Mesenchymal stem cells protect islets from hypoxia/reoxygenation‐induced injury

Transplantation of pancreatic islets has become a promising clinical option to treat patients with type 1 diabetes, alternative to the standard therapy with insulin injections. Islet transplantation is a minimally invasive therapeutic approach, and it allows a better metabolic control and a long-term insulin independence in more than 80% of patients (Ryan et al., 2002). However this therapeutic treatment has some side effects, such as the poor yield of pancreatic islet explants and even more the immune graft rejection, which have as a consequence the very limited lifespan of transplanted pancreatic islets. To avoid these side effects several strategies have been proposed and, besides the treatment with immunosuppressive drugs, promising results have been obtained with the use of Mesenchymal Stem cells (MSCs), already known in literature to be able to support the survival of many cell types (Scuteri et al., 2006). Several in vivo studies have demonstrated that the concurrent transplantation of pancreatic islets with MSCs reduces the number of islets required to achieve glycemic control in diabetic rats, but the mechanisms of these encouraging results are still unknown (Figliuzzi et al., 2009). For these reasons in this in vitro study we characterized the effect of co-culture of rat MSC on survival and functioning of rat pancreatic islets, by evaluating for 4 weeks: i) MSC adhesion to pancreatic islets; ii) viability of pancreatic islets co-cultured with MSCs; iii) the expression of insulin after co-culture; iv) the ability of co-cultured pancreatic islets to correctly adjust insulin release after variation of glucose concentration. Our results demonstrated that MSCs are able to adhere to pancreatic islets, but to increase only partly the pancreatic islet survival, which retain the ability to express and correctly release insulin after glucose variation in medium culture. Noteworthy that the insulin level in the medium of co-cultured pancreatic islets is always higher with respect to medium of pancreatic islets alone. The immunofluorescence analysis reveals that also MSCs (and not only pancreatic islets) are able to express insulin, but only in co-culture. These results, which justify the in vivo observation reported above, suggest that MSCs undergo to differentiation into a insulin-releasing phenotype after co-culture with pancreatic islets. We are now evaluating the molecular mechanisms which drive this effect, by analyzing the role of soluble factors and of proteins able to induce insulin expression. This study was granted by MIUR – FIRB Futuro in Ricerca 2008 RBFR08VSVI_001.

Mesenchymal stromal cells (MSCs) are adult stromal cells with therapeutic potential in allogeneic islet transplantation for type 1 diabetes patients. The process of islet isolation alone has been shown to negatively impact islet survival and function in vivo. In addition, insults mediated by the instant blood-mediated inflammatory reaction, hypoxia, ischemia and immune response significantly impact the islet allograft post transplantation. MSCs are known to exert cytoprotective and immune modulatory properties and thus are an attractive therapeutic in this context. Herein, the recent progress in the field of MSC therapy in islet transplantation is discussed. MSC can promote islet survival and function in vivo. Importantly, studies have shown that human MSC donors have differential abilities in promoting islet regeneration/survival. Recently, several biomarkers associated with MSC islet regenerative capacity have been identified. Expressions of Annexin A1, Elastin microfibril interface 1 and integrin-linked protein kinase are upregulated in MSC displaying protective effects on islet survival and function in vivo. The discovery of biomarkers associated with MSC therapeutic efficacy represents an important step forward for the utilization of MSC therapy in islet transplantation; however, much remains to be elucidated about the mechanisms utilized by MSC in protection against transplanted islet loss, autoimmune-mediated and alloimmune-mediated rejection.

ABSTRACTPancreatic islets, especially the large islets (> 150µm in diameter) have poor survival rates in culture. Co-culturing with mesenchymal stem cells (MSCs) has been shown to improve islet survival and function. However, most co-culture studies have been comprised of MSC surrounding islets in the media. The purpose of this study was to determine whether islet survival and function was improved when the 2 populations of cells were intermingled with each other in a defined geometry. Hybrid spheroids containing 25, 50 or 75 or 90% islets cells with appropriate numbers of MSCs were created along with spheroids comprised of only islet cells or only MSCs. Spheroids were tested for yield, viability, diameter, cellular composition, and glucose-stimulated insulin secretion. The 25% islet/75% MSC group created the fewest spheroids, with the poorest survival and insulin secretion and the largest diameter. The remaining groups were highly viable with average diameters under 80µm at formation. However, the hybrid spheroid groups preferred to cluster in islet-only spheroids. The 50, 75 and 90% islet cell groups had excellent long-term survival with 90–95% viability at 2 weeks in culture, compared with the islet only group that were below 80% viability. The glucose-stimulated insulin secretion was not statistically different for the 50, 75, or 90 groups when exposed to 2.4, 16.8, or 22.4 mM glucose. Only the spheroids with 25% islet cells had a statistically lower levels of insulin release, and the 100% had statistically higher levels at 22.4 mM glucose and in response to secretagogue. Thus, imbedded co-culture improved long-term viability, but failed to enhance glucose-stimulated insulin secretion in vitro.

