Human embryonic stem cell differentiation into insulin secreting β‐cells for diabetes

hESC (human embryonic stem cells), when differentiated into pancreatic β ILC (islet‐like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five‐step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine β islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti‐hyperglycaemic hormone‐Glp1 (glucagon‐like peptide 1) analogue Liraglutide with prolonged t_½ and Exendin 4. The differentiated islet‐like 3D clusters expressed bonafide mature and functional β‐cell markers‐PDX1 (pancreatic and duodenal homoeobox‐1), C‐peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C‐peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D β islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non‐obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long‐term in vivo functionally viable islets from hESC.     

Formation of islet cell monolayers from fetal human and neonatal rat pancreatic islets: protein biosynthesis, insulin secretion, and binding of islet cell antibodies

The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) has been used to promote monolayer formation in cultured islets isolated from fetal human or neonatal rat pancreas. Immunofluorescence with specific antiserum to insulin revealed B-cells in the outgrown monolayers. The rat islet cells were further characterized by their secretory and biosynthetic response to the action of IBMX. Both glucose-stimulated insulin release and recoverable insulin, i.e. intracellular insulin plus insulin secreted, were increased by the addition of IBMX (0.1 mmol/l) to the medium containing 10 mmol/l glucose. 3H-leucine incorporation into (pro)insulin was significantly higher following culture in 10 mmol/l glucose plus IBMX (0.1 and 1.0 mmol/l) than after cultivation with glucose alone. However, the percentage of (pro)insulin synthesized in relation to total protein synthesis was increased to a lesser extent after an acute incubation for 3 h at 1.5 mmol/l or in the absence of glucose. Moreover, the culture system employed in the present study proved to be useful for detecting islet cell antibodies bound to human as well as rat islet cells after exposure to islet cell antibody-positive sera.     

HPAC,a new human glucocorticoid-sensitive pancreatic ductal adenocarcinoma cell line

Summary A new human pancreatic cancer (HPAC) cell line was established from a nude mouse xenograft (CAP) of a primary human pancreatic ductal adenocarcinoma. In culture, HPAC cells form monolayers of morphologically heterogenous, polar epithelial cells, which synthesize carcinoembryonic antigen, CA 19-9, CA-125, cytokeratins, antigens for DU-PAN-2, HMFG1, and AUA1, but do not express chromogranin A or vimentin indicative of their pancreatic ductal epithelial cell character. In the presence of serum, HPAC cell DNA synthesis was stimulated by insulin, insulin growth factor-I, epidermal growth factor, and TGF-α but inhibited by physiologic concentrations of hydrocortisone and dexamethasone. Dose-dependent inhibition of DNA synthesis was limited to steroids with glucocorticoid activity. The inhibitory effect of dexamethasone was abolished by the glucocorticoid antagonist RU 38486. Binding of [³H]dexamethasone to cytosolic proteins was specific and saturable at 4° C. Scatchard analysis of binding data demonstrated a single class of high-affinity binding sites (K_d=3.8±0.9 nM; B_max=523±128 fmol/mg protein). Western blot analysis revealed a major protein band that migrated at a M_r of 96 kDa. Northern blot analysis identified an mRNA of approximately 7 kilobases which hybridized with a specific glucocorticoid receptor complementary DNA probe (OB7). These findings support a role for glucocorticoids in the regulation of human malignant pancreatic cell function.     

Cortisone-induced islet cell hyperplasia in hamsters

Pancreatic islet cell hyperplasia was studied in hamsters during one to eight weeks of cortisone treatment. Measurement of serum glucose and insulin; pancreatic insulin, glucagon, somatostatin, pancreatic polypeptide as well as islet tissue morphometry were performed. Serum glucose was highest at week 2, followed by mild to moderate hyperglycemia. Serum insulin was increasingly higher from week 1 to week 8. Pancreatic insulin was maximal at week 5 then declined through week 8 in the presence of beta cell neurosis in markedly hyperplastic islets. Pancreatic concentration of somatostatin and pancreatic polypeptide moderately increased more than the control levels; however, compared with the controls, glucagon was reduced by cortisone treatment. Effect of cortisone in the four types of islet cells is discussed, particularly on beta cell hyperplasia, which appears to be a response to decreased insulin binding to the target organs with no changes in receptor concentration.     

