Search

Background: Type 1 diabetes is an autoimmune disorder characterized by the loss of pancreatic islets. Islet allotransplantation is a potentially beneficial therapeutic approach for diabetes. Islets suffer a variety of cellular insults including ischemia and partial vascular loss during isolation, resulting in a significant reduction in viability prior to transplantation. The present study aimed to investigate the effect of angiogenic microRNA (miRNA)-126 and -210 on islet function and viability in an indirect way. Methods: Poly Ethylenimine (PEI)-miRNA-126 and -210 polyplexes were constructed at various Nitrogen/Phosphate (N/P) ratios. After confirmation by gel retardation and ethidium bromide dye exclusion assay, its cytotoxicity and transfection efficiency were analyzed by MTT and fluorescent assays, respectively. After that, the selected polyplexes were used to transfect Human Umbilical Vein Endothelial Cells (HUVECs) in vitro and were indirectly co-cultured with islet cells for three days. Real-time polymerase chain reaction and enzyme-linked immunoassay were conducted to verify the regulation of target genes and the functionality of the islets. Results: The findings showed that PEI could condense miRNAs at N/P=5. The viability of the HUVECs was decreased by increasing the amount of PEI. Additionally, ployplex-126 and -210 led to a decrease in the expressions of target genes, phosphoinositol-3 kinase regulatory subunit 2, sprouty-related EVH1 domain-containing protein 1, and ephrin-A3 in the islets. Moreover, the expressions of Bax and Bcl2 and their ratio in the treated groups by polyplex-126 and -210 led to better survival and function of the islets, with a higher expression of insulin and response to glucose stimulations. Furthermore, polyplex-210 could downregulate the anti-angiogenic protein, thrombospondin 1, compared to the other groups. Finally, the secretion of C-peptide was higher in polyplex-210 than in the other groups, adjusted for insulin secretion. Conclusion: The results indicated that angiogenic miRNAs could promote the survival and function of islet cells by interacting with their targets.

Prior to transplantation into individuals with type 1 diabetes, in vitro assays are used to evaluate the quality, function and survival of isolated human islets. In addition to the assessments of these parameters in islet, they can be evaluated by multiparametric morphological scoring (0–10 points) and grading (A, B, C, D, and F) based on islet characteristics (shape, border, integrity, single cells, and diameter). However, correlation between the multiparametric assessment and transplantation outcome has not been fully elucidated. In this study, 55 human islet isolations were scored using this multiparametric assessment. The results were correlated with outcomes after transplantation into immunodeficient diabetic mice. In addition, the multiparametric assessment was compared with oxygen consumption rate of isolated islets as a potential prediction factor for successful transplantations. All islet batches were assessed and found to score: 9 points ( n = 18, Grade A), 8 points ( n = 19, Grade B), and 7 points ( n = 18, Grade B). Islets that scored 9 (Grade A), scored 8 (Grade B) and scored 7 (Grade B) were transplanted into NOD/SCID mice and reversed diabetes in 81.2%, 59.4%, and 33.3% of animals, respectively ( P < 0.0001). Islet scoring and grading correlated well with glycemic control post-transplantation ( P < 0.0001) and reversal rate of diabetes ( P < 0.05). Notably, islet scoring and grading showed stronger correlation with transplantation outcome compared to oxygen consumption rate. Taken together, a multiparametric assessment of isolated human islets was highly predictive of transplantation outcome in diabetic mice.

Abstract Islets transplantation, as a treatment of type 1 diabetes, faces challenges, including the loss of islets in the process of isolation and pre-transplantation due to cellular stresses-induced apoptosis. Accordingly, the optimization of culture plays a decisive role in the transplantation success. In this study, we evaluated the effect of nobiletin on the cultured human islets. Isolated human islets were treated by different concentrations of nobiletin and cultured for 24 and 72 hours. Then, the islets viability, apoptosis, insulin and C-peptide secretion, and apoptosis markers were evaluated. Also, the production of reactive oxygen species (ROS), hypoxia inducible factor 1 alpha (HIF-1α), and its target genes in the islets were examined. Our findings showed that the islets were encountered with hypoxia and oxidative stress after isolation and during culture. These insults induced apoptosis and reduced viability during culture period. Moreover, the secretion of insulin and C-peptide decreased. Nobiletin treatments significantly improved the islets survival through reduction of HIF-1α and ROS production and suppression of apoptosis, along with increased islets function. Islet protective effect of nobiletin might be related to its anti-oxidant, anti-apoptotic and insulinotropic properties. Hence, in order to achieve viable and functional islets for clinical transplantation, the application of nobiletin during pre-transplantation period is useful.

Abstract We developed an optimized Dipheylthiocarbazone or Dithizone (DTZ) with improved physical and chemical properties to characterize human islets and insulin-producing cells differentiated from embryonic stem cells. Application of the newly formulated iDTZ (i stands for islet) over a range of temperatures, time intervals and cell and tissue types found it to be robust for identifying these cells. Through high transition zinc binding, the iDTZ compound concentrated in insulin-producing cells and proved effective at delineating zinc levels in vitro.

