Cell-based treatments for diabetes

In Type 1 diabetes mellitus the insulin-secreting beta-cells in pancreatic islets of Langerhans are selectively destroyed by autoimmune assault. Because diabetes is caused by the loss of a single cell type it is amenable to treatment by cell replacement therapy. Advances in islet transplantation procedures have demonstrated that people with Type 1 diabetes can be cured by human islet transplantation, but the severely limited availability of donor islets has restricted the widespread application of this approach, and driven the search for substitute transplant tissues. Recent experimental studies suggest that three separate sources of tissue show therapeutic potential - xenografts from other species, tissue stem cells and embryonic stem cells. Of these, xenografts are closest to clinical application but there are still major obstacles to be overcome. Insulin-expressing cells have been derived from a number of different stem cell populations but embryonic stem cells offer the major advantage of being able, in principle, to provide the vast numbers of cells required for transplantation therapy.     

Radionuclide probes for molecular imaging of pancreatic beta-cells

Islet transplantation is a promising treatment option for patients with type 1 diabetes (T1D); however, the fate of the graft over time remains difficult to follow, due to the lack of available tools capable of monitoring graft rejection and inflammation prior to islet graft loss. Due to the challenges imposed by the location of the pancreas and the sparsely dispersed beta-cell population within the pancreas, currently, the clinical verification of beta-cell abnormalities can only be obtained indirectly via metabolic studies, which typically is not possible until after a significant deterioration in islet function has already occurred. The development of non-invasive imaging methods for the assessment of the pancreatic beta-cells, however, offers the potential for the early detection of beta-cell dysfunction prior to the clinical onset of T1D and type 2 diabetes (T2D). Ideal islet imaging agents would have an acceptable residence time in the human body, be capable of providing high-resolution images with minimal uptake in surrounding tissues (e.g., the liver), would not be toxic to islets, and would not require pre-treatment of islets prior to transplantation. A variety of currently available imaging techniques, including magnetic resonance imaging (MRI), bioluminescence imaging (BLI), and nuclear imaging have been tested for the study of beta-cell diseases. In this article, we summarize the recent advances made in nuclear imaging techniques for non-invasive imaging of pancreatic beta-cells. The use of radioactive probes for islet imaging is also discussed.     

Ex vivo gene transfer for improvement of transplanted pancreatic islet viability and function

Human pancreatic islet transplantation has recently been shown to be successful in replacing pancreatic endocrine function into type 1 diabetic recipients. A major drawback, however, is the high amount of pancreatic ss cells required to render one single patient insulin-independent. Given the shortage of human beta cell donors, the majority of type 1 diabetic patients remain excluded from this therapeutic option. High number of islets are requested since substantial islet cell death and dysfunction occur within the first few hours and days after islet transplantation. Impaired vascularization of the engraft, the non-specific inflammatory reaction at the site of transplantation, together with the presence of active or memory autoimmune responses to islet autoantigens and allogeneic recognition contribute to apoptosis of ss cells and subsequent early graft function loss. This review will focus on ex vivo engineering of the islet graft by gene transfer to improve islet engraftment. An overview of currently used gene transfer techniques will be given and their potential will be discussed.     

Lotus (Nelumbo nucifera Gaertn) plumule polysaccharide ameliorates pancreatic islets loss and serum lipid profiles in non-obese diabetic mice

To unravel possible protective effects of a newly isolated lotus plumule polysaccharide (LPPS) on type 1 diabetes (T1D), this study isolated LPPS and administered it to non-obese diabetic (NOD) female mice for 15 weeks. Oral glucose tolerance, serum ketone body, glucose, insulin, and lipid levels, as well as pancreatic islet cell numbers and the insulin secretion ability of the experimental mice were determined. The results showed that LPPS administration in vivo significantly (P < 0.05) increased pancreatic islet cell numbers and slightly enhanced the basal insulin secretion ability compared to the control group. LPPS administration improved serum lipid profiles in the diabetic mice via relatively increasing serum high density lipoprotein-cholesterol, but decreasing low density lipoprotein-cholesterol and total cholesterol levels. The present study suggests that LPPS supplementation may ameliorate T1D progress and its complications through protecting pancreatic islets and modulating serum lipid profiles.     

