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Biomedical subjects

A Hayek

Publications and source records attributed to A Hayek.

At least 19 recordsLinked to original sources
PubMed1999

Transplantation of human fetal pancreas: fresh vs. cultured fetal islets or ICCS.

The paucity of human adult islets available for transplantation in IDDM makes the use of human fetal pancreas a potential alternative. Fetal pancreatic endocrine cells grow and differentiate over time when fresh explants or cultured islet-like cell clusters (ICCs) are transplanted under the kidney capsule in athymic nude mice. We have recently developed a procedure to isolate fetal islets, which differ from ICCs in their beta-cell content. This study was undertaken to compare the maturation and growth of grafts from purified fetal islets, containing mostly beta-cells, to grafts of mostly undifferentiated endocrine cell precursors, cultured as ICCs, and fresh, uncultured tissue. Total insulin content was highest in the fresh tissue pre-transplant while insulin levels fell precipitously during culture as either fetal islets or ICCs. Although 500 fetal islets contained more insulin than 500 ICCS before transplantation, the insulin content of the resulting grafts was the same 3 months post-transplantation. The degree of stimulation following glucose challenge was comparable, as was the histological appearance. However 70 mg of fresh tissue was needed to generate the fetal islets while only 30 mg was needed for the ICCs. Grafts of 30 mg fresh tissue also had similar total insulin contents and stimulation following glucose challenge, but, when normalized to DNA there was a significantly higher concentration of insulin in the grafts from ICCs or fetal islets. Moreover there were distinct morphological differences, with fibrous and ductal elements prominent in the grafts from fresh tissue, which were also much larger and more diffuse, with cystic elements evident macroscopically. Quantitative immunohistochemical analysis showed that grafts from cultured tissue were 48.3+/-5% positive for immunoreactive insulin compared with grafts from fresh tissue which were only 13.3+/-1.4% positive for insulin. In conclusion cultured ICCs, a heterogeneous mixture of hormone-containing and undifferentiated endocrine cells, are a preferable source for transplantation than either purified fetal islets or uncultured tissue.

Animals↗
PubMed1999

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.

Adenoviridae Infections↗
PubMed1998

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.

Adult↗
PubMed1998

Microfabricated immunoisolating biocapsules.

A microfabricated silicon-based biocapsule for the immunoisolation of cell transplants is presented. The biocapsule-forming process employs bulk micromachining to define cell-containing chambers within single crystalline silicon wafers. These chambers interface with the surrounding biological environment through polycrystalline silicon filter membranes. The membranes are surface micromachined to present a high density of uniform pores, thus affording sufficient permeability to oxygen, glucose, and insulin. The pore dimensions, as small as 20 nm, are designed to impede the passage of immune molecules and graft-borne viruses. The underlying filter-membrane nanotechnology has been successfully applied in controlled cell culture systems (Ferrari et al., 1995), and is under study for viral elimination in plasma fractionation protocols. Here we report the encouraging results of in vitro experiments investigating the biocompatibility of the microfabricated biocapsule, and demonstrate that encapsulated rat neonatal pancreatic islets significantly outlive and outperform controls in terms of insulin-secretion capability over periods of several weeks. These results appear to warrant further investigations on the potential of cell xenografts encapsulated within microfabricated, immunoisolating environments for the treatment of insulin-dependent diabetes.

Animals↗
PubMed1997

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.

Androstadienes↗
PubMed1997

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.

Adult↗
PubMed1997

Experimental transplantation of human fetal and adult pancreatic islets.

We examined morphology and function following transplantation of human fetal islet-like clusters (ICCs) in nude mice and compared the functional efficiency of human adult islets and fetal ICCs after transplantation. To assess the optimal site we first transplanted ICCs under the kidney capsule, pancreas, lung, and liver in nude mice. Grafts to the kidney and pancreas matured functionally and morphologically, as evidenced by a 4-fold increase in C peptide after glucose stimulation and the presence of insulin in the grafts of all animals. Grafts to the lung, liver, and spleen did poorly; C peptide was only measurable in two of eight, two of five, or three of five of mice grafted to the lung, liver, or spleen, respectively. Using chemically diabetic nude rats as recipients, we were able to restore normoglycemia using 15,000 ICCs/kg. Lastly, when transplanted under the kidney capsule of normal nude mice, ICCs had significantly higher insulin contents and C peptide release than equivalent grafts of adult islets. In summary, ICCs are an efficient source of insulin-producing cells of potential use in clinical transplantation. In nude mice, both the kidney and the pancreas provide suitable environments for the growth and maturation of undifferentiated human beta-cells.

Animals↗
PubMed1997

Functional beta-cell mass after transplantation of human fetal pancreatic cells: differentiation or proliferation?

The scarcity of human adult islets available for transplantation in IDDM makes the use of human fetal pancreatic cells desirable. Human fetal pancreatic cells grow and differentiate after transplantation in nude mice. It is unclear whether proliferation of preexisting endocrine cells or differentiation of precursor cells is mainly responsible for the increased islet mass and if beta-cell enrichment before transplantation enhances the functional outcome of the graft. To answer these questions, we transplanted purified human fetal islets, islet-like cell clusters (ICCs), and fresh tissue under the kidney capsule of nude mice. Insulin content was highest in the fresh tissue but fell rapidly during culture as either fetal islets or ICCs. Although fetal islets contained fourfold more insulin than ICCs before transplantation, the insulin content of the resulting grafts was the same after 3 months in vivo. The degree of stimulation after glucose challenge was comparable; however, more tissue was needed to generate the fetal islets. Grafts of fresh tissue also had similar total insulin contents, but when normalized to DNA, insulin concentration was significantly higher in the grafts from cultured tissue. Moreover, there were distinct morphological differences; the grafts from fresh tissue were more fibrous, with prominent ductal and cystic elements. Grafts from cultured tissue were two- to threefold enriched in endocrine tissue when compared with grafts originating from fresh tissue. These results suggest that islet cells identified in the grafted ICCs are mainly derived through differentiation of endocrine precursors and that cultured ICCs are more preferable than either fetal islets or uncultured tissue for transplantation.

