Effect of mesenchymal stem cells on the vascularization of the artificial site for islet transplantation in rats.

An adequate vascularization of the artificially created cavity is crucial for subsequent transplantation of isolated pancreatic islets. In a reported study, dynamic contrast-enhanced magnetic resonance imaging was used to assess the effect of mesenchymal stem cells on neoangiogenesis within connective tissue surrounding an implantable biocompatible device. The signal increase detected after injection of magnetic resonance contrast agent in each target region was considered to be an effect of contrast agent, which was related to the blood supply. To minimize the influence of variability in contrast agent application, all outcomes measured in the implanted devices were normalized to the signal intensity of kidney tissue. When supported by mesenchymal stem cells, the mean signal increase intra-abdominally was 42%, 41%, and 64% and within subcutaneously implanted devices was 23%, 54%, and 52% of that measured in kidney.

The effects of DPP-IV inhibition in NOD mice with overt diabetes.

Sitagliptin is a dipeptidyl peptidase IV (DPP-IV) inhibitor that exerts an anti-hyperglycaemic effect by preventing degradation of glucagon-like peptide 1 with subsequent ß-cell stimulation and potential regeneration. We tested whether sitagliptin therapy in symptomatic non-obese diabetic (NOD) mice would lead to changes in the immune cell profile, improve ß-cell survival and induce diabetes remission. Flow cytometry analysis of immune cells in the spleen and peripheral lymph nodes, immunohistology of the pancreas and DPP-IV activity were investigated in diabetic NOD mice, either treated or non-treated with sitagliptin, at 0, 7, 14 and 28 days after hyperglycaemia onset, and in non-diabetic NOD controls. While compared to diabetic controls sitagliptin prevented increase of the CD8+/CD4+ ratio in pancreatic nodes after four weeks (0.443 ± 0.067 vs. 0.544 ± 0.131; P < 0.05), the population of Tregs in lymph nodes increased from day 0 to 28 in both treated and non-treated diabetic groups (8 ± 1.76 vs. 13.45 ± 5.07 % and 8 ± 1.76 vs. 13.19 ± 5.58 %, respectively). The severity of islet infiltration was similar in both diabetic groups and decreased in parallel with ß-cell loss. Surprisingly, sitagliptin blocked the DPP-IV activity only temporarily (on day 7, 277.68 ± 89.2 vs. 547.40 ± 94.04 ng/ml in the diabetic control group) with no apparent effect later on. In conclusion, sitagliptin administered after the onset of overt hyperglycaemia in NOD mice had only a marginal immunological effect and did not lead to diabetes remission. Failure to block DPP-IV over time represents an important finding that requires further explanation.

Gene expression changes in rat pancreas transplant model after long-term cold storage of the graft in perfluorohexyloctane.

BACKGROUND: Perfluorohexyloctane (PFH) is a promising storage solution that has been successfully used for pancreas preservation before islet isolation. This hyperoxygen carrier has been designed to prevent ischemic injury to the pancreas graft during cold storage. In our storage, we aimed to evaluate the impact of this solution on long-term cold storage in a rat whole pancreas transplantation model. METHOD: Brown-Norway rats were used for syngeneic heterotopic pancreas transplantation. The procured organs were cold-stored for 18 hours in preoxygenated PFH (PFH group; n = 8) or in the University of Wisconsin solution (UW group; n = 8), or were transplanted immediately in the control group (n = 8). Two hours after reperfusion, we obtained blood and pancreas tissue samples for biochemistry and gene analyses (real-time polymerase chain reaction). RESULTS: A significant difference between the UW and PFH group was observed in the tumor necrosis factor (TNF)ß and endothelin 1 genes, which was overexpressed more than twofold in the UW group. In the blood samples, the UW group compared with the PFH group showed significantly higher levels of pancreatic amylase and lipase (94.2 ± 25.2 vs 67.7 ± 13.4 µkat/L and 5.5 ± 2.8 vs 3 ± 0.7 µkat/L, respectively; P < .05). CONCLUSION: We found significantly lower expression levels of the endothelin 1 and TNFß genes and lower concentrations of pancreatic amylase and lipase in the PFH group. All these findings suggest lower rate of ischemic reperfusion injury in the PFH group. These findings may result in better post-transplant outcomes after long-term cold storage in PFH compared with the UW solution. Further research in this area is required.

