Human Pancreatic Beta Cell Line Research Articles

760-P: Discovery of GSBR-1290, a Highly Potent, Orally Available, Novel Small Molecule GLP-1 Receptor Agonist

Peptidic glucagon-like peptide 1 receptor agonists (GLP-1RAs) are established treatments for patients with Type 2 Diabetes Mellitus (T2DM) and obesity. However, the need for injection and cold chain storage may limit the utility of most peptidic GLP-1RAs. An oral small molecule GLP-1RA that offers enhanced bioavailability and stability could be more convenient and accessible to patients. Here we report the discovery of GSBR-1290, a highly potent, orally available, novel small molecule GLP-1RA and the characterization of its in vitro and in vivo pharmacology profiles. GSBR-1290 is a small molecule GLP-1RA with high binding affinity to human GLP-1R. GSBR-1290 strongly activated GLP-1R Gαs cAMP pathway without inducing measurable β-arrestin recruitment signaling, which indicates it is a fully biased agonist. The insulin secretion stimulation effect of GSBR-1290 was evaluated in a functional human pancreatic beta cell line, in which GSBR-1290 showed dose dependent induction of insulin secretion. The in vivo efficacy of GSBR-1290 on insulin secretion, glucose control and food intake were evaluated in nonhuman primates (NHPs). In an acute intravenous glucose tolerance test (ivGTT), a single dose of GSBR-1290 strongly induced insulin secretion and glucose clearance. In a repeated dosing study, GSBR-1290 administered orally once daily for 7-day demonstrated robust increase in insulin secretion and glucose clearance in ivGTT and a dose dependent reduction of food intake and body weight. Overall, GSBR-1290 demonstrated potent in vivo efficacy in stimulating insulin secretion, improving glucose tolerance, and reducing food intake and body weight. In conclusion, GSBR-1290 is a highly potent, orally available, fully biased GLP-1RA. Human clinical trials are underway to further evaluate GSBR-1290 as a potential therapy for T2DM and obesity. Disclosure T.Mao: Employee; Structuretx. X.Lin: Employee; Structure Therapeutics. Q.Meng: Employee; Beijing Stonewise Technology, Structure Therapeutics Inc. H.Zhang: Employee; Structuretx. J.J.Zhang: Employee; Structure Therapeutics Inc. S.Shi: Employee; Structure Therapeutics Inc. Z.Guan: Employee; Structure Therapeutics. X.Jiang: Employee; Structure Therapeutics. F.Zhang: Employee; Structure Therapeutics, Eternity BioSciences, Stock/Shareholder; Novo Nordisk. H.Lei: Employee; Structure Therapeutics.

A genome-wide CRISPR screen identifies CALCOCO2 as a regulator of beta cell function influencing type 2 diabetes risk

Identification of the genes and processes mediating genetic association signals for complex diseases represents a major challenge. As many of the genetic signals for type 2 diabetes (T2D) exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of-function screen in a human pancreatic beta cell line. We assessed the regulation of insulin content as a disease-relevant readout of beta cell function and identified 580 genes influencing this phenotype. Integration with genetic and genomic data provided experimental support for 20 candidate T2D effector transcripts including the autophagy receptor CALCOCO2. Loss of CALCOCO2 was associated with distorted mitochondria, less proinsulin-containing immature granules and accumulation of autophagosomes upon inhibition of late-stage autophagy. Carriers of T2D-associated variants at the CALCOCO2 locus further displayed altered insulin secretion. Our study highlights how cellular screens can augment existing multi-omic efforts to support mechanistic understanding and provide evidence for causal effects at genome-wide association studies loci.

Investigation of the utility of the 1.1B4 cell as a model human beta cell line for study of persistent enteroviral infection

