoma cell line 804G, 5'-bromo-2'-deoxyuridine (BrdU) labeling increased from 4.7 +/- 2.5 to 13.2 +/- 2.2%, while cells simultaneously labeled for insulin and BrdU increased from 0 to 32%. Addition of the growth factor hepatocyte growth factor/scatter (HGF/SF) increased BrdU labeling to 17.5 +/- 1.8 and the percentage of double positive (BrdU + insulin) cells to 69%. This is the first in vitro demonstration that human beta-cells grown in monolayer culture are able to replicate when exposed to selected matrices and growth factors. These experiments add further evidence that HGF/SF is an important mitogenic agent for human beta-cells.