Antioxidant treatment may protect pancreatic beta cells through the attenuation of islet fibrosis in an animal model of type 2 diabetes

Islet fibrosis could be important in the progression of pancreatic beta cell failure in type 2 diabetes. It is known that oxidative stress is involved in the pancreatic fibrosis through the activation of pancreatic stellate cells. However, no study has investigated the in vivo effects of antioxidants on islet fibrogenesis in type 2 diabetes. In this study, antioxidants (taurine or tempol) were administered in drinking water to Otsuka Long-Evans Tokushima Fatty rats, an animal model of type 2 diabetes, for 16 weeks. An intraperitoneal glucose tolerance test revealed that the blood glucose levels after the glucose injection were decreased by the antioxidants. The insulin secretion after the glucose injection, which was markedly reduced in the rats, was also restored by the antioxidants. Beta cell mass and pancreatic insulin content were greater in the rats treated with the antioxidants than in the untreated rats. Beta cell apoptosis was attenuated in the rats by the antioxidants. Finally, islet fibrosis and the activation of pancreatic stellate cells were markedly diminished in the rats by the antioxidants. Our data suggest that antioxidants may protect beta cells through the attenuation of both islet fibrosis and beta cell apoptosis in type 2 diabetes.     

Glucagon secretion by dispersed alpha cell enriched islets from streptozotocin treated hamsters in perifusion

Streptozotocin (70 mg/kg) was administered intravenously to female Syrian hamsters. The hamsters received insulin (5U/animal/day). Insulin treatment was withdrawn 3 days before sacrifice in one group, while another group was maintained on insulin until sacrifice. Ten to 14 days following streptozotocin administration the animals were killed, and the pancreatic islets isolated and subsequently dispersed. Islet DNA content was decreased while the glucagon content was elevated by streptozotocin treatment. The glucagon secretory responsiveness of the dispersed alpha cells of control animals was stimulated by glucopenia and decreased by glucose. Alpha cells of streptozotocin hamsters were not only suppressed but were actually stimulated by high glucose concentrations. Treatment with insulin in vivo but not in vitro, resulted in a restoration of the alpha cells responsiveness to glucose suppression. Dispersed alpha cells from control and streptozotocin treated animals were stimulated by arginine. Basal and total glucagon secretion was greatest in dispersed alpha cells from streptozotocin treated animals. We concluded: that the paradoxical response of alpha cells to glucose noted in diabetes is not due to short term insulin deprivation or the lack of morphologic contact with beta cells; that the alpha cells require and insulin stimulated islet metabolite and extra islet materials to respond appropriately to glucose; and that the alpha cells response to arginine is mediated independently of glucose regulation.     

Metabolic and secretory effects of methylamine in pancreatic islets

The metabolic and secretory effects of methylamine in rat pancreatic islets were investigated. Methylamine accumulated in islet cells, was incorporated into endogenous islet proteins, and inhibited the incorporation of [2,5-³H] histamine into either N,N-dimethylcasein or endogenous islet proteins. Methylamine (2 mM ) did not affect the oxidation of glucose or endogenous nutrients or the intracellular pH in islet cells. Glucose did not affect the activity of transglutaminase in islet homogenates, the uptake of ¹⁴C-methylamine by intact islets or its incorporation into endogenous islet proteins. Methylamine inhibited insulin release evoked by glucose, other nutrient secretagogues, and non-nutrient insulinotropic agents such as L -arginine or gliclazide. The inhibitory effect of methylamine upon insulin release was diminished in the presence of cytochalasin B or at low extracellular pH. Methylamine retarded the conversion of proinsulin to insulin. Trimethylamine (0.7 mM ) was more efficiently taken up by islet cells than methylamine (2.0 mM ), and yet caused only a modest inhibition of insulin release. These findings suggest that methylamine interferes with a late step in the secretory sequence, possibly by inhibiting the access of secretory granules to their exocytotic site.     