The generation of a human pancreatic beta cell line which reproduces the responses seen in primary beta cells, but is amenable to propagation in culture, has long been an important goal in diabetes research. This is particularly true for studies focussing on the role of enteroviral infection as a potential cause of beta-cell autoimmunity in type 1 diabetes. In the present work we made use of a clonal beta cell line (1.1B4) available from the European Collection of Authenticated Cell Cultures, which had been generated by the fusion of primary human beta-cells with a pancreatic ductal carcinoma cell, PANC-1. Our goal was to study the factors allowing the development and persistence of a chronic enteroviral infection in human beta-cells. Since PANC-1 cells have been reported to support persistent enteroviral infection, the hybrid 1.1B4 cells appeared to offer an ideal vehicle for our studies. In support of this, infection of the cells with a Coxsackie virus isolated originally from the pancreas of a child with type 1 diabetes, CVB4.E2, at a low multiplicity of infection, resulted in the development of a state of persistent infection. Investigation of the molecular mechanisms suggested that this response was facilitated by a number of unexpected outcomes including an apparent failure of the cells to up-regulate certain anti-viral response gene products in response to interferons. However, more detailed exploration revealed that this lack of response was restricted to molecular targets that were either activated by, or detected with, human-selective reagents. By contrast, and to our surprise, the cells were much more responsive to rodent-selective reagents. Using multiple approaches, we then established that populations of 1.1B4 cells are not homogeneous but that they contain a mixture of rodent and human cells. This was true both of our own cell stocks and those held by the European Collection of Authenticated Cell Cultures. In view of this unexpected finding, we developed a strategy to harvest, isolate and expand single cell clones from the heterogeneous population, which allowed us to establish colonies of 1.1B4 cells that were uniquely human (h1.1.B4). However, extensive analysis of the gene expression profiles, immunoreactive insulin content, regulated secretory pathways and the electrophysiological properties of these cells demonstrated that they did not retain the principal characteristics expected of human beta cells. Our data suggest that stocks of 1.1B4 cells should be evaluated carefully prior to their use as a model human beta-cell since they may not retain the phenotype expected of human beta-cells.

A functional genomic approach to identify reference genes for human pancreatic beta cell real-time quantitative RT-PCR analysis

ABSTRACT Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression. We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-βH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| <1.5) and a coefficient of variation <10%. Candidate reference genes were validated by quantitative real-time PCR in independent samples. We identified a total of 264 genes stably expressed in EndoC-βH1 cells and human islets following cytokines – or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells.

Regulated expression and function of the GABAB receptor in human pancreatic beta cell line and islets

G protein-coupled receptors are seven transmembrane signaling molecules that are involved in a wide variety of physiological processes. They constitute a large protein family of receptors with almost 300 members detected in human pancreatic islet preparations. However, the functional role of these receptors in pancreatic islets is unknown in most cases. We generated a new stable human beta cell line from neonatal pancreas. This cell line, named ECN90 expresses both subunits (GABBR1 and GABBR2) of the metabotropic GABAB receptor compared to human islet. In ECN90 cells, baclofen, a specific GABAB receptor agonist, inhibits cAMP signaling causing decreased expression of beta cell-specific genes such as MAFA and PCSK1, and reduced insulin secretion. We next demonstrated that in primary human islets, GABBR2 mRNA expression is strongly induced under cAMP signaling, while GABBR1 mRNA is constitutively expressed. We also found that induction and activation of the GABAB receptor in human islets modulates insulin secretion.

SUN-645 Cornus Officinalis Promotes IGFBP2 and Autophagy in Human 1.1B4 Pancreatic Cell Line as Revealed by Employing a Global Proteomic Approach via Mass Spectrometry

Type 1 diabetes (T1D) results in the loss of pancreatic beta cells and subsequent loss of insulin production. Exogenous insulin is the only effective treatment, but there is still no cure or interventional therapy available to inhibit progression of T1D. Successful T1D interventional therapy must protect pancreatic beta cells from autoimmunity while enhancing beta cell survival and function. Our data suggests Cornus officinalis (CO) may be a candidate for interventional therapy to protect pancreatic beta cells from autoimmune attack and increase their function. CO has been used in traditional Chinese medicine (TCM) for over 2,000 years and has shown characteristics of anti-diabetic effects in vitro and in vivo but never examined in the application of T1D. Our prior publication (Mol. and Cell. Endo.2019:494:110491), has shown increased proliferation and protection against Th1 cytokine attack upon CO treatment using a human pancreatic beta cell line, 1.1B4. From this, we sought to define precise molecular mechanism by employing a global and phosphorylation mass spectrometry (MS) approach. We applied CO to 1.1B4 cells for 2, 6, 12, and 24h then collected the cell lysates for MS analysis. Our global MS analysis revealed a 12-fold increase in beta cell functional regulator, IGFBP2, at multiple time points. IGFBP2 has been shown to display a T2D protective effect and regulate glucose metabolism. The ingenuity pathway analysis program (IPA) predicted an increase in insulin starting at 2h and the NRF2-mediated oxidative stress pathway at 12h and 24h. Furthermore, NRF2 is an upstream regulator of P62 which was significantly hyperphosphorylated at multiple timepoints from our MS analysis. Nrf2 is responsible for activating antioxidant enzymes upon oxidative stress, which is caused by proinflammatory cytokines in T1D. P62 aids in this pathway by targeting proteins for autophagy upon oxidative stress in order to keep cellular homeostasis within beta cells rather than cells progressing through apoptosis. Autophagy is critical for beta cell function and survival as it promotes survival under beta cell stress which would otherwise lead to cell death. The recovery and protection of autophagy in beta cells of patients in the pre-diagnosed stages of T1D could provide a beneficial interventional therapy in order to delay or inhibit the onset of T1D. Altogether, our proteomic analysis revealed an increase in IGFBP2 and predicted an increase in the NRf2-mediated oxidative stress pathway upon CO induction. Further analysis will examine the IGFBP2 and Nrf2-mediated oxidative pathway as a mechanism of CO induced protective and proliferative effects in pancreatic beta cells.