Background. Low engraftment and adverse immune reactions hamper the success rate of clinical islet transplantation. In this study, we investigated the capacity of human mesenchymal stem cells (MSCs) to adhere to human islets of Langerhans and their effects in immune modulation and during blood interactions in vitro.Methods. Composite MSC–islets were formed by suspension co-culture, and the phenotype was evaluated by confocal microscopy. Islet function was assessed by dynamic insulin release in response to glucose in vitro. Mixed lymphocyte–islet reactions (MLIR) and the tubing blood loop model were utilized as in vitro tools to analyse the effect of MSCs on the innate and adaptive immune reactions triggered by the islets.Results. MSCs rapidly adhered to islets and spread out to cover the islet surface. Insulin expression and secretion were sustained with the MSC coating. MSC-coated islets showed unaffected reactions with blood in vitro in comparison to control islets. Furthermore, MSCs suppressed lymphocyte proliferation induced by islet cells in MLIR.Conclusion. We conclude that it is possible to create composite MSC–islets to enable delivery of the MSCs by utilizing the adhesive capacity of the MSCs. This could have beneficial immunosuppressive effects in optimizing pancreatic islet transplantation.

Introduction: Pancreatic islet transplantation has recently emerged as one of the most promising therapeutic approaches for improving glycemic control in type 1 diabetes patients. However, one of the problems with islet transplantation is that it is impossible to culture the isolated islet for extended periods to while recipients are selected, tested and prepared for surgery. To address this problem, we developed the islet preservation solution, and to improve the function of the preservation solution, we added taurine and conducted a preservation experiment. Taurine is antioxidant activity and it is not a classical free radical scavenger. Therefore, its mechanism remains unclear but it controls osmotic pressure. Our hypothesis is that taurine supplementation can improve islet recovery and enhance islet function after low temperature preservation. Methods: CMRL 1066 medium (Corning) was used as a control group, and a comparison group was prepared some compounds OPTI(Optipharm presevation) solution (with taurine) and OPTI-T solution (without taurine). To confirm the preservation effect of these solutions, we isolated islet from adult Yucatan pig’s pancreas using standard enzymatic digestion (Nordmark) followed by Ficoll purification, these islets were preserved in a humidified tissue culture incubator with preservation solutions and preserved in 4℃ for up to 4 weeks. We compared islet survival in groups of islets. To islet viability and islet functionality, we utilized Glucose Stimulated Insulin Secretion (GSIS) for measuring insulin release, calculated stimulation index (SI) and AO/PI viability staining for during the preservation period. Results: Highest islet recovery was preserved in the group of pig islets cultured with Taurine as compared to the group of islets hypothermically preserved in standard tissue culture media over the 4 week preservation period (p<0.001 using ANOVA). The porcine islet viability/cell number ratio in the OPTI solution maintained a high survival rate for up to 2 weeks (94%). In contrast, porcine islet in OPTI-T solution and CMRL 1066 supplemented media significantly reduced viability after 1 weeks. OPTI solution (with taurine) was significantly higher than OPTI-T solution (without taurine) and CMRL 1066 during 4 weeks after preservation. Glucose stimulation insulin secretion test was performed for functional analysis. As a result, a level of porcine insulin was higher during 2 weeks in the OPTI solution than OPTI-T solution and CMRL 1066. But after 3 weeks preservation, insulin level in high glucose, significantly decreased. Therefore, the viability and functionality of islets were improved by adding taurine to the developed preservation solution. Conclusion: Taurine supplementation has been shown to highest islet recovery, viability and insulin secretion level for 2 weeks. Based on this data, we have performed 2 weeks cold storage of isolated islets for current clinical islet transplantation. Ministry of trade industry and energy, KEIT 20011276, NTIS 1415172664.

