Islet Cell Suspensions Research Articles

1450-P: Identifying Hidden Sources of Tissue Plasticity in Pancreatic Islets with a Novel Multicolor Flow Cytometry Approach

Identifying pancreatic islet cells that acquire a beta-cell like phenotype is important for developing new diabetes therapies. Our studies have revealed a novel serpinB13 protease inhibitor antibody, that augments cathepsin L protease activity, resulting in Notch1 receptor cleavage and mobilization of endocrine progenitor cells to become beta cells in the embryonic pancreas. It is unclear, however, whether a similar induction of additional beta cells occurs with exposure to serpinB13 monoclonal antibody (mAb) in later life. To explore the exact source (s) of new beta cells in response to serpinB13 mAb after birth we developed a novel, multicolor flow cytometry analysis approach to study pancreatic islets. Five-week old normal Balb/c mice were injected with serpinB13 mAb (or control IgG) , and sacrificed at 8 weeks for islet isolation. Islet cell suspensions were stained with a viability dye, and a panel of antibodies to CD31, CD45, cytokeratin 19, EpCAM, insulin (Ins) , glucagon (Glu) , somatostatin (SS) , c-peptide, and four transcription factors, Maf-A, Nkx6.1, NeuroD1, and Pdx-1. Exposure to the serpinB13 mAb caused a significant increase in the number of hormonal-negative (e.g., Ins-Glu-SS-c-peptide-) cells (p=0.0011) , and hormonal negative, but c-peptide positive, cells (p=0.002) . At least one of four transcription factors examined were positive in 5-10% and 10-30% of these populations, respectively. At the same time, there was a trend in the drop of Glu+ and SS+ cells. The average number of pancreatic islets, and the total islet cellularity per mouse, was augmented in animals receiving serpinB13 mAb. Although additional studies are necessary to characterize the hormonal negative cells and assess whether Glu+ and SS+ cells transdifferentiate to beta cells, these findings demonstrate that enhanced tissue plasticity and beta-cell output can be achieved in the pancreas following treatment with antibodies to protease inhibitors. Disclosure Y.Kryvalap: None. S.Z.Meng: None. J.Czyzyk: None.

Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets.

sRodent islets are widely used to study the pathophysiology of beta cells and islet function, however, structural and functional differences exist between human and rodent islets, highlighting the need for human islet studies. Human islets are highly variable, deteriorate during culture, and are difficult to genetically modify, making mechanistic studies difficult to conduct and reproduce. To overcome these limitations, we tested whether pseudoislets, created by dissociation and reaggregation of islet cell suspensions, allow for assessment of dynamic islet function after genetic modulation. Characterization of pseudoislets cultured for 1 week revealed better preservation of first‐phase glucose‐stimulated insulin secretion (GSIS) compared with cultured‐intact islets and insulin secretion profiles similar to fresh islets when challenged by glibenclamide and KCl. qPCR indicated that pseudoislets are similar to the original islets for the expression of markers for cell types, beta cell function, and cellular stress with the exception of reduced proinflammatory cytokine genes (IL1B, CCL2, CXCL8). The expression of extracellular matrix markers (ASPN, COL1A1, COL4A1) was also altered in pseudoislets compared with intact islets. Compared with intact islets transduced by adenovirus, pseudoislets transduced by lentivirus showed uniform transduction and better first‐phase GSIS. Lastly, the lentiviral‐mediated delivery of short hairpin RNA targeting glucokinase (GCK) achieved significant reduction of GCK expression in pseudoislets as well as marked reduction of both first and second phase GSIS without affecting the insulin secretion in response to KCl. Thus, pseudoislets are a tool that enables efficient genetic modulation of human islet cells while preserving insulin secretion.

Patient and Procedural Factors Associated With Increased Islet Cell Yield in Total Pancreatectomy With Islet Autotransplantation.

