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Evaluating the quality of isolated human islets before transplantation is crucial for predicting the success in treating Type 1 diabetes. The current gold standard involves time-intensive in vivo transplantation into diabetic immunodeficient mice. Given the susceptibility of isolated islets to hypoxia, we hypothesized that hypoxia present in islets before transplantation could indicate compromised islet quality, potentially leading to unfavorable outcomes. To test this hypothesis, we analyzed expression of 39 hypoxia-related genes in human islets from 85 deceased donors. We correlated gene expression profiles with transplantation outcomes in 327 diabetic mice, each receiving 1200 islet equivalents grafted into the kidney capsule. Transplantation outcome was post-transplant glycemic control based on area under the curve of blood glucose over 4 weeks. In linear regression analysis, DDIT4 (R = 0.4971, P < 0.0001), SLC2A8 (R = 0.3531, P = 0.0009) and HK1 (R = 0.3444, P = 0.0012) had the highest correlation with transplantation outcome. A multiple regression model of 11 genes increased the correlation (R = 0.6117, P < 0.0001). We conclude that assessing pre-transplant hypoxia in human islets via gene expression analysis is a rapid, viable alternative to conventional in vivo assessments. This approach also underscores the importance of mitigating pre-transplant hypoxia in isolated islets to improve the success rate of islet transplantation., (© 2024. The Author(s).)

miRNAs are critical for pancreas development and function. However, we found that there are discrepancies regarding pancreatic miRNA abundance in published datasets. To obtain a more relevant profile that is closer to the true profile, we profiled small RNAs from human islets cells, acini, and four rodent pancreatic cell lines routinely used in diabetes and pancreatic research using a bias reduction protocol for small RNA sequencing. In contrast to the previous notion that miR-375-3p is the most abundant pancreatic miRNA, we found that miR-148a-3p and miR-7-5p were also abundant in islets. In silico studies using predicted and validated targets of these three miRNAs revealed that they may work cooperatively in endocrine and exocrine cells. Our results also suggest, compared to the most-studied miR-375, that both miR-148a-3p and miR-7-5p may play more critical roles in the human pancreas. Moreover, according to in silico-predicted targets, we found that miR-375-3p had a much broader target spectrum by targeting the coding sequence and the 5' untranslated region, rather than the conventional 3' untranslated region, suggesting additional unexplored roles of miR-375-3p beyond the pancreas. Our study provides a valuable new resource for studying miRNAs in pancreata.

Progenitor cells capable of self-renewal and differentiation in the adult human pancreas are an under-explored resource for regenerative medicine. Using micro-manipulation and three-dimensional colony assays we identify cells within the adult human exocrine pancreas that resemble progenitor cells. Exocrine tissues were dissociated into single cells and plated into a colony assay containing methylcellulose and 5% Matrigel. A subpopulation of ductal cells formed colonies containing differentiated ductal, acinar, and endocrine lineage cells, and expanded up to 300-fold with a ROCK inhibitor. When transplanted into diabetic mice, colonies pre-treated with a NOTCH inhibitor gave rise to insulin-expressing cells. Both colonies and primary human ducts contained cells that simultaneously express progenitor transcription factors SOX9, NKX6.1, and PDX1. In addition, in silico analysis identified progenitor-like cells within ductal clusters in a single-cell RNA sequencing dataset. Therefore, progenitor-like cells capable of self-renewal and tri-lineage differentiation either pre-exist in the adult human exocrine pancreas, or readily adapt in culture., Competing Interests: Conflict of interests H.T.K. maintains a patent no. 9,783,784 titled “Methods for establishing and improving the survival of a population of pancreatic progenitor or stem cells.” The other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hypothermic (cold) preservation is a limiting factor for successful cell and tissue transplantation where cell swelling (edema) usually develops, impairing cell function. University of Wisconsin (UW) solution, a standard cold preservation solution, contains effective components to suppress hypothermia-induced cell swelling. Antifreeze proteins (AFPs) found in many cold-adapted organisms can prevent cold injury of the organisms. Here, the effects of a beetle AFP from Dendroides canadensis (DAFP-1) on pancreatic β-cells preservation were first investigated. As low as 500 µg/mL, DAFP-1 significantly minimized INS-1 cell swelling and subsequent cell death during 4 °C preservation in UW solution for up to three days. However, such significant cytoprotection was not observed by an AFP from Tenebrio molitor (TmAFP), a structural homologue to DAFP-1 but lacking arginine, at the same levels. The cytoprotective effect of DAFP-1 was further validated with the primary β-cells in the isolated rat pancreatic islets in UW solution. The submilligram level supplement of DAFP-1 to UW solution significantly increased the islet mass recovery after three days of cold preservation followed by rewarming. The protective effects of DAFP-1 in UW solution were discussed at a molecular level. The results indicate the potential of DAFP-1 to enhance cell survival during extended cold preservation.