Activation of the Jak/Stat signalling pathway by leukaemia inhibitory factor stimulates trans-differentiation of human non-endocrine pancreatic cells into insulin-producing cells.

Differentiation of pancreatic ß-cells is regulated by a wide range of signalling pathways. The aim of our current work was to evaluate the effect of the Jak/Stat signalling pathway on the differentiation of human non-endocrine pancreatic cells into insulin-producing cells. Activation of the Jak/Stat signalling pathway by leukaemia inhibitory factor (LIF) stimulated differentiation of C-peptide-negative human non-endocrine pancreatic cells into insulin-producing cells in 6.3 ± 2.0 % cells (N = 5) and induced expression of pro-endocrine transcription factor neurogenin 3, Notch signalling pathway suppressor HES6 and stimulator of ß-cell neogenesis REG3A. The expression of the REG3A gene and increased rate of differentiation into insulin-producing cells (10.2 ± 2.1 %) were further stimulated by a combination of LIF with nicotinamide and dexamethasone. Glucose-stimulated (5 vs. 20 mM) C-peptide secretion confirmed proper insulin secretory function of trans-differentiated insulin-producing cells (0.51 vs. 2.03 pmol C-peptide/µg DNA, P < 0.05). Our results indicate that Jak/Stat signalling critically contributes to trans-differentiation of non-endocrine pancreatic cells into functional insulin-producing cells. The positive effect of the Jak/Stat signalling pathway on trans-differentiation is mediated by the key genes that activate differentiation of pancreatic ß-cells.

The effect of epigenetic factors on differentiation of pancreatic progenitor cells into insulin-producing cells.

Differentiation of pancreatic progenitors into insulin-producing ß cells is regulated by various transcription factors. To be expressed the genes coding these transcription factors need to be in accessible DNA. Whether a particular gene is present in a form of active euchromatin structure with accessible DNA or in an inactive heterochromatin structure with inaccessible DNA is determined by various epigenetic modifications. We studied the effect of epigenetic modifiers on differentiation of human nonendocrine cells into insulin-producing cells with the aim to evaluate the effect of epigenetic modifications in that process. Within 3 days of cultivation nonendocrine cells form isletlike cell clusters (ILCCs) containing mainly cytokeratin-19-positive cells. After cultivation with epigenetic modifiers and further differentiation, the highest number of C-peptide-positive cells (10.3% ± 2.9%) as well as glucagon-positive cells (7.2% ± 2.8%) was observed in a sample supplemented with a combination of 5-Aza-2'-deoxycytidine modifiers, BIX01294 and MC1568. In response to glucose stimulation (5 vs 20 mmol/L) these ILCCs secreted increased amounts of C-peptide (0.45 vs 1.05 pmol C-peptide/µg DNA). Control samples treated without any epigenetic modifiers showed significantly lower numbers of C-peptide-positive cells (3.5% ± 1.6%). These results showed that a combination of epigenetic modifiers 5-Aza-2'-deoxycytidine (BIX01294 and MC1568) significantly improved reproducible differentiation of nonendocrine pancreatic cells into insulin-producing cells.

Assessment of mitochondrial DNA as an indicator of islet quality: an example in Goto Kakizaki rats.

