The effect of hydroxyethyl starch as a cryopreservation agent during freezing of mouse pancreatic islets.

Islet transplantation is the most effective treatment strategy for type 1 diabetes. Long-term storage at ultralow temperatures can be used to prepare sufficient islets of good quality for transplantation. For freezing islets, dimethyl sulfoxide (DMSO) is a commonly used penetrating cryoprotective agent (CPA). However, the toxicity of DMSO is a major obstacle to cell cryopreservation. Hydroxyethyl starch (HES) has been proposed as an alternative CPA. To investigate the effects of two types of nonpermeating CPA, we compared 4 % HES 130 and HES 200 to 10 % DMSO in terms of mouse islet yield, viability, and glucose-stimulated insulin secretion (GSIS). After one day of culture, islets were cryopreserved in each solution. After three days of cryopreservation, islet recovery was significantly higher in the HES 130 and HES 200 groups than in the DMSO group. Islet viability in the HES 200 group was also significantly higher than that in the DMSO group on Day 1 and Day 3. Stimulation indices determined by GSIS were higher in the HES 130 and 200 groups than in the DMSO group on Day 3. After three days of cryopreservation, HES 130 and HES 200 both reduced the expression of apoptosis- and necrosis-associated proteins and promoted the survival of islets. In conclusion, the use of HES as a CPA improved the survival and insulin secretion of cryopreserved islets compared with the use of a conventional CPA.

Magnetic resonance imaging of pancreatic islets using tissue-adhesive particles containing iron oxide nanoparticles.

Post-transplantation tracking of pancreatic islets is a prerequisite for advancing cell therapy to treat type 1 diabetes. Magnetic resonance imaging (MRI) has emerged as a safe and non-invasive technique for visualizing cells in clinical applications. In this study, we proposed a novel MRI contrast agent formulation by encapsulating iron oxide nanoparticles (IONPs) in poly(lactic-co-glycolic acid) (PLGA) particles functionalized with a tissue adhesive polydopamine (PD) layer (IONP-PLGA-PD MS). Intriguingly, our particles facilitated efficient and robust labeling through a one-step process, allowing for the incorporation of a substantial amount of IONPs without detrimental impacts on the viability and functionality of pancreatic islets. The MRI signals emanating from islets labeled using our particles were found to be stable over 30 days in vitro and 60 days when transplanted under kidney capsules of diabetic mice. These results suggest that our approach provides a potential platform for monitoring the fate of pancreatic islets after transplantation.

DAMP-modulating nanoparticle for successful pancreatic islet and stem cell transplantation.

Cell therapy is targeted at many organs, but locally or systemically delivered cells are shortly able to survive resulting from the immune/inflammation reactions and irregular cell targeting. Here we explore the multimodal nanoparticle having anti-inflammation and magnetic guidance for successful cell transplantation. We design magnetic resonance (MR)-active glycyrrhizin-chitosan coated superparamagnetic iron oxide nanoparticle (SPIO@Chitosan-GL) to inhibit release of inflammatory damage-associated molecular pattern (DAMP) protein and to offer noninvasive monitoring after intrahepatic transplantation of pancreatic islets and mesenchymal stem cell (MSC) spheroids. Intracellular delivered SPIO@Chitosan-GL is not cytotoxic to pancreatic islets and MSC spheroids and attenuate DAMP release from them. Also, therapeutic cells labeled with SPIO@Chitosan-GL are magnetically localized to the intended lobe of liver during transplantation procedure. If necessary, partial hepatectomy can be performed to remove the localized therapeutic cells for protection of the remaining liver lobes from systemic inflammation. Therapeutically, the cells selectively localized in the liver can treat blood glucose in diabetic mice to normal levels with DAMP modulation, and are visualized using in vivo MR imaging for over 4 weeks. Collectively, DAMP-modulating SPIO@Chitosan-GL can be used in multimodal nanomedince for attenuating the inflammation reaction by transplanted cells and for noninvasively long-term monitoring of transplanted cells.

Optimal allogeneic islet dose for transplantation in insulin-dependent diabetic Macaca fascicularis monkeys.