We investigated the effect of chronic marijuana use, defined as 4 times weekly for more than 3 years, on human pancreatic islets. Pancreata from deceased donors who chronically used marijuana were compared to those from age, sex and ethnicity matched non-users. The islets from marijuana-users displayed reduced insulin secretion as compared to islets from non-users upon stimulation with high glucose (AUC, 3.41 ± 0.62 versus 5.14 ±0.47, p<0.05) and high glucose plus KCl (AUC, 4.48 ± 0.41 versus 7.69 ± 0.58, p<0.001). When human islets from chronic marijuana-users were transplanted into diabetic mice, the mean reversal rate of diabetes was 35% versus 77% in animals receiving islets from non-users (p<0.01). Immunofluorescent staining for cannabinoid receptor type 1 (CB1R) was shown to be colocalized with insulin and enhanced significantly in beta cells from marijuana-users vs. non-users (CB1R intensity/islet area, 14.95 ± 2.71 vs. 3.23 ± 0.87, p<0.001). In contrast, CB1R expression was not co-localized with glucagon or somatostatin. Furthermore, isolated islets from chronic marijuana-users appeared hypertrophic. In conclusion, excessive marijuana use affects islet endocrine phenotype and function in vitro and in vivo. Given the increasing use of marijuana, our results underline the importance of including lifestyle when evaluating human islets for transplantation or research., Competing Interests: The authors have declared that no competing interests exist.

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.

Ischemia reperfusion injury (IRI) is a condition that occurs wherever blood flow and oxygen is reduced or absent, such as trauma, vascular disease, stroke, and solid organ transplantation. This condition can lead to tissue damage, especially during organ transplantation. Under such circumstances, some signaling pathways are activated, leading to up- or down- regulation of several genes such as microRNAs (miRNAs) that might attenuate or ameliorate this status. Therefore, by manipulating miRNAs level, they can be used as a biomarker for early diagnosis of IRI or suggestive to be therapeutic agents in clinical situation in future., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Islet cell death and loss of function after isolation and before transplantation is considered a key barrier to successful islet transplantation outcomes. Mesenchymal stem cells (MSCs) have been used to protect isolated islets owing to their paracrine potential partially through the secretion of vascular endothelial growth factor (VEGF). The paracrine functions of MSCs are also mediated, at least in part, by the release of extracellular vesicles including exosomes. In the present study, we examined (i) the effect of exosomes from human MSCs on the survival and function of isolated mouse islets and (ii) whether exosomes contain VEGF and the potential impact of exosomal VEGF on the survival of mouse islets. Isolated mouse islets were cultured for three days with MSC-derived exosomes (MSC-Exo), MSCs, or MSC-conditioned media without exosomes (MSC-CM-without-Exo). We investigated the effects of the exosomes, MSCs, and conditioned media on islet viability, apoptosis and function. Besides the expression of apoptotic and pro-survival genes, the production of human and mouse VEGF proteins was evaluated. The MSCs and MSC-Exo, but not the MSC-CM-without-Exo, significantly decreased the percentage of apoptotic cells and increased islet viability following the downregulation of pro-apoptotic genes and the upregulation of pro-survival factors, as well as the promotion of insulin secretion. Human VEGF was observed in the isolated exosomes, and the gene expression and protein production of mouse VEGF significantly increased in islets cultured with MSC-Exo. MSC-derived exosomes are as efficient as parent MSCs for mitigating cell death and improving islet survival and function. This cytoprotective effect was probably mediated by VEGF transfer, suggesting a pivotal strategy for ameliorating islet transplantation outcomes., (Copyright © 2020 Keshtkar et al.)