It is unknown whether a scaffold containing both small intestinal submucosa (SIS) and mesenchymal stem cells (MSCs) for transplantation may improve pancreatic islet function and survival. In this study, we examined the effects of a SIS-MSC scaffold on islet function and survival in vitro and in vivo. MSCs and pancreatic islets were isolated from Sprague-Dawley rats, and SIS was isolated from Bamei pigs. The islets were apportioned among 3 experimental groups as follows: SIS-islets, SIS-MSC-islets and control-islets. In vitro, islet function was measured by a glucose-stimulated insulin secretion test; cytokines in cultured supernatants were assessed by enzyme-linked immunosorbent assay; and gene expression was analyzed by reverse transcription-quantitative PCR. In vivo, islet transplantation was performed in rats, and graft function and survival were monitored by measuring the blood glucose levels. In vitro, the SIS-MSC scaffold was associated with improved islet viability and enhanced insulin secretion compared with the controls, as well as with the increased the expression of insulin 1 (Ins1), pancreatic and duodenal homeobox 1 (Pdx1), platelet endothelial cell adhesion molecule 1 [Pecam1; also known as cluster of differentiation 31 (CD31)] and vascular endothelial growth factor A (Vegfa) in the islets, increased growth factor secretion, and decreased tumor necrosis factor (TNF) secretion. In vivo, the SIS-MSC scaffold was associated with improved islet function and graft survival compared with the SIS and control groups. On the whole, our findings demonstrate that the SIS-MSC scaffold significantly improved pancreatic islet function and survival in vitro and in vivo. This improvement may be associated with the upregulation of insulin expression, the improvement of islet microcirculation and the secretion of cytokines.

Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number of clinical applications, e.g., non-healing bone fractures and defects and also non-skeletal degenerative diseases like heart failure. Currently, the numbers of clinical trials that employ MSC are increasing. However, several biological and biotechnological challenges need to be overcome to benefit from the full potential of hMSC. In this current review, we present some of the most important and recent advances in understanding of the biology of hMSC and their current and potential use in therapy.

Intraportal islet transplantation has the risk of severe complication, such as portal embolization. On the other hand, intramuscular islet transplantation is prior to the safety and easy accessibility. However, the outcome is poor. In the islet transplantation into the liver or kidney, previous reports showed that mesenchymal stem cells (MSCs) had some preferable effects. In this study, we examined about the effects on the islet co-transplantation with MSCs, and investigated the influences on co-cultured islet gene expression in the rodent model. The viability and glucose sensitivity of cultured islets were evaluated. Additionally, the influence of co-culturing MSCs on the islets was investigated by the microarray analysis and quantitative PCR. Islets were transplanted into the muscle without MSCs (group A) or with MSCs (group B). Blood glucose level, glucose tolerance test, and plasma IL-6 level of recipients were measured. Furthermore, anti-inflammatory effect, anti-apoptotic effect, and neovascularization were evaluated histologically. In vitro study, glucose sensitivity of group B was significantly increased, and qPCR revealed that the 3 gene expressions of islets, which were possibly correlated in the islet function and engraftment, were significantly up-regulated by co-culturing with MSCs. Although co-transplanted MSCs could not significantly improve the glycemic control, the glucose level of group B in the IP-GTT was decreased more after the stimulation. The plasma IL-6 level of group B was decreased more than that of group A (p = 0.0002). In the histological exams, while anti-inflammatory effect of MSCs was showed, the effects on anti-apoptotic effect and promotion of neovascularization could not be demonstrated. In summary, although effectiveness of MSCs was clarified in vitro assessments, that was inadequate in vivo study. It might be needed to promote neovascularization.

Co-transplantation of islets with mesenchymal stem cells in microcapsules demonstrates graft outcome can be improved in an isolated-graft model of islet transplantation in mice.

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.

Pre-culturing islets with mesenchymal stromal cells using a direct contact configuration is beneficial for transplantation outcome in diabetic mice.