The aim of this study was to investigate the effects of elevated donor HbA1c levels (type 2 diabetes, T2D) on the islet yield and functionality postisolation. In this retrospective analysis, donors for islet isolations were classified into two groups: T2D group (HbA1c ≥ 6.5%, n = 18) and normal group (HbA1c < 6.5%, n = 308). Optimum pancreas digestion time (switch time) was significantly higher in the T2D group compared to the normal group (13.7 ± 1.2 vs. 11.7 ± 0.1 min, respectively, p = 0.005). Islet yields were significantly lower in the T2D group compared to the control (T2D vs. control): islet equivalent (IEQ)/g (prepurification 2,318 ± 195 vs. 3,713 ± 114, p = 0.003; postpurification 1,735 ± 175 vs. 2,663 ± 89, p = 0.013) and islet particle number (IPN)/g (prepurification, 2,519 ± 336 vs. 4,433 ± 143, p = 0.001; postpurification, 1,760 ± 229 vs. 2,715 ± 85, p = 0.007). Islets from T2D pancreata had significantly lower viability (T2D vs. control: 91.9 ± 1.6 vs. 94.4 ± 0.3%, p = 0.004) and decreased oxygen consumption rate (DOCR) (T2D vs. control: 0.09 ± 0.01 and 0.21 ± 0.03 nmol O 2 100 islets −1 min −1 , p = 0.049). The islets isolated from T2D donor pancreata reversed diabetes in NOD-SCID mice in 9% (2/22) compared to islets from control donor pancreata, which reversed diabetes in 67% (175/260, p < 0.001). In conclusion, this study demonstrates that elevated HbA1c (≥6.5%) is associated with impairment of islet function and lower islet yield; however, these islets could not be suitable for clinical applications.

The choice of enzyme blend is critical for successful islet isolation. Islet yield, viability, integrity, and function are important factors that influence the outcome of islet transplantation. Liberase HI has been used as a standard enzyme for pancreas digestion and has successfully produced islets that reversed diabetes. However, the replacement of Liberase HI with collagenase NB1 has significantly influenced the process outcome, both in quality and quantity of the isolated islets. The assessment of islet cells by Flow Cytometry (FC) has been reported to be useful for evaluating islet quality. The aim of this study was to assess the isolation outcomes and islet quality when comparing human islet cell processed with Liberase HI and NB1. A total of 66 islet isolations, 46 processed using Liberase HI and 20 using Serva NB1, were retrospectively analyzed. Islet yield, function in vitro, islet cell viability by FC, as well as isolation-related factors were compared. There was no significant difference in donor characteristics such as age and height; however, body mass index (BMI) in the Liberase HI group was significantly higher. There was also no significant difference in prepurification, postisolation, or postculture IEQ or percent recovery between the two groups. Flow data showed Liberase HI preparations had a significantly higher percent of live cells (DAPI - ) and NG + / TMRE + when compared to NB1. Stimulation Indices (SI) for Liberase HI ( n = 45) showed 3.17 and NB1 ( n = 18) 2.71 ( p = NS). The results of Annexin V/DAPI staining for live, apoptotic, and necrotic cells were 50.7 ± 2.24%, 14.4 ± 1.02%, and 27.8 ± 1.92% for Liberase HI versus 48.1 ± 1.93%, 12.3 ± 0.92%, and 33.9 ± 2.28% for NB1. Islets isolated using Liberase HI showed higher viable β cells by NG/TMRE staining and decreased necrosis by Annexin V/DAPI staining. FC assessment may be useful for determining the choice of digestion enzyme to maximize viable islets.

Transplantation of unpurified islets into the liver, unlike that of purified islets, causes portal hypertension and coagulopathy. The aim of this project was to determine the most suitable alternative site for transplantation of unpurified pancreatic islets in autotransplanted dogs. Twenty-five female mongrel dogs were divided into 5 groups depending on the site of islet transplantation: liver (3), spleen (7), skeletal muscle (5), omental pouch (6), and renal subcapsule (4). Pancreatic digestion of the total pancreatectomized specimen was carried out by distension of the pancreas with 1.5 mg/mL collagenase suspended in 250 mL Hanks' balanced salt solution using a semiautomatic method. The total number of islets equivalent isolated from 25 dogs was 90948 ± 6053. Only islets > 60 μm in diameter were counted, and the mean islet equivalent transplanted per kg body wt was 6762 ± 429. Islet function was achieved with transplantation into spleen in 71%, omental pouch in 50%, and muscle in 20%, but none in the renal subcapsule or liver groups. Glucose tolerance test at 30 d showed a mean K Value (decline in glucose, %/min) of 1.94 ± 0.73,0.79 ± 0.15 and 1.02 in the splenic, omental pouch and muscle groups, respectively. All animals in the liver group, 2 from the splenic group, and 2 from the renal subcapsule group died of diffuse bleeding. Four out of 5 dogs in the muscle group developed necrosis at the site of transplantation and the islets never functioned. This study demonstrates that in dogs, spleen and omental pouch appear to be suitable sites for transplantation of unpurified islets.