The first step in differentiation of pluripotent stem cell toward endoderm-derived cell/organ is differentiation to definitive endoderm (DE) which is the central issue in developmental biology. Based on several evidences, we hypothesized that activin-A optimization as well as replacement of fetal bovine serum (FBS) with knockout serum replacement (KSR) is important for differentiation of induced pluripotent stem cell (iPSC) line into DE. Therefore, a stepwise differentiation protocol was applied on R1-hiPSC1 cell line. At first, activin-A concentration (30, 50, 70 and 100 ng/ml) was optimized. Then, substitution of FBS with KSR was evaluated across four treatment groups. The amount of differentiation of iPSC toward DE was determined by quantitative gene expression analyses of pluripotency ( NANOG and OCT4 ), definitive endoderm ( SOX17 and FOXA2 ) and endoderm-derived organs ( PDX1 , NEUROG3, and PAX6 ). Based on gene expression analyses, the more decrease in concentrations of activin-A can increase the differentiation of iPSC into DE, therefore, 30 ng/ml activin-A was chosen as the best concentration for the differentiation of R1-hiPSC1 line toward endoderm-derived organ. Moreover, complete replacement of FBS with gradually increased KSR improved the differentiation of iPSC toward DE. For this reason, the addition of 0% KSR at day 1, 0.2% at day 2 and 2% for the next 3 days was the best optimal protocol of the differentiation of iPSC toward DE. Overall, our results demonstrate that optimization of activin-A is important for differentiation of iPSC line. Furthermore, the replacement of FBS with KSR can improve the efficiency of iPSC differentiation toward DE., Competing Interests: Conflict of interestOn behalf of all authors, the corresponding author states that there is no conflict of interest., (© King Abdulaziz City for Science and Technology 2020.)

Objectives: Type 1 diabetes (T1D) is an autoimmune disease resulting from inflammatory destruction of islets β-cells. Nowadays, progress in cell therapy, especially mesenchymal stem cells (MSCs) proposes numerous potential remedies for T1D. We aimed to investigate the combination therapeutic effect of these cells with insulin and metformin on neuropeptide Y, melanocortin-4 receptor, and leptin receptor genes expression in TID., Materials and Methods: One hundreds male rats were randomly divided into seven groups: the control, diabetes, insulin (Ins.), insulin+metformin (Ins.Met.), Wharton's Jelly-derived MSCs (WJ-MSCs), insulin+metformin+WJ-MSCs (Ins.Met.MSCs), and insulin+WJ-MSCs (Ins.MSCs). Treatment was performed from the first day after diagnosis as diabetes. Groups of the recipient WJ-MSCs were intraportally injected with 2× 10⁶ MSCs/kg at the 7th and 28th days of study. Fasting blood sugar was monitored and tissues and genes analysis were performed., Results: The blood glucose levels were slightly decreased in all treatment groups within 20 th and 45 th days compared to the diabetic group. The C-peptide level enhanced in these groups compared to the diabetic group, but this increment in Ins.MSCs group on the 45th days was higher than other groups. The expression level of melanocortin-4 receptor and leptin receptor genes meaningfully up-regulated in the treatment groups, while the expression of neuropeptide Y significantly down-regulated in the treatment group on both times of study., Conclusion: Our data exhibit that infusion of MSCs and its combination therapy with insulin might ameliorate diabetes signs by changing the amount of leptin and subsequent changes in the expression of neuropeptide Y and melanocortin-4 receptor.

