Exosomes derived from human mesenchymal stem cells preserve mouse islet survival and insulin secretion function (original) (raw)
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Journal of Cellular Physiology, 2019
Type 1 diabetes, has been recognized as an autoimmune disease. Like other immunological conditions, regulation of immune response is a key strategy to control the autoimmunity in diabetic patients. Mesenchymal stem cells have been shown to have a distinct potential in modulating the immune reactions. However, treatment with stem cells is combined with concerns about safety issues. To overcome these concerns, in this study, we have utilized the regenerative potential of exosomes isolated from menstrual blood-derived mesenchymal stem cells to restore the β-cell mass and insulin production in type 1 diabetes. Exosomes are nanovesicles containing various cargos involved in cellular communications. Streptozotocin was used to induce islet destruction and diabetes in male Wistar rats. Then, exosomes were intravenously injected into animals at different time points and in a single or repeated therapeutic doses. After about 6 weeks, animals were euthanized and the pancreas was analyzed for the presence of the regenerated β islets as well as the insulin secretion. The non-fasting blood glucose and the serum insulin level were also monitored during the study. Our results represented that menstrual blood-derived mesenchymal stem cell-derived exosomes enhance the β-cell mass and insulin production in the pancreas of diabetic animals that received repeated doses of exosomes. Immunohistochemistry analysis also confirmed the presence of insulin in the islets of treated animals. Further investigations proposed that exosomes induce the islet regeneration through pancreatic and duodenal homeobox 1 pathway. The exosome tracking also revealed the homing of injected exosomes to the pancreas. K E Y W O R D S β cell, exosome, menstrual blood, mesenchymal stem cell, pancreas, type 1 diabetes 1 | INTRODUCTION Type 1 diabetes is an autoimmune disease in which the immunemediated loss of β-cell mass results in a long-life insulin dependence. Therefore, any therapeutic, which is capable to overcome autoimmunity and to regenerate the damaged β cells, can be the ultimate goal and a cure for this type of diabetes. Mesenchymal stem cells (MSCs) encompass great regenerative potentials. These cells are characterized by their capacity to self-renew and to differentiate into other mesenchymal tissues such as bone, fat, and cartilage (Dominici et al., 2006). MSCs have a tropism towards inflammation and injured tissues (Bernardo & Fibbe, 2013; Ji, He, Dheen, & Tay, 2004). Once there they are capable to regulate the immune response and help to restore the damage (Bernardo & Fibbe, 2013). There are two possible mechanisms by which MSCs can promote tissue repair. First, through direct differentiation into cell types required for the tissue restoration and
Stem cell-derived exosomes: a novel vector for tissue repair and diabetic therapy
Journal of Molecular Endocrinology, 2017
Exosomes are extracellular vesicles (EVs) secreted from a majority of cell types. Exosomes play a role in healthy and pathogenic intercellular interactions via the transfer of proteins, lipids and RNA. The contents and effects of exosomes vary depending on the properties of the originating cell. Exosomes secreted from some cell types, including stem cells, carry biological factors implicated in the protection, regeneration and angiogenesis of damaged tissues. Due to these properties, exosomes have attracted attention as a novel vector for regenerative therapies. Exosomes as a therapeutic tool could have applications for the treatment of many disorders characterized by chronic tissue damage. Exosomes derived from stem cells could be applied to repair or prevent damage from the complications of diabetes mellitus. The immunomodulatory and reparative properties of stem cell-derived exosomes could protect or even restore an early-stage type 1 diabetic patient’s original islets from autoi...
Stem Cells International
It is unclear whether extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) have a direct protective effect on pancreatic islets. In addition, whether culturing MSCs in three dimensions (3D) instead of a monolayer (2D) can induce changes in the cargo of EVs that facilitate the polarization of macrophages into an M2 phenotype has not been investigated. We sought to determine whether EVs from MSCs cultured in 3D can prevent inflammation and dedifferentiation in pancreatic islets and, if so, whether the protective effect is superior to that of EVs from 2D MSCs. Human umbilical cord blood- (hUCB-) MSCs cultured in 3D were optimized according to cell density, exposure to hypoxia, and cytokine treatment based on the ability of the hUCB-MSC-derived EVs to induce the M2 polarization of macrophages. Islets isolated from human islet amyloid polypeptide (hIAPP) heterozygote transgenic mice were cultured in serum-deprived conditions with hUCB-MSC-derived EVs. EVs derived from 3D hUCB-...