Many groups are working to improve the results of clinical allogeneic islet transplantation in a primate model. However, few studies have focused on the optimal islet dose for achieving normal glycemia without exogenous insulin after transplantation in primate models or on the relationship between rejection and islet amyloid polypeptide (IAPP) expression. We evaluated the dose (10,000, 20,000, and > 25,000 islet equivalents (IEQ)/kg) needed to achieve normal glycemia without exogenous insulin after transplantation using eleven cynomolgus monkeys, and we analyzed the characteristics exhibited in the islets after transplantation. 10,000 IEQ/kg (N = 2) failed to control blood glucose level, despite injection with the highest dose of exogenous insulin, and 20,000 IEQ/kg group (N = 5) achieved unstable control, with a high insulin requirement. However, 25,000 IEQ/kg (N = 4) achieved normal glycemia without exogenous insulin and maintained it for more than 60 days. Immunohistochemistry results from staining islets found in liver biopsies indicated that as the number of transplanted islets decreased, the amount of IAPP accumulation within the islets increased, which accelerated CD3+ T cell infiltration. In conclusion, the optimal transplantation dose for achieving a normal glycemia without exogenous insulin in our cynomolgus monkey model was > 25,000 IEQ/kg, and the accumulation of IAPP early after transplantation, which depends on the transplanted islet dose, can be considered one factor in rejection.

Protective effect of a novel clinical-grade small molecule necrosis inhibitor against oxidative stress and inflammation during islet transplantation.

Inhibition of mitochondrial reactive oxygen species (ROS) and subsequent damage-associated molecular patterns (DAMPs)-induced inflammatory responses could be a novel target in clinical islet transplantation. We investigated the protective effects of NecroX-7, a novel clinical-grade necrosis inhibitor that specifically targets mitochondrial ROS, against primary islet graft failure. Islets from heterozygote human islet amyloid polypeptide transgenic (hIAPP+/- ) mice and nonhuman primates (NHPs) were isolated or cultured with or without NecroX-7 in serum-deprived medium. Supplementation with NecroX-7 during hIAPP+/- mouse islet isolation markedly increased islet viability and adenosine triphosphate content, and attenuated ROS, transcription of c-Jun N-terminal kinases, high mobility group box 1, interleukin-1beta (IL-1 ß ), IL-6, and tumor necrosis factor-alpha. Supplementation of NecroX-7 during serum-deprived culture also protected hIAPP+/- mouse and NHP islets against impaired viability, serum deprivation-induced ROS, proinflammatory response, and accumulation of toxic IAPP oligomer. Supplementation with NecroX-7 during isolation or serum-deprived culture of hIAPP+/- mouse and NHP islets also improved posttransplant glycemia in the recipient streptozotocin-induced diabetic hIAPP-/- mice and BALB/c-nu/nu mice, respectively. In conclusion, pretransplant administration of NecroX-7 during islet isolation and serum-deprived culture suppressed mitochondrial ROS injury, generation of DAMPs-induced proinflammatory responses, and accumulation of toxic IAPP oligomers ex vivo, and improved posttransplant glycemia in vivo.

Integrated whole liver histologic analysis of the allogeneic islet distribution and characteristics in a nonhuman primate model.

The most obvious method to observe transplanted islets in the liver is direct biopsy, but the distribution and location of the best biopsy site in the recipient's liver are poorly understood. Islets transplanted into the whole liver of five diabetic cynomolgus monkeys that underwent insulin-independent survival for an extended period of time after allo-islet transplantation were analyzed for characteristics and distribution tendency. The liver was divided into segments (S1-S8), and immunohistochemistry analysis was performed to estimate the diameter, beta cell area, and islet location. Islets were more distributed in S2 depending on tissue size; however, the number of islets per tissue size was high in S1 and S8. Statistical analysis revealed that the characteristics of islets in S1 and S8 were relatively similar to other segments despite various transplanted islet dosages and survival times. In conclusion, S1, which exhibited high islet density and reflected the overall characteristics of transplanted islets, can be considered to be a reasonable candidate for a liver biopsy site in this monkey model. The findings obtained from the five monkey livers with similar anatomical features to human liver can be used as a reference for monitoring transplanted islets after clinical islet transplantation.

Multi-layer surface modification of pancreatic islets for magnetic resonance imaging using ferumoxytol.