The therapeutic potential of islet cell transplant in the treatment of diabetes

Diabetic patients requiring insulin treatment are at an increased risk of developing chronic complications affecting the eye, kidney and heart as well as other disabilities, such as neurological and vascular disease. The Diabetes Control and Complications Trial (DCCT) showed that the development of these complications can be profoundly reduced or even prevented by strict glycaemic control through intensive insulin therapy. The DCCT study, which was concluded in 1993, administered intensive insulin treatment either by multiple (three or four per day) insulin injections or by an external insulin pump. However, it is both stressful and difficult to achieve effective glycaemic control by these methods. Furthermore, intensive insulin administration imposes a significant danger of hypoglycaemia to patients receiving this treatment. Biological replacement of the destroyed insulin-producing B-cells of pancreatic islets, with normal functioning islet transplants, remains the best option with which to achieve strict glycaemic control in diabetic patients requiring insulin treatment. The ultimate goal in transplantation is the unlimited availability of organs/tissues to be transplanted in a simple procedure that requires little or no immunosuppression. Other obstacles have also contributed to the delay in islet transplantation becoming a clinical reality. There are now good methods for isolating long-term functional islets, and it has been proposed that these islets can be immunoisolated by microencapsulation to prevent transplant rejection, an approach that could easily lead to the use of islet xenografts in human diabetic patients. Therefore, the development of microencapsulated islets promises to solve the two major obstacles to clinical islet transplantation, transplant rejection and shortage of human islets. This technique has enormous potential as a viable treatment for diabetic patients requiring insulin therapy to achieve glycaemic control.     

The therapeutic potential of islet cell transplant in the treatment of diabetes

Diabetic patients requiring insulin treatment are at an increased risk of developing chronic complications affecting the eye, kidney and heart as well as other disabilities, such as neurological and vascular disease. The Diabetes Control and Complications Trial (DCCT) showed that the development of these complications can be profoundly reduced or even prevented by strict glycaemic control through intensive insulin therapy. The DCCT study, which was concluded in 1993, administered intensive insulin treatment either by multiple (three or four per day) insulin injections or by an external insulin pump. However, it is both stressful and difficult to achieve effective glycaemic control by these methods. Furthermore, intensive insulin administration imposes a significant danger of hypoglycaemia to patients receiving this treatment. Biological replacement of the destroyed insulin-producing B-cells of pancreatic islets, with normal functioning islet transplants, remains the best option with which to achieve strict glycaemic control in diabetic patients requiring insulin treatment. The ultimate goal in transplantation is the unlimited availability of organs/tissues to be transplanted in a simple procedure that requires little or no immunosuppression. Other obstacles have also contributed to the delay in islet transplantation becoming a clinical reality. There are now good methods for isolating long-term functional islets, and it has been proposed that these islets can be immunoisolated by microencapsulation to prevent transplant rejection, an approach that could easily lead to the use of islet xenografts in human diabetic patients. Therefore, the development of microencapsulated islets promises to solve the two major obstacles to clinical islet transplantation, transplant rejection and shortage of human islets. This technique has enormous potential as a viable treatment for diabetic patients requiring insulin therapy to achieve glycaemic control.     

Immunomodulation of cell-penetrating tat-metallothionein for successful outcome of xenotransplanted pancreatic islet

Pancreatic islet transplantation is a promising treatment for treatment of type 1 diabetes; however, transplantation outcomes have been disappointing due to early graft loss that is mediated by many immune responses. Immune cells not only directly damaged islet but also produced reactive oxygen species (ROS), which is highly toxic to islet cells. Metallothionein (MT) can provide protection against oxidative stress by scavenging various ranges of ROS including superoxide, hydroxyl radical, hydrogen peroxide and nitric oxide. For scavenging immune response-induced ROS, cell-penetrating Tat peptide-metallothionein (Tat-MT) was delivered into islets. The viability of Tat-MT-treated islets was not damaged during co-culture with macrophages or ROS-generating paraquat. When Tat-MT-treated islets were xenotransplanted, ROS production was significantly attenuated at the islets. Eventually, the survival time of Tat-MT-treated islets was significantly enhanced without any immunosuppressant medicine. Additionally, we confirmed that the survival time of Tat-MT-treated islets in all animals was dramatically improved when accompanied with low dose immunosuppressive agents (tacrolimus and anti-CD154 monoclonal antibody), indicating that Tat-MT delivery could have synergistic effect with immunosuppressants. Collectively, this new combination therapy of Tat-MT delivery with low dose immunosuppressant would be a powerful remedy for successful outcome of islet xenotransplantation.     