BACKGROUND: Diabetic Goto Kakizaki (GK) rats represent an established model of type 2 diabetes that exhibit an onset of pancreatic islet (PI) pathology characterized by islet hypertrophy with a decreased number of insulin-secreting ß-cells. Among the remaining ß-cells, oxidative phosphorylation (OXPHOS) and consequently glucose-stimulated insulin secretion (GSIS) are impaired, perhaps owing to a deficit in mitochondrial DNA (mtDNA). We sought to identify this abnormality. METHODS: ß-Cells were obtained from Accutase-dissolved PI isolated from GK or Wistar rats and sorted based on the positive Zn(2+) signal of Newport Green. The mtDNA copy number per cell was quantified as the amplicon ratio by polymerase chain reaction using specific primers against the rat ND5 mt gene and UCP2 nuclear gene. RESULTS: The 12-month-old GK rats exhibited drastically reduced copy numbers per remaining ß-cell, from 7,400 ± 600 in 12-month old Wistar rats (100%) to 24 ± 4%; mtDNA content in heart and liver was 70 ± 25% and 60 ± 20%, respectively. Versus age-paired Wistar rats, 6- and 4-month-old GK rats showed reductions to 60 ± 15% and 50 ± 20%, respectively. CONCLUSIONS: OXPHOS of remnant ß-cells in diabetic GK was drastically impaired due to the lack of sufficient mtDNA levels. We suggest the use of mtDNA quantification to quickly assess PI quality before transplantation.

Immunoregulatory effect of anti-thymocyte globulin monotherapy on peripheral lymphoid tissues of non-obese diabetic mice.

OBJECTIVE: Experimental and clinical studies have shown that autoimmunity-causing diabetes may be abrogated by immune intervention. Several anti-T-lymphocyte antibodies focus on distinct T-cell targets. We tested the effect of murine anti-thymocyte globulin (ATG; Genzyme, Framingham, MA) in peripheral lymphoid organs of non-obese diabetic (NOD) mice after the onset of hyperglycemia. METHODS: Diabetic NOD mice were treated with two doses of ATG (1 mg totally) or maintained without treatment as controls. Blood glucose levels were monitored twice a week. The mice were terminated at day 0, 7, 14, or 28 after the initiation of the study. Subpopulations of T-lymphocytes and FoxP3+ (forkhead box P3 positive) regulatory T-cells were analyzed among elements isolated from the spleen and pancreatic lymph nodes. RESULTS: Mice with blood glucose levels greater than 13 mmol/L were included in the study. Diabetes remission occurred in 16% (3/19) of mice treated with ATG. Only one case of remission was observed in the control group (6%; 1/16). ATG therapy a significantly decreased the CD8+/CD4+ T-lymphocyte ratio. Among splenocytes, a significant difference was detected only on day 7 (0.069 versus 0.198 T-lymphocyte ratio); in lymph nodes, a decrease was observed on day 28 (0.21 versus 0.51 T-lymphocytes ratio). The regulatory T-cells population increased after ATG administration compared with the control group at day 7 (16.2% versus 10.8% in CD4+ splenocytes; 20.7% versus 10.3% in CD4+ lymph node cells). However, the increased FoxP3+ cell population was not durable. CONCLUSIONS: ATG treatment of diabetic NOD mice showed an immunoregulatory effect in peripheral lymphoid tissue with a significantly deceased CD8+/CD4+ ratio, which, however, did not normalize the metabolic parameters in a short period after the onset of overt diabetes.

An acidic pH and activation of phosphoinositide 3-kinase stimulate differentiation of pancreatic progenitors into insulin-producing cells.

Adult pancreatic nonendocrine cells represent a potential alternative source of insulin-producing tissue for the treatment of diabetes. Differentiation of these cells is regulated by various signaling pathways including the phosphoinositide 3-kinase (PI3K) pathway. Therefore, we evaluated the effect of PI3K on this process. Compared with untreated cells the differentiation of human nonendocrine pancreatic cells into insulin-producing elements was increased after treatment with IGF-1, EGF, and Exendin-4, growth factors known to be activators of the PI3K pathway (12.2 +/- 3.2% vs 9.1 +/- 3.2%). Treatment with PI3K pathway inhibitor wortmannin reduced the number of differentiated beta cells from 9.1 +/- 3.2 to 0.7 +/- 0.4%. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and Exendin-4 significantly increased the expression of the transcription factor neurogenin-3, whereas the expressions of pancreatic and duodenal homeobox 1 (PDX-1), neurogenic differentiation 1 (NeuroD) were increased only among samples treated with ZnCl2 and not significantly affected by treatment with the tested growth factors. Successful differentiation of IGF-1, EGF-, and Exendin-4-treated cells into functional beta cells was confirmed by C-peptide secretion in response to 5 versus 20 mmol glucose stimulation (0.24 vs 0.91 pmol C-peptide/microg DNA). These results showed that activation of the PI3K signaling pathway might be used to stimulate the differentiation of nonendocrine pancreatic cells into insulin-producing elements.