2164-P: Paired Box 6 Modulates Pancreatic Beta-Cell Insulin Secretion through Regulation of Munc18-1/2 Expression: Involvement of CREB Signaling

The Paired box 6 (PAX6) transcription factor is crucial for normal pancreatic islet development and function. Heterozygous mutations of PAX6 are associated with impaired insulin secretion and early-onset diabetes mellitus in human. Previous studies demonstrated that PAX6 plays critical roles in insulin biosynthesis, glucose-sensing and glucose-induced ATP synthesis (the proximal signaling of insulin secretion). Yet the molecular mechanism of PAX6 in controlling beta-cell insulin secretion, especially the involvement of distal signaling, remains ambiguous. This study investigated the regulatory role of PAX6 in exocytotic proteins, the core components of distal signaling of insulin secretion, by using human pancreatic beta-cell line EndoC-bH1. The correlation between PAX6 and type 2 diabetes mellitus (T2DM)-associated beta-cell dysfunction was also assessed. Loss-of-function and gain-of-function studies showed that PAX6 positively regulated the expression of exocytotic proteins Munc18-1 and Munc18-2 in beta-cells by modulating the activity of cAMP response element-binding protein (CREB). The PAX6/CREB/Munc18-1/2 axis in turn controlled glucose-stimulated insulin secretion. On the other hand, EndoC-bH1 cells under diabetic condition displayed diminished PAX6 expression, blunted CREB phosphorylation and Munc18-1/2 down-regulation which were rescued by PAX6 overexpression. We conclude that PAX6 regulates distal signaling of insulin secretion through CREB/Munc18-1/2 axis; while maintenance of normal PAX6 expression or activity in human beta-cells may serve as a promising strategy in preserving beta-cell function and delaying the onset of T2DM. Disclosure W. So: None. W. Han: None. Funding National Medical Research Council of Australia

Studies of cell surface and soluble HLA class I expression in pancreatic beta cells exposed to interferons or Poly I:C

HLA class I (HLA-I) molecules play a crucial role in cell-mediated immunity by presenting peptide antigens to cytotoxic CD8+T cells. A pathological hallmark of Type 1 diabetes (T1D) is the hyperexpression of HLA-I in pancreatic islets that contain residual insulin producing beta cells. The expression of HLA-I can be induced following exposure to interferons (IFN). Previous studies have implicated enteroviruses (EV) as major players in triggering an autoimmune response against beta cells and it seems likely that the hyperexpression of HLA-I contributes to the recognition and targeting of beta cells by CD8+T cells. Whilst HLA-I molecules are expressed on the surface of nucleated cells, there is increasing evidence that HLA-I can also be found in a soluble form in the plasma, including in patients with viral infections. Intriguingly, soluble HLA-I (sHLA-I) levels are significantly elevated in the serum of patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and T1D, yet the function of sHLA-I is still unclear. In this project, we are investigating the impact of interferons and poly I:C, a viral dsRNA mimetic, on the expression of surface and soluble HLA-I in the human pancreatic beta cell line EndoC-βH1, the human pancreatic ductal cell line PANC1 and in HeLa cells. We show that surface HLA-I is upregulated in response to these stimuli in each of the cell lines, with greatest magnitude in EndoC-βH1. We also show for the first time that release of sHLA-I is significantly increased in response to IFNγ in EndoC-βH1 cell line.