Total pancreatectomy with islet autotransplantation (TPIAT) offers symptom relief to highly selected patients with recurrent acute and/or chronic pancreatitis. However, with variable clinical response, it is important to refine islet manipulation technique and patient selection criteria. This study explores the variables associated with high islet cell yield, a driver of success in TPIAT. This study evaluated patients who underwent TPIAT at Dartmouth-Hitchcock Medical Center from 2012 to 2016. Odds ratios were calculated for various patient and procedural characteristics. The primary clinical outcome was the number of isolated islet equivalents per kilogram body weight. Thirty-eight patients met inclusion criteria. Patients with no computed tomography or magnetic resonance imaging evidence of chronic pancreatitis, without pancreatic duct stones, and without parenchymal stones were associated with higher odds of success (P = 0.02, P = 0.02, and P = 0.002, respectively). Patients with preoperative glycated hemoglobin greater than 5.6, with islet cell suspensions positive for cultures, and with positive gram stains were associated with lower odds of success (P = 0.02, P = 0.01, and P = 0.02, respectively). Factors that diminish a successful islet cell harvest during TPIAT include the presence of infected islets, an elevated preoperative glycated hemoglobin, and the presence of pancreatic duct stones.

Patient and Procedural Factors Associated With Increased Islet Cell Yield in Total Pancreatectomy With Islet Autotransplantation

Introduction: Total pancreatectomy with islet autotransplantation (TP-IAT) provides pain relief to highly select patients with recurrent acute and/or chronic pancreatitis. However with variable outcomes and no standardized guideline for patient selection, it is important to refine islet manipulation procedures and patient selection characteristics to optimize outcomes. Success of the procedure depends on a high number of isolated islet equivalents. This study explores the patient and procedural characteristics associated with high islet cell yield. Methods: This study evaluated patients who underwent TP-IAT at Dartmouth Hitchcock Medical Center from 2012 to 2016. 38 patients met inclusion criteria. Odds ratios with 95% confidence intervals were found for various patient and procedural characteristics listed in Table 1. The primary clinical outcome was the number of isolated islet equivalents per kilogram body weight (IEQ/Kg), defined as IEQ/Kg >2,500.Table: Table. Factors Associated with Successful Islet Cell Yield During TPIATResults: All patient factors and procedural fields evaluated are listed in Table 1. Patients with no CT/MRI evidence of chronic pancreatitis showed statistically significant higher odds of success (OR=29, P=0.02). Patients without pancreatic duct stones or parenchymal stones were associated with higher odds of success (OR=23, P=0.02 and OR=55, P=0.002, respectively). Islet cell suspensions positive for cultures or positive gram stains were associated with lower chances of success (OR=0.06, P=0.02, and OR=0.48, P=0.01, respectively). Patients with preoperative HgbA1c greater than 5.6 were associated with lower odds of success (OR=0.13, P=0.02). Conclusion: This investigation found that patients without CT/MRI evidence of chronic pancreatitis, without bacterial infections, and without pancreatic duct or parenchymal stones are more likely to attain successful islet cell yields. Additionally, patients with preoperative HgbA1c less than 5.6 were associated with higher outcomes of success. These factors should be considered when selecting patients for TP-IAT.

Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus

The present study was designed to establish a novel tissue engineering approach for diabetes mellitus (DM) by fabricating a tissue sheet composed of pancreatic islet cells for in vivo transplantation. Pancreatic islet cell suspensions were obtained from Lewis rats, and plated onto temperature-responsive culture dishes coated with extracellular matrix (ECM) proteins. After the cells reached confluency, islet cells cultured on laminin-5 coated dishes were successfully harvested as a uniformly spread tissue sheet by lowering the culture temperature to 20°C for 20min. The functional activity of the islet cell sheets was confirmed by histological examination and Insulin secretion assay prior to in vivo transplantation. Histological examination revealed that the harvested islet cell sheet was comprised of insulin- (76%) and glucagon- (19%) positive cells, respectively. In vivo functionality of the islet cell sheet was maintained even 7 days after transplantation into the subcutaneous space of Lewis rats. The present study describes an approach to generate a functional sheet of pancreatic islet cells on laminin-5 coated temperature-responsive dishes, which can be subsequently transplanted in vivo. This study serves as the foundation for the creation of a novel cell-based therapy for DM to provide patients an alternative method.