PLoS ONE, 2014
The cross-talk between beta cells and endothelium plays a key role in islet physiopathology and in the revascularization process after islet transplantation. However, the molecular mechanisms involved in this cross-talk are not fully elucidated. Extracellular vesicles (EVs) are secreted membrane nanoparticles involved in inter-cellular communication through the transfer of proteins and nucleic acids. The aims of this study were: 1) isolation and characterization of EVs from human islets; 2) evaluation of the pro-angiogenic effect of islet-derived EVs on human islet endothelial cells (IECs). EVs were isolated by ultracentrifugation from conditioned medium of human islets and characterized by nanotrack analysis (Nanosight), FACS, western blot, bioanalyzer, mRNA/microRNA RT-PCR array. On IECs, we evaluated EV-induced insulin mRNA transfer, proliferation, resistance to apoptosis, in vitro angiogenesis, migration, gene and protein profiling. EVs sized 236654 nm, expressed different surface molecules and islet-specific proteins (insulin, C-peptide, GLP1R) and carried several mRNAs (VEGFa, eNOS) and microRNAs (miR-27b, miR-126, miR-130 and miR-296) involved in beta cell function, insulin secretion and angiogenesis. Purified EVs were internalized into IECs inducing insulin mRNA expression, protection from apoptosis and enhancement of angiogenesis. Human islets release biologically active EVs able to shuttle specific mRNAs and microRNAs (miRNAs) into target endothelial cells. These results suggest a putative role for islet-derived EVs in beta cell-endothelium cross-talk and in the neoangiogenesis process which is critical for engraftment of transplanted islets.
Human Mesenchymal Stem Cells Protect Human Islets from Pro-Inflammatory Cytokines
PLoS ONE, 2012
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 1.0610 6 human MSCs derived from bone marrow or pancreas for 24 hours followed by 48 hour exposure to interferon-c, tumor necrosis factor-a and interleukin 1b. Controls include islets cultured alone (6 cytokines) and with human dermal fibroblasts (6 cytokines). For all conditions, glucose stimulated insulin secretion (GSIS), total islet cellular insulin content, islet b 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 1.0610 6 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 b cell apoptosis was observed in the presence of cytokines; however, culture of bMSCs with islets prevented b 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.
Stem Cells and Development, 2021
The secretome of mesenchymal stromal cells (MSCs) is enriched for biotherapeutic effectors contained within and independent of extracellular vesicles (EVs) that may support tissue regeneration as an injectable agent. We have demonstrated that the intrapancreatic injection of concentrated conditioned media (CM) produced by bone marrow MSC supports islet regeneration and restored glycemic control in hyperglycemic mice, ultimately providing a platform to elucidate components of the MSC secretome. Herein, we extend these findings using human pancreas-derived MSC (Panc-MSC) as “biofactories” to enrich for tissue regenerative stimuli housed within distinct compartments of the secretome. Specifically, we utilized 100 kDa ultrafiltration as a simple method to debulk protein mass and to enrich for EVs while concentrating the MSC secretome into an injectable volume for preclinical assessments in murine models of blood vessel and islet regeneration. EV enrichment (EV+) was validated using nanoscale flow cytometry and atomic force microscopy, in addition to the detection of classical EV markers CD9, CD81, and CD63 using label-free mass spectrometry. EV+ CM was predominately enriched with mediators of wound healing and epithelial-to-mesenchymal transition that supported functional regeneration in mesenchymal and nonmesenchymal tissues. For example, EV+ CM supported human microvascular endothelial cell tubule formation in vitro and enhanced the recovery of blood perfusion following intramuscular injection in nonobese diabetic/severe combined immunodeficiency mice with unilateral hind limb ischemia. Furthermore, EV+ CM increased islet number and β cell mass, elevated circulating insulin, and improved glycemic control following intrapancreatic injection in streptozotocin-treated mice. Collectively, this study provides foundational evidence that Panc-MSC, readily propagated from the subculture of human islets, may be utilized for regenerative medicine applications.