Perfluorocarbon improves post-transplant survival and early kidney function following prolonged cold ischemia.

BACKGROUND: The two-layer organ preservation method (TLM) based on oxygenated perfluorocarbon overlaid with University of Wisconsin (UW) solution has been successfully used in clinical islet and experimental heart and intestine transplantation. We tested whether this technique would prevent tissue damage and improve kidney function in a model of syngeneic kidney transplantation with prolonged ischemia time. METHODS: Kidneys were stored for 24 h either in UW solution (n = 16), with TLM (n = 16) or transplanted immediately (control group, n = 12). In half of the animals, survival was observed and in the other animals grafts were procured for semiquantitative histological scoring and TUNEL apoptosis assessment 24 h after transplantation. RESULTS: One-month survival rates in the UW, TLM and control groups were 12.5, 62.5 and 100%, respectively (UW vs. TLM, p < 0.01). Median creatinine levels 24 h after transplantation were 381, 299 and 121 microM, respectively (UW vs. TLM, p < 0.02). Histological scoring showed more severe tissue damage in the UW group than in the TLM group (p < 0.05). Apoptosis was more frequent in the UW group than in the TLM group (p < 0.05). CONCLUSION: We demonstrated for the first time that conservation with TLM significantly improves the outcome of kidney transplantation in a rat model and should therefore be further studied in larger animals.

In vivo differentiation of human umbilical cord blood-derived cells into insulin-producing beta cells.

In our study we confirmed the potential of human umbilical cord blood cells to differentiate into insulin-producing cells following transplantation into immunocompromised mice. The average number of C-peptide-positive human cells per animal was 18 +/- 13 as assessed by immunofluorescence staining and fluorescence in situ hybridization specific for human ALU sequence. Differentiation into insulin-producing cells was further confirmed by reverse transcription-polymerase chain reaction specific for human insulin mRNA. Successful differentiation required sublethal irradiation of xenogeneic recipient at least at a dose of 3 Gy. However, transplantation of human umbilical cord blood cells did not improve hyperglycaemia in diabetic animals. The results of our study show that human umbilical cord blood may be considered as a potential source of stem cells for treatment of diabetes mellitus.

Differentiation of CD133-positive pancreatic cells into insulin-producing islet-like cell clusters.

Adult pancreatic stem and progenitor cells could represent an alternative source of insulin-producing tissue for diabetes treatment. In order to identify these cells, we have focused on the human pancreatic cells expressing cell surface molecule CD133, a marker of adult stem cells. We found that population of human CD133-positive pancreatic cells contains endocrine progenitors expressing neurogenin-3 and cells expressing human telomerase, ABCG2, Oct-3/4, Nanog, and Rex-1, markers of pluripotent stem cells. These cells were able to differentiate into insulin-producing cells in vitro and secreted C-peptide in a glucose-dependent manner. Based on our results, we suppose that the CD133 molecule represents another cell surface marker suitable for identification and isolation of pancreatic endocrine progenitors.

Isolation and characterization of human CXCR4-positive pancreatic cells.