Receptors for insulin and insulin-like growth factor-1 and insulin receptor substrate-1 mediate pathways that regulate islet function

The insulin/insulin-like growth factor-1 (IGF-1) signalling pathways are present in most mammalian cells and play important roles in the growth and metabolism of tissues. Most proteins in these pathways have also been identified in the beta-cells of the pancreatic islets. Tissue-specific knockout of the insulin receptor (betaIRKO) or IGF-1 receptor (betaIGFRKO) in pancreatic beta-cells leads to altered glucose-sensing and glucose intolerance in adult mice, and betaIRKO mice show an age-dependent decrease in islet size and beta-cell mass. These data indicate that these receptors are important for differentiated function and are unlikely to play a major role in the early growth and/or development of the pancreatic islets. Conventional insulin receptor substrate-1 (IRS-1) knockouts manifest growth retardation and mild insulin resistance. The IRS-1 knockouts also display islet hyperplasia, defects in insulin secretory responses to multiple stimuli both in vivo and in vitro, reduced islet insulin content and an increased number of autophagic vacuoles in the beta-cells. Re-expression of IRS-1 in cultured beta-cells is able to partially restore the insulin content indicating that IRS-1 is involved in the regulation of insulin synthesis. Taken together, these data provide evidence that insulin and IGF-1 receptors and IRS-1, and potentially other proteins in the insulin/IGF-1 signalling pathway, contribute to the regulation of islet hormone secretion and synthesis and therefore in the maintenance of glucose homeostasis.     

Effect of fibroblast growth factor and nerve growth factor on the cellular proliferation and functional activity of isolated islands of Langerhans

The effects of fibroblast growth factor (FGF) and nerve growth factor (NGF) on DNA synthesis and insulin secretion were studied in 4-5-day cultures of the isolated neonatal rat islets. FGF (0.1 ng/ml) stimulated significantly the incorporation of 3H-thymidine into DNA of the isolated islets, but failed to change either insulin content in the islets or the rate of insulin secretion. NGF (0.1-1000 ng/ml) did not affect the above parameters. The responses of the islets of Langerhans to increasing concentrations of glucose and isobutylmethylxanthine were not modified after prolonged exposure to NGF. The role of FGF and NGF in the regulation of proliferation and secretory process in pancreatic islet cells is discussed.     

Insulin secretion from perifused rat pancreatic pseudoislets

Summary Isolated adult rat pancreatic islets were dispersed into single cells and cultured free-floating for 3 to 4 d, during which time islet cells reaggregated spontaneously into spherical clusters or pseudoislets. The gross morphology of these tissues resembled nondissociated islets. Electron microscopy revealed well-preserved cell ultrastructure and intercellular membrane connections. Immunofluorescent localization of islet cell types showed that A cells tended to be peripherally distributed around a B cell core, with D cells scattered throughtout the aggregate, mass. The dynamics of insulin release from pseudoislets were evaluated in vitro by perifusion techniques. Pseudoislets exhibited clear biphasic dose-dependent insulin responses to 30 min glucose stimulation over the range 5.5 to 30 mM. Repeated 2-min pulses with 22 mM glucose elicited brief monophasic spikes of insulin release of, consistent magnitude.l-Arginine (5 to 20 mM) evoked biphasic insulin release but these responses were not dose-dependent. These data indicate that islet cells reaggregate into structures with close morphologic similarities to intact islets, and that pseudoislet B cells continue to secrete insulin in response to nutrient secretagogues, comparable to that seen with islets in vitro and in situ. This work was supported by grants from the Medical Research Council of New Zealand. D. W. H. was the recipient of a Novo Diabetes Research Scholarship.     