Beneficial effect of 17β-estradiol on hyperglycemia and islet β-cell functions in a streptozotocin-induced diabetic rat model

The modulating effect of estrogen on glucose homeostasis remains a controversial issue at present. In this study, we sought to determine the beneficial effect of 17β-estradiol (E₂) on hyperglycemia and islet β-cell functions in streptozotocin (STZ)-induced diabetic rats. Male Sprague-Dawley rats were injected i.p. with STZ to induce a relatively mild diabetic condition. The rats were then treated with E₂ orally at 500 μg/kg body weight/day for 15 days to evaluate the modulating effect on hyperglycemia, insulin secretion, and islet β-cell proliferation. E₂ administration for 10 days significantly lowered plasma glucose levels, increased plasma insulin levels, and improved glucose tolerance by attenuating insulin response to oral glucose loading. These beneficial effects of E₂ were accompanied by increases in islet number and volume, rate of islet cell proliferation, and the amount of insulin secreted. The growth-stimulatory effect of E₂ on islet cells was linked to the functions of the estrogen receptor α. Notably, these protective effects of E₂ on diabetic conditions were basically not observed when the STZ-treated rats had a more severe degree of islet damage and hyperglycemia. Taken together, we conclude that E₂ can promote the regeneration of damaged pancreatic islets by stimulating β-cell proliferation in diabetic rats, and this effect is accompanied by improvements in glucose tolerance and a decrease in plasma glucose levels. These findings suggest that oral administration of E₂ may be beneficial in diabetic patients with an accelerated loss of islet β-cells.     

Genetically modified mesenchymal stem cells for improved islet transplantation

The use of adult stem cells for therapeutic purposes has met with great success in recent years. Among several types of adult stem cells, mesenchymal stem cells (MSCs) derived from bone marrow (BM) and other sources have gained popularity for basic research and clinical applications because of their therapeutic potential in treating a variety of diseases. Because of their tissue regeneration potential and immune modulation effect, MSCs were recently used as cell-based therapy to promote revascularization, increase pancreatic β-cell proliferation, and avoid allograft rejection in islet transplantation. Taking advantage of the recent progress in gene therapy, genetically modified MSCs can further enhance and expand the therapeutic benefit of primary MSCs while retaining their stem-cell-like properties. This review aims to gain a thorough understanding of the current obstacles to successful islet transplantation and discusses the potential role of primary MSCs before or after genetic modification in islet transplantation.     

Biological and biomaterial approaches for improved islet transplantation

Islet transplantation may be used to treat type I diabetes. Despite tremendous progress in islet isolation, culture, and preservation, the clinical use of this modality of treatment is limited due to post-transplantation challenges to the islets such as the failure to revascularize and immune destruction of the islet graft. In addition, the need for lifelong strong immunosuppressing agents restricts the use of this option to a limited subset of patients, which is further restricted by the unmet need for large numbers of islets. Inadequate islet supply issues are being addressed by regeneration therapy and xenotransplantation. Various strategies are being tried to prevent beta-cell death, including immunoisolation using semipermeable biocompatible polymeric capsules and induction of immune tolerance. Genetic modification of islets promises to complement all these strategies toward the success of islet transplantation. Furthermore, synergistic application of more than one strategy is required for improving the success of islet transplantation. This review will critically address various insights developed in each individual strategy and for multipronged approaches, which will be helpful in achieving better outcomes.     

Vascular Endothelial Growth Factor Gene Delivery for Revascularization in Transplanted Human Islets

PURPOSE: Islet transplantation is limited by islet graft failure because of poor revascularization, host immune rejection, and nonspecific inflammatory response. Human vascular endothelial growth factor (hVEGF) gene delivery is likely to promote islet revascularization and survival. METHODS: We evaluated gene expression from a bicistronic plasmid encoding hVEGF and enhanced green fluorescent protein (EGFP) (pCMS-EGFP-hVEGF). Glucose responsiveness of islets was evaluated both in vitro and in vivo, and revascularization in islet graft was evaluated by immunohistochemistry. RESULTS: After transfection, hVEGF and EGFP expression levels were comparable with original monocistronic plasmids in Jurkat cells but higher and prolonged hVEGF expression in islets transfected with the bicistronic plasmid was observed, possibly as the result of differences in promoter strength and hypoxia response. The 3:1 w/w complexes showed little toxicity to islets at a dose of 5 microg DNA per 2000 islets. On glucose challenge, insulin release from transfected islets as well as secretion from islets after transplantation under the mouse kidney capsules in response to glucose stimulation, increased with time. Immunohistochemical staining of transplanted islets using mouse anti-human insulin, mouse anti-human von Willebrand factor, and rat anti-mouse CD31 antibodies suggests that islets are functional and there is new blood vessel formation. CONCLUSIONS: These findings suggest that transient hVEGF gene expression by the islets may promote islet revascularization and prolong islet survival after transplantation.     