The existence of an adult PSC that may be used in the treatment of diabetes is still a matter of scientific debate as conclusive evidence of such a stem cell in the adult pancreas has not yet been presented. The main reason why putative PSC has not yet been identified is the lack of specific markers that may be used to isolate and purify them. In order to increase the list of potential PSC markers we have focused on the human pancreatic cells that express cell surface receptor CXCR4, a marker of stem cells derived from different adult tissues. Here we report that CXCR4-positive pancreatic cells express markers of pancreatic endocrine progenitors (neurogenin-3, nestin) and markers of pluripotent stem cells (Oct-4, Nanog, ABCG2, CD133, CD117). Upon in vitro differentiation, these cells form ILCC and produce key islet hormones including insulin. Based on our results, we assume that CXCR4 marks pancreatic endocrine progenitors and in combination with other cell surface markers may be used in the attempt to identify and isolate PSC.

In vitro assessment of pancreatic islet vitality by oxymetry.

In order to assess the quality of freshly isolated and cultivated pancreatic islets designed for experimental transplantation in rats we combined the vitality staining test, in vitro measurement of insulin secretion capacity, and assessment of islet respiration. Oxygen consumption was measured using the respirometer Oxygraph 2K equipped with polarographic oxygen sensors. The results of oxymetry demonstrated a linear correlation between islet number and oxygen consumption. Respiration per unit of viable islet tissue was constant. Oxygen consumption tests were in good correlation with the results of insulin release assays, with a correlation coefficient of 0.82. We found no significant differences in all three vitality-testing methods performed with fresh and 24-hour cultivated islets (P > .05). We conclude that polarographic oxymetry provides a fast and easy evaluation test of islet quality. After appropriate standardization, the oxymetric technique can be used for routine clinical pretransplant islet quality testing. In addition, cell membrane integrity and mitochondrial function could be assessed after addition of specific respiration inhibitors or stimulators.

Magnetic resonance imaging of intrahepatically transplanted islets using paramagnetic beads.

Superparamagnetic agents can be reliably used for magnetic resonance imaging (MRI) of pancreatic islets located in the liver sinusoids. However, the main disadvantages seemed to be the rather long culture time necessary for islet labeling and the low specificity of these agents. In the present study we investigated a more specific approach with a shorter labeling time using immunomagnetic particles. Isolated rat islets were cultivated with immunomagnetic beads coated with antibody against rat MHC class I antigen. Labeled islets were transplanted into the livers of syngeneic rats. The animals were examined weekly by MRI or livers explanted 10 minutes after islet transplantation for in vitro experiments. In both in vitro and in vivo studies, labeled transplanted islets were imaged as hypointensive spots, diffusely distributed throughout the liver. This experiment represents an alternative way of islet imaging by magnetic resonance, which is as effective as the use of known superparamagnetic contrast agents and more specific owing to targeting to specific donor antigens.

Vitality of pancreatic islets labeled for magnetic resonance imaging with iron particles.

We previously described an in vivo method for pancreatic islet visualization using magnetic resonance imaging with the aid of superparamagnetic nanoparticles of iron oxide (Resovist) or by magnetic beads precoated with antibodies (Dynabeads). The aim of this study was to investigate the in vitro effect of islet labeling on their quality. Isolated rat islets were cultivated for 48 hours with a contrast agent or, in the case of magnetic antibody-coated beads, for only 2 hours. The ability to secrete insulin was tested by a static insulin release assay and the results were expressed as a stimulation index. Staining with propidium iodide and acridine orange was performed to determine the ratio of live to dead cells. Stimulation indices in the Resovist islets (n = 23) vs controls (n = 14) were 15.3 and 15.0, respectively, and in the Dynabeads islets (n = 15) vs controls (n = 12) 21.3 and 19.9, respectively. The vitality of the Resovist islets vs controls determined by live/dead cells ratio was 90.8% and 91.1%, respectively (n = 20), and in the Dynabeads islets vs controls was 89.4% and 91.8%, respectively (n = 11). Islet labeling with the contrast agent as well as with specific antibodies with iron beads did not change the vitality and insulin-secreting capacity assessed in vitro (P > .05). Magnetic resonance using iron nanoparticles represents the only method for in-vivo visualization of transplanted islets so far. Our data represent an important contribution for its clinical use.