The pancreas of brain-dead donors is the primary source of islets for transplantation. However, brain death mediates systemic inflammation, which may affect the quantity and quality of isolated islets. Our aim was to identify inflammatory biomarkers in donor blood and/or pancreatic tissue capable of predicting islet isolation success. Blood samples were collected from 21 pancreas donors and 14 healthy volunteers. Pancreatic tissue samples were also collected from the corresponding donor during organ procurement. Six serum cytokines were measured by a fluorescent bead-based immunoassay, and the expression of fifteen inflammatory target genes was quantified by quantitative reverse transcription polymerase chain reaction (RT-qPCR). There was no correlation between serum inflammatory cytokines and mRNA expression of the corresponding genes in peripheral blood mononuclear cells (PBMCs) or pancreatic tissue. The IL6 expression in pancreatic tissue correlated negatively with post-isolation islet yield. Islets isolated from donors highly expressing IFNG in PBMCs and MAC1 in pancreatic tissue functioned poorly in vivo when transplanted in diabetic NOD scid mice. Furthermore, the increased MAC1 in pancreatic tissue was positively correlated with donor hospitalization time. Brain death duration positively correlated with higher expression of IL1B in PBMCs and TNF in both PBMCs and pancreatic tissue but failed to show a significant correlation with islet yield and in vivo function. The study indicates that the increased inflammatory genes in donor pancreatic tissues may be considered as biomarkers associated with poor islet isolation outcome.

Background: Human pancreata contain many types of cells, such as endocrine islets, acinar, ductal, fat, and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions., Methods: We investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore, whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated., Results: IPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5% human platelet lysate (hPL). IPTD-MSCs were found to be highly pure, with > 95% positive for CD90, CD105, and CD73, and negative for CD45, CD34, CD14, and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105 + cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes, chondrocytes, and osteoblasts in vitro, underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-α, gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased, compared to control. In contrast, treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly, a combination of TNF-α and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α contained higher levels of pro-angiogenic (VEGF, IL-6, and IL-8) compared to controls, promoting angiogenesis of human endothelial cells in vitro. In contrast, levels of MCP-1, a pro-inflammatory cytokine, were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α., Conclusions: The results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application.

Ginsenoside Rd (GS-Rd), one of the main pharmacologically active components of ginseng, has shown the potential to stabilize mitochondrial membrane integrity and decrease apoptotic death in neuronal and non-neuronal cells. The present study aimed to evaluate the effect of this bioactive molecule on the apoptosis-associated cell death in human pancreatic islets. In this regard human pancreatic islets were isolated and grouped for the treatment with GS-Rd. The isolated islets were treated with different concentrations of GS-Rd. After 24 and 72 h of incubation, the islets were evaluated in terms of viability, BAX , BCL2 , and insulin gene expression, BAX, BCL2, and caspase-3 protein expression, apoptosis, and glucose-induced insulin/C-peptide secretion. Our results revealed the islet survival was significantly decreased in the control group after 72 h of incubation. However, GS-Rd inhibited the progress of the islet death in the treated groups. TUNEL staining revealed that the preventive effect of this molecule was caused by the inhibition of apoptosis-associated death. In this regard, the activation of caspase-3 was down-regulated in the presence of GS-Rd. GS-Rd did not exhibit undesirable effects on glucose-induced insulin and C-peptide stimulation secretion. In conclusion, GS-Rd inhibited the progress of death of cultured human pancreatic islets by diminishing the apoptosis of the islet cells., (Copyright © 2019 Kaviani et al.)

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.

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.