Bioavailability of a Small Unilamellar Low-Clearance Liposomal Amikacin Formulation after Extravascular Administration

Rapid and adequate revascularization of transplanted islets is important for their survival and function during transplantation. Vascular endothelial growth factor (VEGF) could play a critical role with respect to islet revascularization. The aim of this study was to compare two strategies that are used to overexpress VEGF in β-cells: (1) gene therapy through adenoviral infection and (2) a pharmacological approach using deferoxamine (DFO). β-Cell lines from rat insulinoma (RINm5F) were either infected using an adenovirus encoding the gene of human VEGF 165 or incubated with DFO. One day after treatment, the viability of RINm5F cells was preserved with 10 μmol/L of DFO (103.95?±?5.66% toward control; n?=?4). In addition, adenoviral infection maintained the viability of cells for all the concentrations used. In both treatments, overexpression of VEGF was in a comparable level. Finally, the ratio of Bax/Bcl-2 indicated that the apoptosis increased in infected β-cells whereas treatment with DFO seems to be antiapoptotic. Our results suggest that the use of DFO could be a realistic approach to improve the vascularization of islets during transplantation.     

Novel role of curcumin combined with bone marrow transplantation in reversing experimental diabetes: Effects on pancreatic islet regeneration, oxidative stress, and inflammatory cytokines

Therapeutic utility of bone marrow transplantation in diabetes is an attractive approach. However, the oxidative stress generated by hyperglycemia may hinder β-cell regeneration. The present study was undertaken to investigate the therapeutic potential of curcumin, a dietary spice with antioxidant activity, bone marrow transplantation, and their combined effects in the reversal of experimental diabetes. Diabetes was induced in mice by multiple low doses of streptozotocin. After the onset of diabetes, mice were treated with curcumin (10 mM; 100 μl/mouse, i.p., for 28 days) or received a single bone marrow transplantation (10⁶ un-fractionated bone morrow cells), or both. Parameters of diabetes, integrity of pancreatic islets, pancreatic oxidative stress markers, and serum pro-inflammatory cytokines, were evaluated. Treatment with either curcumin or bone marrow transplantation significantly reversed streptozotocin-induced hyperglycemia/glucose intolerance, hypoinsulinemia, and damage of pancreatic islets. Interestingly, combination of curcumin and bone marrow transplantation elicited the most profound alleviation of such streptozotocin-evoked anomalies; including islet regeneration/insulin secretion. On the other hand, curcumin, either alone or combined with bone marrow transplantation, blunted the pancreatic lipid-peroxidation, up-regulated activities of the antioxidant enzymes, and suppressed serum levels of TNF-α and IL-1β. Curcumin and single bone marrow transplantation proved their therapeutic potential in reversing diabetes when used in combination. Curcumin, via its antioxidant and anti-inflammatory effects, evidently enhanced the ability of bone marrow transplantation to regenerate functional pancreatic islets. Hence, the use of natural antioxidants combined with other therapeutic regimens to induce pancreatic regeneration is a promising strategy in the management of diabetes.     

Toward a cell-based cure for diabetes: advances in production and transplant of beta cells

Type 1 diabetes results from autoimmune destruction of the insulin-producing beta cells of the pancreatic islets of Langerhans. Although developments in exogenous insulin therapy have greatly improved clinical outcomes in patients with diabetes, the ability of the pancreatic beta cell to exquisitely regulate the delivery of insulin and maintain normal levels of blood glucose is still far superior to what can be achieved by external delivery of insulin. As a result, the majority of patients with type 1 diabetes still experience the complications of chronic hyperglycemia or serious and potentially life-threatening hypoglycemia. The shortcomings of medical therapy have driven research toward more direct approaches of beta cell replacement. Indeed, the specificity of beta cell loss in type 1 diabetes makes this disease a particularly attractive candidate for cell-based therapies. In order for significant progress to be made, however, a thorough understanding of beta cell biology and more broadly islet biology is necessary. This review addresses recent advances in developmental biology that have expanded our understanding of islet cell differentiation, assesses the promise and limitations of islet transplantation, and discusses the future of alternative sources of beta cells, including directed differentiation of stem cells, replication of adult beta cells, and transdifferentiation of nonislet cells to a beta cell fate. =     