Ferumoxytol is the only clinically available ultrasmall superparamagnetic iron oxide. However, the labeling efficacy of islet magnetic resonance imaging (MRI) using ferumoxytol is not suitable for use in clinical pancreatic islet transplantation (PIT). We evaluated the feasibility of pancreatic islet MRI using ferumoxytol through multi-layer surface modification. A four-layer nanoshield with poly (ethylene) glycol (PEG, 2 layers), ferumoxytol, and heparin was formed on the pancreatic islets. We compared pancreatic islet function, viability, and labeling efficacy of control, ferumoxytol alone-labeled, heparin-PEGylated, and ferumoxytol-heparin-PEGylated islets. With optimization of the ferumoxytol concentration during the ferumoxytol-heparin-PEGylation process, the labeling contrast in ex vivo MRI of ferumoxytol-heparin-PEGylated pancreatic islets was stronger than that of pancreatic islets labeled with ferumoxytol alone, without decreasing ex vivo islet viability or function. In a syngeneic mouse renal subcapsular PIT model, heparin-PEGylation and ferumoxytol-heparin-PEGylation delayed the revascularization of pancreatic islet grafts but did not impair glucose tolerance or revascularization of pancreatic islet grafts four weeks post-transplantation. Pancreatic islet visibility after labeling was also confirmed in a syngeneic mouse intraportal PIT model and in preliminary analysis of a non-human primate intraportal PIT model. In conclusion, multi-layer islet surface modification is a promising option for pancreatic islet MRI in intraportal PIT.

Polymeric nano-shielded islets with heparin-polyethylene glycol in a non-human primate model.

Intraportal pancreatic islet transplantation incurs huge cell losses during its early stages due to instant blood-mediated inflammatory reactions (IBMIRs), which may also drive regulation of the adaptive immune system. Therefore, a method that evades IBMIR will improve clinical islet transplantation. We used a layer-by-layer approach to shield non-human primate (NHP) islets with polyethylene glycol (nano-shielded islets, NSIs) and polyethylene glycol plus heparin (heparin nano-shielded islets; HNSIs). Islets ranging from 10,000 to 20,000 IEQ/kg body weight were transplanted into 19 cynomolgus monkeys (n = 4, control; n = 5, NSI; and n = 10, HNSI). The mean C-peptide positive graft survival times were 68.5, 64 and 108 days for the control, NSI and HNSI groups, respectively (P = 0.012). HNSI also reduced the factors responsible for IBMIR in vitro. Based on these data, HNSIs in conjunction with clinically established immunosuppressive drug regimens will result in superior outcomes compared to those achieved with the current protocol for clinical islet transplantation.

Highly Angiogenic, Nonthrombogenic Bone Marrow Mononuclear Cell-Derived Spheroids in Intraportal Islet Transplantation.

Highly angiogenic bone marrow mononuclear cell-derived spheroids (BM-spheroids), formed by selective proliferation of the CD31+CD14+CD34+ monocyte subset via three-dimensional (3D) culture, have had robust angiogenetic capacity in rodent syngeneic renal subcapsular islet transplantation. We wondered whether the efficacy of BM-spheroids could be demonstrated in clinically relevant intraportal islet transplantation models without increasing the risk of portal thrombosis. The thrombogenic potential of intraportally infused BM-spheroids was compared with that of mesenchymal stem cells (MSCs) and MSC-derived spheroids (MSC-spheroids). The angiogenic efficacy and persistence in portal sinusoids of BM-spheroids were examined in rodent syngeneic and primate allogeneic intraportal islet transplantation models. In contrast to MSCs and MSC-spheroids, intraportal infusion of BM-spheroids did not evoke portal thrombosis. BM-spheroids had robust angiogenetic capacity in both the rodent and primate intraportal islet transplantation models and improved posttransplant glycemic outcomes. MRI and intravital microscopy findings revealed the persistence of intraportally infused BM-spheroids in portal sinusoids. Intraportal cotransplantation of allogeneic islets with autologous BM-spheroids in nonhuman primates further confirmed the clinical feasibility of this approach. In conclusion, cotransplantation of BM-spheroids enhances intraportal islet transplantation outcome without portal thrombosis in mice and nonhuman primates. Generating BM-spheroids by 3D culture prevented the rapid migration and disappearance of intraportally infused therapeutic cells.

Role of Human CD200 Overexpression in Pig-to-Human Xenogeneic Immune Response Compared With Human CD47 Overexpression.