Background and Purpose: Islet transplantation is considered as a promising approach in the treatment of diabetes type 1. In this regard, optimal culture of the pancreatic islets is promising in the success of transplantation. In the present study, the effect of olesoxime, as an antiapoptotic substance, was evaluated on human islet culture., Experimental Approach: The pancreatic islets were isolated by mechanical and enzymatic techniques. After overnight recovery, the islets were treated by different concentrations of olesoxime for 24 and 72 h. Then, they were examined in terms of viability, apoptosis, genes and proteins expression including BAX, BCL2, active caspase-3, and insulin. Moreover, the islets function was evaluated through the glucose-induced insulin and C-peptide secretion assay., Key Results: Our findings showed that the islets increased in apoptosis and the decreased in viability after 72 h; also, insulin and C-peptide secretion reduced. However, in the presence of olesoxime, BAX/BCL2 ratio and the activation of caspase-3 were decreased. Therefore, olesoxime could improve the viability of the islets with the decrease of apoptosis., Conclusion: The application of olesoxime can reduce the stressful condition for the islets in vitro and subsequently improve their viability and functionality., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Selection of enzymes for optimal pancreas digestion is essential for successful human islet isolations. The aim of this study was to evaluate the efficacy and outcome of using Collagenase Gold plus BP protease (VitaCyte) (n = 8) by comparing it to two commercially available enzymes, Liberase MTF C/T (Roche) (n = 48) and Collagenase NB1/NP (Serva) (n = 15). The isolation outcomes were assessed by islet counting, viability, glucose-stimulated oxygen consumption rate (OCR), and successful graft-rate following transplantation in diabetic NOD scid mice. The pancreas donor characteristics were not significantly different between the tested enzyme groups regarding their BMI, pancreas weight, cold ischemia time (CIT) and HbA1c. The results show that digested tissue volume was not statistically significant between the VitaCyte enzyme (34.25 ± 5.4 mL) and the Roche enzyme (55.25 ± 3.42 mL, p = 0.073), however, this was significant with Serva enzyme (64.07 ± 7.95 mL, p = 0.020). Interestingly, the islet yields were not statistically different between all enzyme groups. Moreover, when islets were transplanted into NOD scid mice, the reversal rate of diabetes for the VitaCyte enzyme group was similar to all enzyme groups. In conclusion, the effectiveness of Collagenase Gold plus BP protease is comparable to the MTF C/T and the Collagenase NB1/NP enzymes; the low cost could facilitate the use of more pancreata for islet isolations.

There is growing evidence that transplantation of cadaveric human islets is an effective therapy for type 1 diabetes. However, gauging the suitability of islet samples for clinical use remains a challenge. We hypothesized that islet quality is reflected in the expression of specific genes. Therefore, gene expression in 59 human islet preparations was analyzed and correlated with diabetes reversal after transplantation in diabetic mice. Analysis yielded 262 differentially expressed probesets, which together predict islet quality with 83% accuracy. Pathway analysis revealed that failing islet preparations activated inflammatory pathways, while functional islets showed increased regeneration pathway gene expression. Gene expression associated with apoptosis and oxygen consumption showed little overlap with each other or with the 262 probeset classifier, indicating that the three tests are measuring different aspects of islet cell biology. A subset of 36 probesets surpassed the predictive accuracy of the entire set for reversal of diabetes, and was further reduced by logistic regression to sets of 14 and 5 without losing accuracy. These genes were further validated with an independent cohort of 16 samples. We believe this limited number of gene classifiers in combination with other tests may provide complementary verification of islet quality prior to their clinical use.

A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay. The peptide substrate was cleaved by collagenase class I, II, Liberase MTF C/T, collagenase NB1, and thermolysin/neutral protease, which was significantly enhanced in the presence of CaCl 2 . However, the activities of these enzymes were significantly decreased in the presence of ZnSO 4 or ZnCl 2 . Collagenase I, II, Liberase MTF C/T, thermolysin/neutral protease share similar cleavage sites, L ↓ G and P ↓ G. However, collagenase NB1 cleaves the peptide substrate at G ↓ P and P ↓ L, in addition to P ↓ G. The enzyme activity is pH dependent, within a range of 6.8 to 7.5, but was significantly diminished at pH 8.0. Interestingly, the peptide substrate was not cleaved by endogenous pancreatic protease such as trypsin, chymotrypsin, and elastase. In conclusion, the novel peptide substrate is collagenase, thermolysin/neutral protease specific and can be applied to quantify enzyme activities from different microbes. Furthermore, the assay can be used for fine-tuning reaction mixtures of various agents to enhance the overall activity of a cocktail of multiple enzymes and achieve optimal organ/tissue digestion, while protecting the integrity of the target cells.