Characterization of an encapsulated insulin secreting human pancreatic beta cell line in a modular microfluidic device

Type I diabetes mellitus is characterised by the destruction of the insulin producing beta cells within the pancreas by the immune system. After the success of Edmonton protocol, islet transplantation has shown to be a promising therapy, but with the Achilles´ heel of the need of using immunosuppressive drugs. Currently, cell encapsulation technology represents a real alternative to protect transplanted islets from the host´s immune attack. Although preliminary in vitro studies with encapsulated cells have been traditionally performed under static conditions in terms of viability and efficiency, these static cultures do not represent a close approach to in vivo environments. We have developed and characterised different alginate-poly-l-lysine-alginate (APA) microcapsules loaded with the insulin producing 1.1B4 cell line. Static in vitro studies confirmed a constant insulin secretion and a boost of the secretion when the medium was enriched with glucose. Nevertheless, these results were not completely reproduced in a dynamic system by APA liquefied microcapsules containing 1.1B4 cells. The dynamic culture setting created by a microfluidic device, allowed the determination of the glucose response in APA liquefied microcapsules, showing that dynamic conditions can mimic better physiological in vivo environments.

Molecular Mechanisms of Toxicity and Cell Damage by Chemicals in a Human Pancreatic Beta Cell Line, 1.1B4.

Mechanisms of toxicity and cell damage were investigated in novel clonal human pancreatic beta cell line, 1.1B4, after exposure to streptozotocin, alloxan, ninhydrin, and hydrogen peroxide. Viability, DNA damage, insulin secretion/content, [Ca]i, and glucokinase/hexokinase, mRNA expression were measured by MTT assay, comet assay, radioimmunoassay, fluorometric imaging plate reader, enzyme-coupled photometry, and real-time polymerase chain reaction, respectively. Chemicals significantly reduced 1.1B4 cell viability in a time/concentration-dependent manner. Chronic 18-hour exposure decreased cellular insulin, glucokinase, and hexokinase activities. Chemicals decreased transcription of INS, GCK, PCSK1, PCSK2, and GJA1 (involved in secretory function). Insulin release and [Ca]i responses to nutrients and membrane-depolarizing agents were impaired. Streptozotocin and alloxan up-regulated transcription of genes, SOD1 and SOD2 (antioxidant enzymes). Ninhydrin and hydrogen peroxide up-regulated SOD2 transcription, whereas alloxan and hydrogen peroxide increased CAT transcription. Chemicals induced DNA damage, apoptosis, and increased caspase 3/7 activity. Streptozotocin and alloxan decreased transcription of BCL2 while increasing transcription of BAX. Chemicals did not affect transcription of HSPA4 and HSPA5 and nitrite production. 1.1B4 cells represent a useful model of human beta cells. Chemicals impaired 1.1B4 cell secretory function and activated antioxidant defense and apoptotic pathways without activating endoplasmic reticulum stress response/nitrosative stress.

A human beta cell line with drug inducible excision of immortalizing transgenes.

ObjectivesAccess to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen.MethodsWe used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion.ResultsWe showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture.ConclusionsEndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture.

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1μM, 1μM, 10μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE.

Human fucci pancreatic Beta cell lines: new tools to study Beta cell cycle and terminal differentiation.

Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation.

Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4

BackgroundMolecular mechanisms of toxicity and cell damage were investigated in the novel human beta cell line, 1.1B4, after exposure to proinflammatory cytokines — IL-1β, IFN-γ, TNF-α. MethodsMTT assay, insulin radioimmunoassay, glucokinase assay, real time reverse transcription PCR, western blotting, nitrite assay, caspase assay and comet assay were used to investigate mechanisms of cytokine toxicity. ResultsViability of 1.1B4 cells decreased after 18h cytokine exposure. Cytokines significantly reduced cellular insulin content and impaired insulin secretion induced by glucose, alanine, KCl, elevated Ca2+, GLP-1 or forskolin. Glucokinase enzyme activity, regulation of intracellular Ca2+ and PDX1 protein expression were significantly reduced by cytokines. mRNA expression of genes involved in secretory function — INS, GCK, PCSK2 and GJA1 was downregulated in cytokine treated 1.1B4 cells. Upregulation of transcription of genes involved in antioxidant defence — SOD2 and GPX1 was observed, suggesting involvement of oxidative stress. Cytokines also upregulated transcriptions of NFKB1 and STAT1, which was accompanied by a significant increase in NOS2 transcription and accumulation of nitrite in culture medium, implicating nitrosative stress. Oxidative and nitrosative stresses induced apoptosis was evident from increased % tail DNA, DNA fragmentation, caspase 3/7 activity, apoptotic cells and lower BCL2 protein expression. ConclusionsThis study delineates molecular mechanisms of cytokine toxicity in 1.1B4 cells, which agree with earlier observations using human islets and rodent beta cells. General significanceThis study emphasizes the potential usefulness of this cell line as a human beta cell model for research investigating autoimmune destruction of pancreatic beta cells.

Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.

