A scalable human islet 3D-culture platform maintains cell mass and function long-term for transplantation.

Present-day islet culture methods provide short-term maintenance of cell viability and function, limiting access to islet transplantation. Attempts to lengthen culture intervals remain unsuccessful. A new method was developed to permit the long-term culture of islets. Human islets were embedded in polysaccharide 3D-hydrogel in cell culture inserts or gas-permeable chambers with serum-free CMRL 1066 supplemented media for up to 8 weeks. The long-term cultured islets maintained better morphology, cell mass, and viability at 4 weeks than islets in conventional suspension culture. In fact, islets cultured in the 3D-hydrogel retained ß cell mass and function on par with freshly isolated islets in vitro and, when transplanted into diabetic mice, restored glucose balance similar to fresh islets. Using gas-permeable chambers, the 3D-hydrogel culture method was scaled up over 10-fold and maintained islet viability and function, although the cell mass recovery rate was 50%. Additional optimization of scale-up methods continues. If successful, this technology could afford flexibility and expand access to islet transplantation, especially single-donor islet-after-kidney transplantation.

Methylcellulose colony assay and single-cell micro-manipulation reveal progenitor-like cells in adult human pancreatic ducts.

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.

Reassessing the Abundance of miRNAs in the Human Pancreas and Rodent Cell Lines and Its Implication.

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.

Thrombospondin-1, CD47, and SIRPα display cell-specific molecular signatures in human islets and pancreata.

Thrombospondin-1 (TSP1) is a secreted protein minimally expressed in health but increased in disease and age. TSP1 binds to the cell membrane receptor CD47, which itself engages signal regulatory protein α (SIRPα), and the latter creates a checkpoint for immune activation. Individuals with cancer administered checkpoint-blocking molecules developed insulin-dependent diabetes. Relevant to this, CD47 blocking antibodies and SIRPα fusion proteins are in clinical trials. We characterized the molecular signature of TSP1, CD47, and SIRPα in human islets and pancreata. Fresh islets and pancreatic tissue from nondiabetic individuals were obtained. The expression of THBS1, CD47, and SIRPA was determined using single-cell mRNA sequencing, immunofluorescence microscopy, Western blot, and flow cytometry. Islets were exposed to diabetes-affiliated inflammatory cytokines and changes in protein expression were determined. CD47 mRNA was expressed in all islet cell types. THBS1 mRNA was restricted primarily to endothelial and mesenchymal cells, whereas SIRPA mRNA was found mostly in macrophages. Immunofluorescence staining showed CD47 protein expressed by ß cells and present in the exocrine pancreas. TSP1 and SIRPα proteins were not seen in islets or the exocrine pancreas. Western blot and flow cytometry confirmed immunofluorescent expression patterns. Importantly, human islets produced substantial quantities of secreted TSP1. Human pancreatic exocrine and endocrine tissue expressed CD47, whereas fresh islets displayed cell surface CD47 and secreted TSP1 at baseline and in inflammation. These findings suggest unexpected effects on islets from agents that intersect TSP1-CD47-SIRPα.NEW & NOTEWORTHY CD47 is a cell surface receptor with two primary ligands, soluble thrombospondin-1 (TSP1) and cell surface signal regulatory protein alpha (SIRPα). Both interactions provide checkpoints for immune cell activity. We determined that fresh human islets display CD47 and secrete TSP1. However, human islet endocrine cells lack SIRPα. These gene signatures are likely important given the increasing use of CD47 and SIRPα blocking molecules in individuals with cancer.

Submilligram Level of Beetle Antifreeze Proteins Minimize Cold-Induced Cell Swelling and Promote Cell Survival.

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 ß-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 ß-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.

A Multiparametric Assessment of Human Islets Predicts Transplant Outcomes in Diabetic Mice.

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.

Chronic marijuana usage by human pancreas donors is associated with impaired islet function.

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.

The potential of the incorporated collagen microspheres in alginate hydrogel as an engineered three-dimensional microenvironment to attenuate apoptosis in human pancreatic islets.