Effect of serum from diabetes-prone BB/OK rats on neonatal rat pancreatic islets and islet cell suspensions.

Humoral-mediated cytolytic activity against neonatal rat pancreatic islet cells as well as islets of Langerhans was detected in sera of newly diagnosed diabetic BB/OK rats by measurement of enhanced 51Cr-release or insulin leakage. Beta-cell-specific functions of islet cell suspensions such as insulin secretion and (pro)insulin biosynthesis can be affected by BB rat serum in vitro.

The Frequency of Islet Cell Surface Antibodies and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) of Mononuclear Cells in HLA-Typed Patients with High Diabetes Risk*

To evaluate the significance of ICSA as a prognostic marker for the development of type I diabetes we investigated 66 subjects with first degree relatives of type I (47) and type II (9) diabetes as well as subjects with anamnestical data suggestive of diabetes. Patients were studied for glucose tolerance (oGTT) and IRI-response, ICSA (indirect fluorescence of living rat islet cell suspensions), ADCC (specific 51Cr-release of serum pretreated neonatal rat islets elicited by mononuclear cells) and HLA-antigens. 23 subjects revealed normal glucose tolerance, 17 impaired glucose tolerance and 26 had a prevoius abnormality. The incidence of ICSA varied between 46 and 53 per cent, that of positive ADCC between 22 and 56 per cent, both being highest in subjects with IGT. 87 per cent of all patients revealed diabetes associated HLA-antigens. We found no correlation of ICSA with glucose tolerance, IRI-response, ADCC and HLA-antigens. In conclusion it can be said that ICSA are present in a high percentage in patients with high diabetes risk but their predictive role as a marker for the manifestation must be elucidated in follow-up studies.

Vascularized composite islet-kidney transplantation in a miniature swine model

Previous work from this laboratory has demonstrated that transplantation of allogeneic thymic tissue as part of a composite vascularized graft is far more successful in terms of both engraftment and long-term survival than transplantation of thymic tissue or cells alone. We have subsequently extended this concept to transplantation of allogeneic islets, comparing survival of islet cell suspensions to that of vascularized composite islet-kidneys (IK), prepared by injection of autologous islets underneath the renal capsule 2-3 months prior to allogeneic transplantation of the composite organ. We have utilized partially inbred miniature swine with defined MHC loci as the experimental large animals for this study, permitting reproducible transplantation across specific MHC barriers. Composite IK have been transplanted successfully across minor and full MHC mismatch barriers, using treatment regimens previously demonstrated to induce long-term tolerance of kidney allografts across these barriers. IK allografts containing > or =5000 islet equivalents (IE)/kg recipient body weight were found capable of reversing surgically induced diabetes, while injection of comparable numbers of purified islets via the portal vein or under the renal capsule did not. Studies are also being directed toward preparation of autologous "thymo-isletkidneys" (TIK), for potential use as xenografts, in which the thymic component is intended to induce tolerance and the islets to reverse diabetic hyperglycemia. The use of both types of composite organ transplants may eventually be applicable to the treatment of type I diabetic patients suffering from end-stage diabetic nephropathy.

Transformed rat pancreatic islet-cell lines established by BK virus infection in vitro.

Single-cell suspensions of primary rat pancreatic islet cells were infected with BK virus (BKV) prototype (Gardner) and the naturally occurring BKV strain (TU), isolated from human urine. These viral strains have different sequences in their non-coding control regions which contain promoter-enhancer elements and origin of DNA replication. Uninfected cell cultures disintegrated within 3 weeks, while a varying number of virus-infected cultures were immortalized and a majority exhibited focal areas of 2-dimensional growth. A significantly higher proportion of the BKV (TU)- than of the BKV (Gardner)-infected cultures were immortalized. Large tumor (T) antigen expression was evident in virus-infected cultures from day 4 post infection (p.i.), while insulin secretion decreased steadily during the first weeks of culture. Two cell lines were established from BKV (TU)-infected cultures. Line 5A4 was contact-inhibited, growing as a dense monolayer, while 6A3 demonstrated foci of 2-dimensional growth. Both cell lines have retained their morphology and T-antigen expression for approximately 130 passages. At high passages a low level of intracellular insulin, but no secretion into media, was detected. Based on standard biological tests (reduced serum requirements, growth at low cell density, anchorage-independent growth and tumor induction in nude mice) both cell lines have a fully transformed phenotype, but 6A3 appears to be more malignantly transformed. Since both cell lines were established simultaneously from the same primary cells with the same virus batch, they provide an opportunity to study the transforming mechanisms of BKV in a relative context.