Age Factors↗
PubMed1997

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.

Adult↗
PubMed1996

Development of a VSV-G protein pseudotyped retroviral vector system expressing dominant oncogenes from a lacO-modified inducible LTR promoter.

We report the development of a retroviral vector system in which two dominant oncogenes are expressed inducibly in human cells using the lac repressor/lac operator regulatable promoter system. First, the parent vector, pLoCRNLo, was constructed to contain a retroviral long terminal repeat (LTR) promoter that has been modified by incorporation of a lac operator sequence (lacO). This promoter, LTRo, was shown to mediate IPTG-inducible cat expression in rat cells expressing the lac repressor. The pLoCRNLo backbone was used to develop the retroviral vector LoTPRRNLo which expresses SV40 T antigen and H-ras val12 oncogenes as a dicistronic unit separated by a poliovirus internal ribosome entry sequence (PO-IRES). LoPRRNLo retrovirus was produced as a VSV-G protein pseudotype and used to infect primary human cells, resulting in the efficient formation of transformed cell lines. Subsequent introduction into the transformed cells of the lac repressor, expressed from a second retroviral vector, MSCV-In(S), resulted in IPTG-responsive oncogene expression and cell growth. This vector system is useful for introducing multiple genes under inducible control into mammalian cells.

Antigens, Polyomavirus Transforming↗
PubMed1996

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.

Adult↗
PubMed1996

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.

Adult↗
PubMed1996

E2A gene products are not required for insulin gene expression.

Transcripts for E2A gene products, ubiquitous basic helix-loop-helix transactivating proteins, are expressed at high levels in the pancreatic epithelium. E2A proteins have been shown to bind the cognate E box sequence (CANNTG) of the insulin promoter/enhancer. E2A gene products dimerize with cell-specific basic helix-loop-helix proteins and synergize with the homeodomain transcription factor, PDX-1, in insulin gene transactivation. PDX-1 is also required for normal pancreatic development in mice. We investigated whether pancreatic development and insulin production could occur in the absence of E2A gene products by studying mice with a null mutation for the gene. E2A(-/-) mice demonstrated normal formation of pancreatic endocrine and exocrine tissue in histochemical sections as well as positive and distinct immunostaining for insulin and glucagon in islet tissue, signifying development of mature beta- and alpha-cells. Moreover, E2A(-/-) mice displayed no significant difference in blood glucose levels or pancreatic insulin content compared with wild-type littermates. These data show that although E2A gene products probably play an important role in insulin gene expression, pancreatic development and insulin production can proceed in their absence.

Adenovirus E2 Proteins↗
PubMed1996

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.

Adult↗
PubMed1996

Regulation of proliferation and differentiation of human fetal pancreatic islet cells by extracellular matrix, hepatocyte growth factor, and cell-cell contact.

Ex vivo expansion of human fetal pancreatic endocrine cells is important for biological studies and as a potential tissue source for transplantation in insulin-deficient states. In tissue culture experiments involving the use of hepatocyte growth factor/scatter factor and selected extracellular matrices, we obtained a 30-fold increase in cell number of human fetal pancreatic epithelial cells. This proliferation in monolayer culture was associated with marked downregulation of insulin and glucagon gene expression. However, gene expression increased when the cells were combined into three-dimensional aggregates, suggesting that cell-cell contact mediated mechanisms regulate the transcription of islet-specific genes, a process enhanced by nicotinamide (NIC). After transplantation into nude mice, either as cell suspensions or aggregates, only the cell aggregates treated with NIC developed into mature functional islet-like structures. These are the first experiments to describe the interactions of specific matrices and growth factors in the ex vivo expansion of human fetal pancreatic cells, and they also show the importance of cell aggregates in the context of cellular and molecular events that might positively influence islet cell transplantation.

Abortion, Induced↗
PubMed1995

Constitution of a biphasic insulin response to glucose in human fetal pancreatic beta-cells with glucagon-like peptide 1.

Insulin release from fetal beta-cells responds only minimally to acute glucose stimulation. Glucagon-like peptide-1 (GLP-1) is able to correct glucose sensitivity in some models of glucose-resistant beta-cells. We have now tested whether GLP-1 can induce glucose-responsive insulin release in human fetal islet-like cell clusters (ICCs). For this purpose we used perifusion and static incubation of ICCs and isolated adult human islets. In perifusion, the fetal ICCs responded with only minimal insulin release to 16.7 mmol/L glucose or 10 nmol/L GLP-1 combined with 1.67 mmol/L glucose. However, in the presence of GLP-1, the insulin response to glucose was markedly potentiated and appeared in a biphasic manner (10-fold first phase and 3-fold second phase). The first phase response was equal to that of adult human islets in similar experiments, whereas the second phase of the fetal cells was significantly lower. In static incubations, the relative insulin release responses to 16.7 mmol/L glucose plus 10 nmol/L GLP-1 were equal in the fetal and adult cells. The cAMP content of the cells was increased by glucose only in the presence of GLP-1. Our studies indicate that the glucoincretin hormone GLP-1 is able to constitute biphasic insulin release in the immature beta-cell, possibly as the result of cAMP-mediated priming of the glucose sensor mechanism.

Adult↗