BACKGROUND: Tissue engineering is considered as a promising tool for remodeling the native cells microenvironment. In the present study, the effect of alginate hydrogel and collagen microspheres integrated with extracellular matrix components were evaluated in the decrement of apoptosis in human pancreatic islets. MATERIALS/METHODS: For three-dimensional culture, the islets were encapsulated in collagen microspheres, containing laminin and collagen IV and embedded in alginate scaffold for one week. After that the islets 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 glucose-induced insulin and C-peptide secretion assay. In order to evaluate the structure of the scaffolds and the morphology of the pancreatic islets in three-dimensional microenvironments, we performed scanning electron microscopy. RESULTS: Our findings showed that the designed hydrogel scaffolds significantly improved the islets viability using the reduction of activated caspase-3 and TUNEL positive cells. CONCLUSIONS: The reconstruction of the destructed matrix with alginate hydrogels and collagen microspheres might be an effective step to promote the culture of the islets.

microRNAs in liver and kidney ischemia reperfusion injury: insight to improve transplantation outcome.

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.

Exosomes derived from human mesenchymal stem cells preserve mouse islet survival and insulin secretion function.

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.

Significant reduction of apoptosis induced via hypoxia and oxidative stress in isolated human islet by resveratrol.

BACKGROUND AND AIMS: Successful islet transplantation as a promising treatment of diabetes type 1 is threatened with the loss of islets during the pre-transplant culture due to hypoxia and oxidative stress-induced apoptosis. Therefore, optimization of culture in order to preserve the islets is a critical point. In this study, we investigated the effect of resveratrol, as a cytoprotective agent, on the cultured human islets. METHODS AND RESULTS: Isolated islets were treated with different concentrations of resveratrol for 24 and 72 h. Islets' viability, apoptosis, apoptosis markers, and insulin and C-peptide secretion, along with the production of reactive oxygen species (ROS), hypoxia inducible factor 1 alpha (HIF-1α), and its target genes in the islets were investigated. Our findings showed that the islets were exposed to hypoxia and oxidative stress after isolation and during culture. This insult induced apoptosis and decreased viability during 72 h. The presence of resveratrol significantly attenuated HIF-1α and ROS production, reduced apoptosis, promoted the VEGF secretion, and increased the insulin and C-peptide secretion. In this regard, resveratrol improved the islet's survival and function in the culture period. CONCLUSIONS: Using resveratrol can attenuate the stressful condition for the islets in the pre-transplant culture and subsequently ameliorate their viability and functionality that lead to successful outcome after clinical transplantation.

The effect of human wharton's jelly-derived mesenchymal stem cells on MC4R, NPY, and LEPR gene expression levels in rats with streptozotocin-induced diabetes.

OBJECTIVES: Type 1 diabetes (T1D) is an autoimmune disease resulting from inflammatory destruction of islets ß-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 20th and 45th 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.

Optimization of activin-A: a breakthrough in differentiation of human induced pluripotent stem cell into definitive endoderm.

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.

Inflammatory biomarkers in the blood and pancreatic tissue of organ donors that predict human islet isolation success and function.

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 NODscid 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.

Intra-pancreatic tissue-derived mesenchymal stromal cells: a promising therapeutic potential with anti-inflammatory and pro-angiogenic profiles.

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.

Cytoprotective effects of ginsenoside Rd on apoptosis-associated cell death in the isolated human pancreatic islets.

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.

Protective effect of nobiletin on isolated human islets survival and function against hypoxia and oxidative stress-induced apoptosis.

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.

Amelioration of the apoptosis-mediated death in isolated human pancreatic islets by minocycline.

Minocycline functions as a therapeutic drug in different diseases because of its cytoprotective properties. In the present study, we examined the potential of minocycline to decrease the islet loss in pre-transplantation culture stage. Pancreatic islets were isolated from the deceased donors and treated by 0, 2, 10, and 20 µM minocycline for 24 and 72 h. After that, the incubated islets were evaluated for viability and function. Apoptosis markers including Bax, Bcl2, and caspase-3 were determined at gene and protein level. On the other hand, TUNEL assay was used to confirm apoptosis. The functionality of the islets was investigated using glucose-induced insulin and c-peptide secretion assay. After 72 h of incubation, the viability of human islet was drastically decreased, whereas supplementation with minocycline inhibited the cells death. In this regard, the expression of Bax and active Caspase-3 was downregulated, whereas the expression of Bcl2 was upregulated. These consequences suggest that pancreatic islets undergo apoptosis in vitro and minocycline can decelerate or inhibit this process. Our findings identified minocycline as a cytoprotective molecule for preventing human islets death in pre-transplantation culture.

Pancreatic human islets and insulin-producing cells derived from embryonic stem cells are rapidly identified by a newly developed Dithizone.

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.

Cytoprotective effects of olesoxime on isolated human pancreatic islets in order to attenuate apoptotic pathway.

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.