BACKGROUND: Macrophages play important roles in xenograft rejection. Here, we investigated whether overexpression of human CD200 or CD47 in porcine endothelial cells (PEC) can suppress macrophages activation in xenogeneic immune responses. METHODS: PECs and human macrophages were incubated together, harvested, and analyzed for in vitro macrophage phagocytic and cytotoxicity activity, and cytokine release. Next, PECs were injected into renal subcapsular space of humanized mice. On day 10 posttransplantation, we analyzed xenograft survival and perigraft inflammatory cell infiltrations in PEC-to-humanized mouse transplantation. RESULTS: PECs highly expressing human CD200, CD47, or both CD47/CD200 were established by lentiviral vector transduction. Both CD200 and CD47 suppressed in vitro macrophage phagocytic and cytotoxic activity against PECs; decreased TNF-α, IL-1ß, and IL-6 secretion; and increased IL-10 secretion. However, simultaneous overexpression of CD200 and CD47 did not show additive effects. Next, PECs were transplanted into NOD-scid IL-2Rg null mice, and human monocytes and lymphocytes were adoptively transferred 1 day after xenotransplantation. PEC xenograft cell death and apoptosis were decreased in the CD200-PEC and CD47/CD200-PEC groups. Perigraft infiltration of human T cells was suppressed by CD47; CD200 suppressed infiltration of human macrophages to a greater extent than CD47; and the CD47/CD200-PEC group exhibited the lowest level of leukocyte infiltration. In summary, overexpression of CD200 in PECs suppressed xenogeneic activation of human macrophages and improved survival of PEC xenografts in humanized mice; however, coexpression of CD200 and CD47 did not show additive effects. CONCLUSIONS: Therefore, overexpression of human CD200 in donor pigs could constitute a promising strategy for overcoming xenograft rejection.

Enhanced effect of human mesenchymal stem cells expressing human TNF-αR-Fc and HO-1 gene on porcine islet xenotransplantation in humanized mice.

BACKGROUND: Porcine islet xenotransplantation is considered an attractive alternative treatment for type 1 diabetes mellitus. However, it is largely limited because of initial rejection due to Instant Blood-Mediated Inflammatory Reaction (IBMIR), oxidative stress, and inflammatory responses. Recently, soluble tumor necrosis factor-ɑ receptor type I (sTNF-αR) and heme oxygenase (HO)-1 genes (HO-1/sTNF-αR) have been shown to improve the viability and functionality of porcine islets after transplantation. METHODS: In this study, genetically modified mesenchymal stem cells (MSCs) expressing the HO-1/sTNF-αR genes (HO-1/sTNF-αR-MSC) were developed using an adenoviral system, and porcine islet viability and function were confirmed by in vitro tests such as GSIS, AO/PI, and the ADP/ATP ratio after coculturing with HO-1/sTNF-αR-MSCs. Subsequently, isolated porcine islets were transplanted underneath the kidney capsule of diabetic humanized mice without MSCs, with MSCs or with HO-1/sTNF-αR-MSCs. RESULTS: According to the results, the HO-1/sTNF-αR-MSC-treated group exhibited improved survival of porcine islets and could reverse hyperglycemia more than porcine islets not treated with MSCs or islets cotransplanted with MSCs. Moreover, the HO-1/sTNF-αR-MSC group maintained its morphological characteristics and the insulin secretion pattern of transplanted porcine islets similar to endogenous islets in immunocompetent humanized mice. CONCLUSIONS: Our results suggest that HO-1/sTNF-αR-MSCs are efficient tools for porcine islet xenotransplantation, and this study may provide basic information for pre-clinical animal models and future clinical trials of porcine islet xenotransplantation.

Xenotransplantation of layer-by-layer encapsulated non-human primate islets with a specified immunosuppressive drug protocol.