Use of liposomes to introduce substances into pancreatic islet cells.

The liposome technique is widely used to transport substances that cannot normally traverse the plasma membrane into the cell. The interactions of liposomes with the plasma membrane of pancreatic islet cells have not previously been studied. We evaluate the suitability of the liposome technique for introducing substances into the pancreatic beta-cell to which the cell membrane is impermeable. Liposomes were synthesized with an ether-injection method, and the cell-liposomal interactions were investigated by means of radioactive labeling and the fluorescent aqueous space marker 6-carboxyfluorescein. Experiments were performed on freshly isolated mouse pancreatic islets and on free islet cell preparations. With fluorescence microscopy, liposomes were observed to fuse spontaneously with islet cells, and the corresponding internalized volumes were quantified with spectrofluorometric measurements. The liposome association with islets and islet cell suspensions, as assessed by radioactive labeling, was found to increase with the liposome concentration. The effects of liposome membrane lipid composition on the fusion rate were found to be decreased in the presence of glucolipid. In addition, polyethylene glycol failed to affect the liposomal uptake. Freshly isolated islets incubated with liposomes containing glucose 6-phosphate were observed to release slightly more insulin than islets incubated with "empty" liposomes. In conclusion, liposomes fuse spontaneously with islet cells in vitro, and the uptake of liposomes is regulated by the lipid composition of the liposomal bilayer and the amount of liposomes present. The function of the beta-cell can be altered with the liposome technique, e.g., by addition of biologically active molecules such as glucose 6-phosphate.

Identification of monoclonal antibodies to pancreatic islet cells by immunoperoxidase staining

Immunoperoxidase staining and enzyme-linked immunosorbent assay (ELISA) were used to identify monoclonal antibodies that reacted with pancreatic islet cells. All monoclonal antibodies produced against isolated human or rat pancreatic islets including one mouse autoantibody reacted with pancreatic islets in formalin-fixed pancreas sections, but not with rat kidney or thyroid. Reactivity was also found with suspensions of normal rat islet cells and rat insulinoma cells using a 3-stage immunoperoxidase procedure and an ELISA technique. Differences were observed in staining intensity between the various antigenic substrates tested suggesting variable cross-reactivity and/or number of epitopes. The sensitivity of the immunoperoxidase technique proved to be favourable for identification of monoclonal antibodies that recognize cellular constituents such as islet cell antigens present in low concentrations.

Preparation of suspensions of pancreatic islet cells: a comparison of methods

A comparative study for preparation of cell suspensions from pancreatic islets has been performed using mechanical or enzymatic dissociation with proteolytic enzymes such as trypsin, dispase, and pronase. Treatment of isolated pancreatic islets from neonatal rats with these enzymes proved to be superior to a mechanical dissociation methods. The enzymatic dissociation was performed by fractioned treatment of pancreatic islets with low concentration of enzymes in Hansk' solution for 2–3 min at room temperature. With the exception of trypsin the percentage of single cells was consistently higher with dispase and pronase treatment, being 83–92%. Cell viability (dye exclusion) was more than 90%. Mechanical disintegration of pancreatic islets resulted in a low yield of single cells, and cell viability was considerably reduced in comparison with enzymatic methods. Labeling of islets cells with Na 2 51CrO 4 and measurement of the basal 15Cr-release demonstrated superior membrane preservation after pronase or dispase treatment. Islet cells isolated either by fractioned dispase or pronase treatment were found to be well preserved and very suitable for the detection of circulating cell surface antibodies and their cytotoxic effects to islet cells.