Long-term pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and rewarming-induced injury by interrogating the proapoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic ("Firefly") Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3-knockout (KO) mice, and 3) cold preservation and rewarming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during rewarming in "Firefly" islets. Silencing Bbc3 by transfecting Bbc3 siRNA into islets in vitro prior to cold preservation improved postpreservation mitochondrial viability. Cold preservation resulted in decreased postisolation islet yield in both wild-type and Bbc3 KO pancreata. However, after culture, the islet viability was significantly higher in Bbc3-KO islets, suggesting that different mechanisms are involved in islet damage/loss during isolation and culture. Furthermore, Bbc3-KO islets from cold-preserved pancreata showed reduced HMGB1 (high-mobility group box 1 protein) expression and decreased levels of 4-hydroxynonenal (4-HNE) protein adducts, which was indicative of reduced oxidative stress. During human islet isolation, BBC3 protein was upregulated in digested tissue from cold-preserved pancreata. Hypoxia in cold preservation increased BBC3 mRNA and protein in isolated human islets after rewarming in culture and reduced islet viability. These results demonstrated the involvement of BBC3/Bbc3 in cold preservation/rewarming-mediated islet injury, possibly through modulating HMGB1- and oxidative stress-mediated injury to islets., (Copyright © 2016 the American Physiological Society.)

Background: Transplanting pancreatic islets into recipients must be safe and effective to treat Type 1 diabetes. Islet quality and quantity are important, however, the final product must also be free from microbial contamination and low endotoxin levels., Methods: This study explored a method to eliminate contamination in manufacturing islets for transplantation. A simple (single antibiotic, n=164) and refined (triple antimicrobial agents, n=279) pancreas decontaminating methods were used to test their effects on reducing the contamination rates in the islet final product. A total of 443 pancreata were processed for islet isolations. Three samples for microbial tests (Gram stain, aerobic, and anaerobic culture) were taken at pre-process (pancreas preservation), post-isolation, and post-culture. Endotoxin levels were measured only for islets considered for transplantation., Results: Out of 443 pancreata used for islet isolation, 79 (17.8%) showed signs of contamination in pre-process samples; 10 (2.3%) were contaminated in both pre-process and in the final product (post-isolation and post-culture) samples. Contamination rates in which pre-process and final product samples were positive for contamination was significantly lower using the refined method (refined vs. simple method: 5% vs. 20.5%, p=0.045). Identical microbial species were present in both pre-process and in the final product., Conclusions: This study demonstrated that the refined method reduces the rate of contamination of the islet final product and is safe for clinical application. Moreover, it may be used as a standard method during human islet manufacturing facilitating the application of a biological license agreement from United States Food and Drug Administration.

Background: We evaluated three commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality post-isolation., Methods: Retrospectively compared and analyzed islet isolations from pancreata using three different enzyme groups: Liberase HI (n=63), Collagenase NB1/Neutral Protease (NP) (n=43), and Liberase Mammalian Tissue Free Collagenase/Thermolysin (MTF C/T) (n=115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate (ΔOCR), and in vivo transplantation model in mice., Results: Donor characteristics were not significantly different among the three enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index (BMI), hemoglobin A1c (HbA1c), cold ischemia time (CIT), and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the Liberase MTF C/T group (73.5 ± 1.5 %) when compared to the Liberase HI group (63.6 ± 2.3 %) (p<0.001) and the Collagenase NB1/NP group (61.7 ± 2.9%) (p<0.001). The stimulation index for GSIS was significantly higher in the Liberase MTF C/T group (5.3 ± 0.5) as compared to the Liberase HI (2.9 ± 0.2) (p<0.0001) and the Collagenase NB1/NP (3.6 ± 2.9) (p=0.012) groups. Furthermore, the Liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic NOD Scid mice (65%), which was significantly higher than the Liberase HI (42%, p=0.001) and the Collagenase NB1/NP enzymes (41%, p<0.001)., Conclusions: Liberase MTF C/T is superior to Liberase HI and Collagenase NB1/NP in terms of digestion efficacy and glucose-stimulated insulin secretion in vitro . Moreover, Liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to Liberase HI and Collagenase NB1/NP enzymes.