Cell Therapy for Diabetes Mellitus

The number of diabetic patients in the world is increasing in recent years and the prevention of diabetes mellitus is therefore one of the urgent medical issues. Exogenous insulin is used for the control of blood glucose in diabetic patients; however, hypoglycemic episodes are unavoidable. Over the last several decades, islet transplantation has been developed as a promising method to achieve strict control of blood glucose and a potential cure for type 1 diabetes. However, due to the shortage of donor pancreata, alternative sources of islets have been sought through the generation of beta cells from stem cells, use of porcine islets, and beta cell expansion with growth factors. However, differentiation and expansion of embryonic and pancreatic stem cells and expansion of differentiated beta cells in vitro is limited. Expansion of primary beta cells by growth factors is also hampered by the senescence of the cells. Thus, we focused on establishing a human pancreatic beta cell line that is functionally equivalent to primary beta cells and can yield large amounts of cells for transplantation. Using Cre/loxP-based reversible immortalization, we constructed a reversibly immortalized pancreatic beta cell clone (NAKT-15). The cells may overcome the limitation of primary pancreatic beta cells for transplantation to control type 1 diabetes. In order to avoid the use of immunosuppressive agents, we are currently engaged in developing an implantable bag-type bioartificial pancreas. In this article, I discuss the hurdles of the current therapy for diabetes and introduce the possible future treatment of diabetes.

A human β-cell line for transplantation therapy to control type 1 diabetes

A human pancreatic beta-cell line that is functionally equivalent to primary beta-cells has not been available. We established a reversibly immortalized human beta-cell clone (NAKT-15) by transfection of primary human beta-cells with a retroviral vector containing simian virus 40 large T-antigen (SV40T) and human telomerase reverse transcriptase (hTERT) cDNAs flanked by paired loxP recombination targets, which allow deletion of SV40T and TERT by Cre recombinase. Reverted NAKT-15 cells expressed beta-cell transcription factors (Isl-1, Pax 6, Nkx 6.1, Pdx-1), prohormone convertases 1/3 and 2, and secretory granule proteins, and secreted insulin in response to glucose, similar to normal human islets. Transplantation of NAKT-15 cells into streptozotocin-induced diabetic severe combined immunodeficiency mice resulted in perfect control of blood glucose within 2 weeks; mice remained normoglycemic for longer than 30 weeks. The establishment of this cell line is one step toward a potential cure of diabetes by transplantation.

TNF-Related Apoptosis-Inducing Ligand Death Pathway-Mediated Human Beta-Cell Destruction

The aim of this study is to investigate whether apoptosis in human beta cells can be related to the induction of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. We examined the expression of TRAIL and TRAIL receptors in two human pancreatic beta-cell lines and in human primary islet cells using RT-PCR assays and flow cytometric analyses and tested TRAIL-mediated beta-cell destruction in (51)Cr release cytotoxicity assays, Annexin-V and APO-DIREC assays. Most of the human beta cells express TRAIL receptors-R1, -R2, -R3, -R4 and/or TRAIL. TRAIL induced much stronger cytotoxicity and apoptosis to beta-cell lines CM and HP62 than did FasL, TNF-alpha, LTalpha1beta2, LTalpha2beta1, LIGHT, and IFN-gamma. The cytotoxicity and apoptosis induced by TRAIL to beta-cell lines CM were inhibited competitively by soluble TRAIL receptors, R1, R2, R3 or R4. Treatment of these beta cells with antibodies against TRAIL receptors was able to block the cytotoxicity of TRAIL to these cells. Beta-cell antigen-specific CTL (CD4(+) and CD8(+)) clones express TRAIL, suggesting that these cells are potential sources of TRAIL-inducing beta-cell destruction. Normal primary islet cells from most donors are resistant to the cytotoxicity mediated by TRAIL. However, treatment with an inhibitor of protein synthesis (cycloheximide) or with an enzyme (PI-PLC) that can remove TRAIL-R3 from the islet-cell membrane was able to increase the susceptibility of TRAIL-resistant primary islet cells to the TRAIL death pathway. The TRAIL death pathway is present and can function in human islet beta cells, but unidentified inhibitors of the TRAIL death pathway are present in normal islet cells.

Analysis of islet cell antibodies reactivity to a human islet cell line.

A human pancreatic beta cell line (HP62) was tested for reactivity with islet cell antibodies (ICA) as compared with previously-established methods. Using indirect immunofluorescence test, we found that HP62 cell line failed to react in a specific way with ICA from type 1 (insulin-dependent) diabetic patients since sera from normal controls showed a reactivity similar to that found in the patients. So, the usefulness of this human beta cell line as a tool of immunological purpose is questioned when indirect immunofluorescence procedures are used.