Characterization of pseudo-islets formed from pancreatic islet cell suspensions of neonatal rats

Well-preserved pancreatic islet cell suspensions were prepared from islets of Langerhans of neonatal rats by gentle trypsin treatment. Within a culture period of 4–6 days the islet cells reaggregate spontaneously and form pseudo-islets of different size and of a variable insulin content. While the ratio of insulin to glucagon in isolated islets of Langerhans is constant (18 ± 1.9), the hormone ratio of the pseudo-islets is strongly variable and increased, indicating an excess of insulin. Glucose enhancement from 1.5 mmoles/l to 15 mmoles/1 results in a significant stimulation of (pro)insulin biosynthesis whereas insulin secretion of the pseudo-islets is only slightly increased. At high glucose concentration (15 mmoles/1) insulin secretion of the pseudo-islets can be potentiated (by a factor of 4.5 ±0.46) by 3-isobutyl-1-methylxanthine (IBMX). Compared with the initial islet cell suspension, the cell aggregation during pseudo-islet formation did not result in an enhanced secretory response on glucose stimulation.

An immunofluorescence study of anti-pancreatic islet cell antibodies in the spontaneously diabetic BB Wistar rat.

Sera from BB Wistar rats and Wistar control rats were evaluated for the presence of islet cell antibodies in a prospective study using an indirect immunofluorescence assay on pancreatic islet cell suspensions from cultured rat islets. Islet cell surface antibodies were detected in sera from all animals of the spontaneously diabetic BB Wistar rat colony. The antibody could be detected well before the clinical onset of the disease and was present throughout the course of study of all diabetic animals. Sera from control Wistar rats were negative when tested for this antibody.

In vitro inhibition of pancreatic B cell function by lymphocytes from diabetics with associated autoimmune diseases: a T cell phenomenon.

Lymphocytes from insulin-dependent diabetic patients were previously shown to suppress insulin release from mouse islet cells in vitro. Glucagon release was not suppressed. In order to further analyze this phenomenon, lymphocytes from three insulin-dependent diabetic patients with associated autoimmune diseases were treated by using the panning method for cell separation before testing on islet cell suspensions. The OKT3+, OKT3-, and OKT4- cell subsets were obtained. Insulin release was suppressed by the OKT3+ (T lymphocyte-enriched) subset, but not by the OKT3- (T lymphocyte-depleted) subset. These results suggest that T lymphocytes are directly involved in the suppression of insulin release in this model. Furthermore, the OKT4- (T helper-depleted) subset also suppressed insulin release.

Flow sorting of mouse pancreatic B cells by forward and orthogonal light scattering

Mouse islet cell suspensions were separated into subpopulations based on forward low angle and orthogonal scattered light. Separation fractions were analyzed for hormone content by radioimmunoassays. The pancreatic B-cells had the highest orthogonal light scattering, while they overlapped in forward low angle light scattering with other endocrine (glucagon, pancreatic polypeptide and somatostatin containing cells) and nonendocrine cells. Living and paraformaldehyde fixed islet cell suspensions showed similar distributions of combined light scattering.

Specific pancreatic beta-cell surface antigens recognized by a xenogenic antiserum.

An antiserum (R4) from a rabbit immunized with suspensions of C57BL/61 ob/ob mouse islet cells contains antibodies which in a 125I-protein A radioligand assay can be demonstrated to bind to single cell suspensions of normal Naval Medical Research Institute (NMRI) mouse islet cells. The binding of 125I-protein A to islet cells was about four times that of normal rabbit serum (NRS) after incubation at a 1/600 dilution of R4 antiserum quantitatively absorbed to mouse spleen lymphocytes (R4A antiserum) and hepatocytes. Subsequent absorption of the R4A antiserum to islet cells significantly reduced the binding of 125I-protein A to islet cells incubated with the doubly absorbed serum. Immunoprecipitation of radiolabeled islet cell lysates followed by SDS polyacrylamide gel electrophoresis and autoradiography suggested that the R4A antiserum recognized a Mr 40,000 glycoprotein. This glycoprotein was not detected in spleen lymphocytes. Electron microscope detection of gold-protein A complexes suggested that the binding of islet cell surface antibodies was cell specific. islet cell suspensions incubated with R4A antiserum and gold-protein A showed that 86 +/- 3 gold particles were bound per 100 beta-cells (mean +/- SE for six experiments). In contrast, the number of gold particles per 100 endocrine non-beta-cells was 8 +/- 1 which was similar to the number achieved with NRS (3 +/- 1) on all endocrine islet cells. Our observations suggest that the pancreatic islet cells, in particular the beta-cells, express a specific antigen.