Human mesenchymal stem cells were exposed to diabetic sera for 7 days. Cell viability and apoptosis rate were detected by MTT and flow cytometry assays. The expression of key genes such as CD63, Alix, Rab27a, Rab27b, and Rab8b was monitored by real-time PCR. We also measured acetylcholinesterase activity and size and zeta potential of exosomes in the supernatant form diabetic cells and control. The cellular distribution of CD63 was shown by immunofluorescence imaging and western blotting. Any changes in the ultrastructure of cells were visualized by electron microscopy. Data showed a slight decrease in survival rate and an increased apoptosis in diabetic cells as compared to control (p < 0.05). By exposing cells to diabetic sera, a significant increase in the level of all genes CD63, Alix, Rab27a, Rab27b, and Rab8b was observed (p < 0.05). Flow cytometry analysis and immunofluorescence imaging confirmed increasing CD63 protein content upon treatment with diabetic sera (p < 0.05). We found an enhanced acetylcholinesterase activity in a diabetic condition which coincided with the increasing size of exosomes and decrease in zeta potential (p < 0.05). The fatty acid profile was not significantly affected by diabetic sera. Ultrastructural examination detected more accumulated cytoplasmic lipid vacuoles in diabetic cells.
Exosomes Released by Islet-Derived Mesenchymal Stem Cells Trigger Autoimmune Responses in NOD Mice
Diabetes, 2013
Exosomes (EXOs) are secreted, nano-sized membrane vesicles that contain potent immunostimulatory materials. We have recently demonstrated that insulinoma-released EXOs can stimulate the autoimmune responses in nonobese diabetic (NOD) mice, a spontaneous disease model for type 1 diabetes. To investigate whether primary islet cells can produce EXOs, we isolated cells from the islet of Langerhans of NOD mice and cultured them in vitro. Interestingly, cultured islets release fibroblast-like, fast-replicating cells that express mesenchymal stem cell (MSC) markers, including CD105 and stem-cell antigen-1. These islet MSC–like cells release highly immunostimulatory EXOs that could activate autoreactive B and T cells endogenously primed in NOD mice. Serum EXO levels and EXO-induced interferon-γ production were positively correlated with disease progression at the early prediabetic stage. Consistent with these observations, immunohistological analysis of pancreata showed that CD105+ cells ar...
A Double Mechanism for the Mesenchymal Stem Cells' Positive Effect on Pancreatic Islets
PLoS ONE, 2014
The clinical usability of pancreatic islet transplantation for the treatment of type I diabetes, despite some encouraging results, is currently hampered by the short lifespan of the transplanted tissue. In vivo studies have demonstrated that cotransplantation of Mesenchymal Stem Cells (MSCs) with transplanted pancreatic islets is more effective with respect to pancreatic islets alone in ensuring glycemia control in diabetic rats, but the molecular mechanisms of this action are still unclear. The aim of this study was to elucidate the molecular mechanisms of the positive effect of MSCs on pancreatic islet functionality by setting up direct, indirect and mixed co-cultures. MSCs were both able to prolong the survival of pancreatic islets, and to directly differentiate into an ''insulin-releasing'' phenotype. Two distinct mechanisms mediated these effects: i) the survival increase was observed in pancreatic islets indirectly co-cultured with MSCs, probably mediated by the trophic factors released by MSCs; ii) MSCs in direct contact with pancreatic islets started to express Pdx1, a pivotal gene of insulin production, and then differentiated into insulin releasing cells. These results demonstrate that MSCs may be useful for potentiating pancreatic islets' functionality and feasibility.