Type 1 Diabetes (T1D) is a chronic autoimmune disease characterized by a gradual destruction of insulin-producing β-cells in the endocrine pancreas due to innate and specific immune responses, leading to impaired glucose homeostasis. T1D patients usually require regular insulin injections after meals to maintain normal serum glucose levels. In severe cases, pancreas or Langerhans islet transplantation can assist in reaching a sufficient β-mass to normalize glucose homeostasis. The latter procedure is limited because of low donor availability, high islet loss, and immune rejection. There is still a need to develop new technologies to improve islet survival and implantation and to keep the islets functional. Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells with high plasticity that can support human pancreatic islet function both in vitro and in vivo and islet co-transplantation with MSCs is more effective than islet transplantation alone in attenuating diabetes progression. The beneficial effect of MSCs on islet function is due to a combined effect on angiogenesis, suppression of immune responses, and secretion of growth factors essential for islet survival and function. In this review, various aspects of MSCs related to islet function and diabetes are described.

Improved human islets’ viability and functionality with mesenchymal stem cells and arg-gly-asp tripeptides supplementation of alginate micro-encapsulated islets in vitro

Background and aim.Transplantation of islets of porcine is a promising technique for treating insulin-dependent diabetes mellitus (type I). Instant blood-mediated inflammatory reaction (IBMIR) causes rapid islet loss in portal vein islet transplantation. Since it has been all known endothelial progenitor cells (EPCs) can aid neovascularization and protect against complement mediated lysis and activation of coagulation. Mesenchymal stem cells (MSCs) can enhace the viability of NICCs and modulate immune response. Thus we'd like to know when transplantation of the NICC coated with EPCs and MSCs would aggravate the IBMIR or alleviate it ? Research Design and methods. Porcine islets were coated with P2-P4 human umbilicl cord derived MSCs and human cord blood derived EPCs in specially modified culture medium composed of EGM2, porcine serum, IBMX and nicotinamide for 5 days. The capacity of EPCs and MSCs adhere to and grow into porcine islets was analyzed by fluorescence microscopy and confocal scanning microscopy with different cell tracers. The survival and functionality of these composite islets were evaluated by AO/EB and static assay. A negative control group, NICC alone group, and the composite islets group were examined with an in vitro tubing loop assay using human blood. Platelet consumption and complement and coagulation activation were assessed by platelet count, C3a, and thrombin-antithrombin complex (TAT), respectively. Results. MSCs can improve the EPCs coverage compared with EPC islets alone. From the first two days the coverage area was increased rapidly and the majority of NICC can be covered more than 80%. Then they were almost no change at all. Islet survival in vitro and the functionality of the composite islets after culture were almost the same to those of control islets. After incubation with human blood, the composite islets group showed platelet consumption inhibition and low C3a and TAT assay results compared to uncoated controls. Conclusions. Compared to the uncoated controls, the MSCs and EPCs coated NICC could alleviate the IBMIR. And this may because the MSCs can help the EPCs covering the NICC then reduce the exposure of NICC to the human blood. These findings might provide directly applicable tool to enhance the efficacy of islet transplantation in clinical practice.

Transplantation of human islets is an attractive alternative to daily insulin injections for patients with type 1 diabetes. However, the majority of islet recipients lose graft function within five years. Inflammation is a primary contributor to graft loss, and inhibiting pro-inflammatory cytokine activity can reverse inflammation mediated dysfunction of islet grafts. As mesenchymal stem cells (MSCs) possess numerous immunoregulatory properties, we hypothesized that MSCs could protect human islets from pro-inflammatory cytokines. Five hundred human islets were co-cultured with 0.5 or 1.0×106 human MSCs derived from bone marrow or pancreas for 24 hours followed by 48 hour exposure to interferon-γ, tumor necrosis factor-α and interleukin 1β. Controls include islets cultured alone (± cytokines) and with human dermal fibroblasts (± cytokines). For all conditions, glucose stimulated insulin secretion (GSIS), total islet cellular insulin content, islet β cell apoptosis, and potential cytoprotective factors secreted in the culture media were determined. Cytokine exposure disrupted human islet GSIS based on stimulation index and percentage insulin secretion. Conversely, culture with 1.0×106 bMSCs preserved GSIS from cytokine treated islets. Protective effects were not observed with fibroblasts, indicating that preservation of human islet GSIS after exposure to pro-inflammatory cytokines is MSC dependent. Islet β cell apoptosis was observed in the presence of cytokines; however, culture of bMSCs with islets prevented β cell apoptosis after cytokine treatment. Hepatocyte growth factor (HGF) as well as matrix metalloproteinases 2 and 9 were also identified as putative secreted cytoprotective factors; however, other secreted factors likely play a role in protection. This study, therefore, demonstrates that MSCs may be beneficial for islet engraftment by promoting cell survival and reduced inflammation.