Sorting of pancreatic islet cell subpopulations by light scattering using a fluorescence-activated cell sorter.

Methods have been developed for the preparation of suspensions of viable rat pancreatic islet cells and their analysis and sorting in the fluorescence-activated cell sorter (FACS III or IV). Histograms of cell number versus light scattering in a near forward angle (1-15 degrees) demonstrated that viable islet cells produce a broad peak that is distinctly separated from the peaks generated by exocrine cells, erythrocytes, and nonviable cells. Electron microscopic examination and radioimmunoassay of hormone content in fractions collected across the peak showed that glucagon-containing (A) cells scatter less intensely and are concentrated within the left side of the islet cell peak, while somatostatin-containing (D) cells are localized to the far right side, indicating a higher intrinsic light scattering property of the D-cells. The more abundant insulin-containing (B) cells define the center of the islet cell peak. Sodium dodecyl sulfate slab gel electrophoresis and radioautography of 35S-methionine labeled cellular proteins confirmed that sorted cells are viable. Cells from the far left region contained increased amounts of labeled 18 Kd proglucagon and its 13-Kd and 10-Kd conversion intermediates, while cells from the right side were relatively enriched in labeled 12.4 Kd prosomatostatin. These results demonstrate that intrinsic light scattering alone can be used to prepare A- or D-cell enriched fractions from islets for biochemical analysis.

Microcarriers: a new approach to pancreatic islet cell culture.

Free islet cell suspensions were prepared from isolated fetal rat islets using a modified enzyme dispersion technique. The islet cells were dispensed into a culture flask containing microcarriers (Cytodex) suspended in culture medium RPMI 1640 by a slowly rotating bar magnet. Microscopical examination of the beads showed that the islet cells attached and then progressively proliferated on the surface of the beads as a monolayer. A highly sustained release of insulin from the beads to the medium was observed during the 7 d culture period. The functional viability of the cultured islet cells was further demonstrated by the ability of batches of the cell-coated beads to synthesize insulin and to increase the insulin release in response to an acute challenge (16.7 mmol/l glucose plus 5 mmol/l theophylline). The results suggest that bead microcarriers may provide a new approach to monolayer islet cell culture providing functional monolayers, which can easily be transferred to different test systems and further manipulated.

45Ca2+ uptake by dispersed pancreatic islet cells: Effects ofd-glucose and the calcium probe, chlorotetracycline

Uptake of 45Ca2+ was studied in dispersed pancreatic islet cells from non-inbred ob/ob-mice. Like whole islets the dispersed cells responded to 20 mM D-glucose with a markedly increased 45Ca2+-labeling of both the lanthanum-nondisplaceable and the lanthanum-displaceable calcium pools. The pronounced effect of D-glucose could not be reproduced with 3-O-methyl-D-glucose, L-glucose, D-mannose, L-leucine, or D-leucine; however, 45Ca2+ uptake was greater in the presence of L-leucine as compared with D-leucine. 45Ca2+ uptake by dispersed cells or whole islets was stimulated severalfold by 100 microM or more chlorotetracycline. At the concentration of only 10 microM, chlorotetracycline had no effect on whole islets and partially inhibited 45Ca2+ uptake by the dispersed cells. The ability of D-glucose to stimulate 45Ca2+ uptake by islets or dispersed cells remained in the presence of 10 microM chlorotetracycline. Islet cell suspensions apparently represent a valid model for studying how Ca2+ interacts with the cells. However, when using chlorotetracycline as fluorescent Ca2+ probe, attention must be paid to it potential ionophoric activity. At only 10 microM, the drug seems to monitor a peripheral pool of Ca2+, some of which may reside in normal transport channels.