Islet transplantation is as effective as but also less immunogenic than pancreas transplantation for the treatment of type 1 diabetes mellitus. However, as the complete elimination of immunogenicity still remains a major obstacle in islet transplantation, layer-by-layer encapsulation (LbL) of pancreatic islets using biocompatible polymers offers a rational approach to reducing host immune response towards transplanted islets. We investigated the effect of LbL of non-human primate (NHP) islets on reducing immunogenicity as a preclinical model since NHPs have close phylogenetic and immunological relationship with humans. LbL with three-layers of polyethylene glycol (PEG) molecules (SH-6-arm-PEG-NHS, 6-arm-PEG-catechol and linear PEG-SH) showed a uniform nano-shielding on islets without the loss of viability or function of islets. An immunosuppressive drug protocol was also combined to improve the survival rate of the transplanted islets in vivo. A xenorecipient (C57BL/6 mice) of LbL islet transplanted along with our immunosuppressive drug protocol showed 100% survival rate for 150days after transplantation. On the other hand, naked islet recipients showed poor survival time of 5.5±1.4days without drugs and 77.5±42days with the drug protocol. Immunohistochemistry of the transplanted grafts and serum cytokine concentration demonstrated less immunogenicity in the LbL islet transplanted recipients compared with the naked islet ones.

Beneficial effects of the transgenic expression of human sTNF-αR-Fc and HO-1 on pig-to-mouse islet xenograft survival.

Both human soluble tumor necrosis factor-α receptor-Fc (sTNF-αR-Fc) and heme oxygenase-1 (HO-1) transgenic pigs have been generated previously for xenotransplantation. Here, we investigated whether overexpression of sTNF-αR-Fc or HO-1 in pig islets prolongs islet xenograft survival. Adult porcine islets were isolated from human sTNF-αR-Fc or HO-1 transgenic and wild type pigs, and were transplanted into diabetic nude mice. Effects of the expression of both genes on islet apoptosis, chemokine expression, cellular infiltration, antibody production, and islet xenograft survival were analyzed. Human sTNF-αR-Fc transgenic pigs successfully expressed sTNF-αR-Fc in the islets; human HO-1 transgenic pigs expressed significant levels of HO-1 in the islets. Pig-to-mouse islet xenograft survival was significantly prolonged in both the sTNF-αR-Fc and HO-1 groups compared with that in the wild type group. Both the sTNF-αR-Fc and HO-1 groups exhibited suppressed intragraft expression of monocyte chemoattractant protein-1 (MCP-1) and decreased perigraft infiltration of immune cells. However, there was no difference in the anti-pig antibody levels between the groups. Apoptosis of islet cells during the early engraftment was suppressed only in the HO-1 group. Porcine islets from both sTNF-αR-Fc and HO-1 transgenic pigs prolonged xenograft survival by suppressing islet cell apoptosis or secondary inflammatory responses following islet death, indicating that these transgenic pigs might have applications in successful islet xenotransplantation.

Coagulation abnormalities in deceased donors are associated with unsuccessful human islet cell isolation.

An equivalent islet number (EIN) greater than 300,000 is necessary for islet cell transplantation for a recipient who weighs about 60 kg. The aim of this study is to identify factors that affect isolation outcome. The most significant independent predictor for successful islet isolation from deceased donors was low international normalized ratio (INR).

Characterization, in vitro cytotoxicity assessment, and in vivo visualization of multimodal, RITC-labeled, silica-coated magnetic nanoparticles for labeling human cord blood-derived mesenchymal stem cells.

Live imaging is a powerful technique that can be used to characterize the fate and location of stem cells in animal models. Here we investigated the characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, MNPs@SiO2(RITC). We also conducted various in vivo-uptake tests with nanoparticle-labeled human MSCs. MNPs@SiO2(RITC) showed photostability against ultraviolet light exposure and were nontoxic to human MSCs, based on the MTT, apoptosis, and cell cycle arrest assays. In addition, MNPs@SiO2(RITC) did not affect the surface phenotype or morphology of human MSCs. We also demonstrated that MNPs@SiO2(RITC) have stable retention properties in MSCs in vitro. Furthermore, using optical and magnetic resonance imaging, we successfully detected a visible signal from labeled human MSCs that were transplanted into NOD.CB17-Prkdc(SCID) (NOD-SCID) mice. These results demonstrate that MNPs@SiO2(RITC) are biocompatible and useful tools for human MSC labeling and bioimaging. FROM THE CLINICAL EDITOR: The characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, RITC were investigated in this study. RITC showed photostability against ultraviolet light exposure and was nontoxic to human MSCs. Using both optical and magnetic resonance imaging, successful detection of signal from labeled human MSCs transplanted into mice is demonstrated.