Allogeneic bone marrow supports human islet beta cell survival and function over six months

In this study, we have established a new strategy increasing human islet longevity utilizing allogeneic whole bone marrow (BM) co-cultured with human islets. The cultured islets' function and survival have been evaluated by analysis of insulin secretion in response to high-glucose-challenge, morphological evaluation of cell growth. Human islet only culture failed to reveal evidence of long term survival, growth or function in terms of insulin release or insulin response to glucose challenge. These results indicate that BM increases islet survival and function with the eventual formation of pancreatic endocrine tissue capable of sustaining beta cell fuction.     

Effects of cyclic AMP on DNA replication and protein biosynthesis in fetal rat islets of Langerhans maintained in tissue culture

The regulatory role of cyclic AMP (cAMP) in the growth and insulin production of the islet organ in vitro has been investigated. The effects of dibutyryl cyclic AMP (dbcAMP), theophylline , and 3-isobutyl-1-methylxanthine (IBMX) on DNA replication and on the biosynthesis of RNA and insulin in fetal rat islets of Langerhans maintained in tissue culture have been studied. Raising the glucose concentration from 2.7 mM to 16.7 mM caused a two-fold increase in DNA replication. Both dbcAMP and theophylline markedly inhibited the DNA replication at all glucose Concentrations studied. Low concentrations of IBMX stimulated DNA synthesis. However, at higher concentrations of this drug, known to considerably increase the islet cAMP levels , a marked inhibition of islet DNA replication was observed. Both (pro)insulin and total protein biosynthesis were stimulated by glucose, whereas dbcAMP stimulated only the (pro)insulin biosynthesis. Since glucose is known to raise islet intracellular levels of cAMP, which is known to be an inhibitor of cellular proliferation, the observed glucose stimulation of both islet-cell DNA replication and insulin production appeared conflicting. It is suggested that this dual effect of glucose may depend on a stimulation of proliferation in a limited pool of islet cells which may not exhibit an increase in cAMP.     

Standardization of insulin secretion from pancreatic islets: validation of a DNA assay

The use of islet DNA content to standardize insulin secretion rates from pancreatic islets of different sizes has been studied. Isolated intact islets were sorted into 4 size categories and perifused with 22 mM glucose, collecting effluent in 5 min fractions for insulin RIA. DNA content of perifused islets was measured by fluorometric assay, and insulin secretion expressed as pmoles/ug DNA/unit time. For islets with diameters less than 300 u (1) insulin secretion was proportional to islet size; (2) insulin release per islet and islet DNA content were strongly correlated; (3) when expressed as a function of DNA content, insulin secretion from different sized islets was not significantly different. These relationships did not continue for very large islets (above 300 u) suggesting a limiting islet size for insulin secretion in vitro. The data demonstrates that expression of insulin secretion from pancreatic islets with diameters less than 300 u, as a function of their DNA content standardizes secretion irrespective of islet size and number, and should allow direct comparison of secretory responses between different islet tissue preparations.     

Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes

Adult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum-free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen-coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, ～80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was ～70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G₀ phase, but that when they formed monolayers, they progressed to the G₁ phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver-specific functions. © 1993 Wiley-Liss, Inc.     

Effect of 2-deoxy-d-glucose on maintenance in culture of neonatal B cell of rat

Summary The effect of 2-deoxy-d-glucose on maintenance in culture of B cells of the neonatal rat was examined by supplementation of Medium 199 containing 5.5 mM glucose with 1 mM 2-deoxy-d-glucose. Islets maintained in medium with 5.5 mM glucose (basal medium) for 7 d underwent remarkable decreases in glucose sensitivity, and the levels of insulin in the medium dropped. By contrast, addition of 2-deoxy-d-glucose promoted a higher insulin content in medium and an increase in the glucose-induced insulin release and biosynthesis. Moreover, the addition of the deoxysugar caused a selective deletion of fibroblasts and prevented the deterioration of islet cells in basal medium, yielding clusters mostly consisting of islet cells at the end of culture.     

A role for polyamines in glucose-stimulated insulin-gene expression.