Organs from hypernatremia (elevated Na+) donors when used for transplantation have had dismal outcomes. However, islet isolation from hypernatremic donors for both transplantation and research applications has not yet been investigated. A retrospective analysis of in vivo and in vitro islet function studies was performed on islets isolated from hypernatremic (serum sodium levels≥160 meq/l) and normal control (serum sodium levels≤155 meq/l) donors. Twelve isolations from 32 hypernatremic and 53 isolations from 222 normal donors were randomly transplanted into diabetic NOD Scid mice. Sodium levels upon pancreas procurement were significantly elevated in the hypernatremia group (163.5±0.6 meq/l) compared with the normal control group (145.9±0.4 meq/l) (P<0.001). The postculture islet recovery rate was significantly lower in the hypernatremia (59.1±3.8%) group compared with the normal (73.6±1.8%) group (P=0.005). The duration of hypernatremia was inversely correlated with the recovery rate (r2=0.370, P<0.001). Furthermore, the percentage of successful graft function when transplanted into diabetic NOD Scid mice was significantly lower in the hypernatremia (42%) group compared with the normal control (85%) group (P<0.001). The ability to predict islet graft function posttransplantation using donor sodium levels and duration of hypernatremia was significant (ROC analysis, P=0.022 and 0.042, respectively). In conclusion, duration of donor hypernatremia is associated with reduced islet recovery postculture. The efficacy of islets from hypernatremia donors diminished when transplanted into diabetic recipients., (Copyright © 2015 the American Physiological Society.)

Objectives: Type 1 diabetes mellitus is an emerging epidemic worldwide and results from autoimmune destruction of insulin-producing β cells. Islet transplanting is a potential treatment for type 1 diabetes mellitus., Materials and Methods: The Shiraz Organ Transplant Center is a leading center for organ transplants, especially pancreatic transplants, in Iran. For this reason, we want to establish an islet transplanting program. Here, we briefly describe our experience with islet isolation on 6 pancreata from deceased donors. We discussed the necessary equipment required for this procedure, as well as the professionals needed and a specially planned facility., Results: Islet yield was ≤ 100 000 (islet equivalent), viability 40% to 45%, and the purity was 30% to 45%. We do not have a refrigerated COBE processor for purification; therefore, the yield was low. Our experience shows that we should improve things, so as to acquire more islets for developing clinical grade cell therapy., Conclusions: Overall, isolation costs are high, and accessing a safer, more economic, and persistent source of material and reagents will improve this technique.

Background: Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression., Methodology/principal Findings: Genomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro., Conclusions/significance: Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

The activation of p38 mitogen-activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum-free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS-p38IH and islet viability was not significantly different in the three groups. beta Cell numbers and function were the highest in islets cryopreserved with ICS-p38IH. Glucose-stimulated human C-peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscid mice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.

This was a pilot study to investigate the possible roles of human lymphocyte antigen (HLA), antipeternal lymphocytotoxic antibodies, and maternal antipaternal mixed lymphocyte reaction (MLR) blocking antibodies in the maintenance of pregnancy following paternal leukocyte immunization for patients with recurrent abortions. A total of 36 patients with unexplained, first trimester, primary recurrent spontenous abortions were investigated for the detection of these two antibodies. There was a 43.3% rate of discordance in the presence of the two antibodies (P <0.05). A total of 26 of these women who lacked either antibody were immunized with paternal leukocyctes on two occasions and the assays were repeated post-immunization. The seroconversion rate was 50% for lymphocytotoxic antibodies and 61.5% for maternal serum (MLR) blocking antibodies. Twenty patients achieved pregnancies post-immunization, 11 completed their pregnancies successfully, and 9 re-aborted. A total of 83.3% of patients who developed MLR blocking antibodies post-immunization had successful pregnancies while those who failed to seroconvert aborted again. This difference is statistically significant (P<.05). A total of 50% of patients who developed lymphocytotoxic antibodies post-immunization had successful pregnancies while only 40% who failed to seroconvert re-aborted. This difference, we felt, was not statistically significant. The development of MLR blocking antibodies psot-immunization is a better indicator of subsequent successful pregnancies than lymphocytotoxic antibodies.