The aim of the present study was to evaluate the possible role for polyamines in the glucose regulation of the metabolism of insulin mRNA of pancreatic islet cells. For this purpose islets were prepared from adult mice and cultured for 2 days in culture medium RPMI 1640 containing 3.3 mM- or 16.7 mM-glucose with or without the addition of the inhibitors of polyamine biosynthesis difluoromethylornithine (DFMO) and ethylglyoxal bis(guanylhydrazone) (EGBG). Culture at the high glucose concentration increased the islet contents of both insulin mRNA and polyamines. The synthesis of total RNA, total islet polyamines and polyamines associated with islet nuclei was also increased. When the combination of DFMO and EGBG was added in the presence of 16.7 mM-glucose, low contents of insulin mRNA, spermine and spermidine were observed. Total islet polyamine synthesis was also depressed by DFMO + EGBG, unlike islet biosynthesis of polyamines associated with nuclei, which was not equally decreased by the polyamine-synthesis inhibitors. Total RNA synthesis and turnover was not affected by DFMO + EGBG. Finally, actinomycin D attenuated the glucose-induced enhancement of insulin mRNA, and cycloheximide counteracted the insulin-mRNA attenuation induced by inhibition of polyamine synthesis. It is concluded that the glucose-induced increase in insulin mRNA is paralleled by increased contents and rates of polyamine biosynthesis and that an attenuation of the increase in polyamines prevents the increase in insulin mRNA. In addition, the results are compatible with the view that polyamines exert their effects on insulin mRNA mainly by increasing the stability of this messenger.     

Growth hormone is a growth factor for the differentiated pancreatic beta-cell

The regulation of the growth of the pancreatic beta-cell is poorly understood. There are previous indications of a role of GH in the growth and insulin production of the pancreatic islets. In the present study we present evidence for a direct long-term effect of GH on proliferation and insulin biosynthesis of pancreatic beta-cells in monolayer culture. In culture medium RPMI 1640 supplemented with 2% normal human serum islets or dissociated islet cells from newborn rats maintained their insulin-producing capacity. When supplemented with 1-1000 ng/ml pituitary or recombinant human GH the islet cells attached, spread out, and proliferated into monolayers mainly consisting of insulin-containing cells. The number of beta-cells in S-phase was increased from 0.9-6.5% as determined by immunochemical staining of bromodeoxyuridine incorporated into insulin-positive cells. The increase in cell number was accompanied with a continuous increase in insulin release to the culture medium reaching a 10- 20-fold increase after 2-3 months with a half-maximal effect at about 10 ng/ml human GH. The biosynthesis of (pro)insulin was markedly increased with a normal rate of conversion of proinsulin to insulin. It is concluded that GH is a potent growth factor for the differentiated pancreatic beta-cell.     

Establishment and immunocharacterization of an immortalized pancreatic cell line derived from the H-2Kb-tsA58 transgenic mouse

Summary This study describes the establishment and characterization of an immortalized cell line derived from the pancreas of an adult H-2K^b-tsA58 transgenic mouse. These cells, designated IMPAN for IMmortalized PANcreatic cells, displayed a cobblestone appearance typical of confluent epithelial cells and a distinct polarity in the organization of their cytoplasmic organelles. Immunocytochemical studies revealed that all IMPAN cells stained positively for a wide range of markers characteristic of pancreatic acinar cells, namely the secretory products α-amylase, chymotrypsinogen, DNAse, the lectinlike secretory protein PAP (pancreatitis associated protein), and the zymogen granule membrane proteins GP-2 and gp300. They also stained positively for carbonic anhydrase II and cytokeratin 19, two proteins characteristic of pancreatic duct cells, as well as for rab3A, a small GTP-binding protein specifically localized in pancreatic islet cells. No reactivity was ever obtained with insulin antibodies. Taken together, these results show that the IMPAN cells exhibit a phenotype comparable to exocrine pancreatic acinar cells. However the expression of some proteins more specific to duct and islet cells make them similar to in vivo or in vitro growing acinar cells. The cell line should be a valuable model to study the mechanisms of growth, differentiation, and transformation of the exocrine pancreatic acinar cell.