体外分步诱导胰腺导管干细胞分化为胰岛β细胞

目的 建立新的体外胰腺导管干细胞分化为胰岛β细胞的分步诱导分化体系.方法 分离纯化SD大鼠的胰腺导管干细胞,体外扩增后进行分步诱导分化,收获的新生类胰岛样细胞团(ILCs)分别行免疫荧光和RT-PCR检测,并通过葡萄糖刺激的胰岛素释放试验(GSIS)评价ILCs的体外功能.结果 分离纯化的胰腺导管干细胞经培养及四步诱导分化后最终可形成ILCs.免疫荧光结果 显示该ILCs胰岛素和胰高血糖素染色均为阳性,RT-PCR检测也有胰岛素和胰高血糖素基因的表达.新生ILCs在低糖和高糖刺激下的胰岛素释放量分别为(1.1±0.2)ng/ml和(2.7±0.2)ng/ml,刺激指数为2.52倍.结论 通过建立新的体外胰腺导管干细胞分化为胰岛β细胞的分步诱导分化体系,获得具有立体结构且有一定胰岛素分泌能力的ILCs,为获得胰岛移植的β细胞来源提供了更有效的途径.     

Human islet cell MORF/cMORF pretargeting in a xenogeneic murine transplant model

Noninvasive measurement of human islet cell mass in pancreas or following islet transplantation by nuclear imaging has yet to be achieved. It has been shown using mouse tumor models that pretargeting imaging strategies are sensitive and can greatly increase target to nontarget signal ratios. The objective now is to demonstrate the specific pretargeting of human islet cells in mice. Our pretargeting strategy uses an anti-human islet cell antibody HPi1, conjugated to a phosphorodiamidate morpholino oligomer (MORF) that binds specifically to a (99m)Tc labeled complementary MORF (cMORF). Sensitivity and specificity of the pretargeting were first validated in culture using a human beta cell line (betalox5) and a negative control human cell line (HEK293). Pretargeting was then used to target and visualize these two cell lines and human islets transplanted subcutaneously in NOD-scid IL2rγ(null) mice. In culture, (99m)Tc accumulation on the betalox5 cells pretargeted by MORF-HPi1 was 100-fold higher than on untreated betalox5 cells or following treatment with native HPi1 and much higher than on the MORF-HPi1 pretargeted control HEK293 cells. Small animal imaging readily localized the transplanted betalox5 cells and human islets, but not the HEK293 cells. Ex vivo counting demonstrated 3-fold higher (99m)Tc accumulation in the transplanted betalox5 cells and human islets than in the control HEK293 cells. The target accumulation was also shown to increase linearly with increased numbers of the implanted betalox5 cells. These results demonstrate specific binding of radioactivity and successful imaging of human betalox5 cells and human islets transplanted in mice. Thus MORF/cMORF pretargeting may be useful to measure noninvasively human islet cell mass within the pancreas or following islet transplantation.     

脐血干细胞治疗1型糖尿病3例

研究证实,干细胞具有趋向分化作用,可分化为胰岛B细胞,在治疗T1DM中是一种安全、有效的治疗手段。目的脐血间充质干细胞治疗T1DM,观察其疗效和安全性。方法3例T1DM患者,年龄分别16岁、20岁、28岁。体质量指数分别是22．3、21．5、21．6。在患者及家属知情同意的情况下于我院介入室行为介入术,脐血间充质干细胞静脉注入胰背动脉。术后随访其临床症状及各项指标的变化,进行综合分析。结果3例患者胰岛素需要量较前下降30％～50％,胰岛功能较前明显改善,糖化血红蛋白从9．85％～13．26％下降到6．7％～7．8％,糖尿病酮症随访期间未发生。结论脐血间充质干细胞作为干细胞的一种是治疗T1DM的新途径,在临床应用中显示出其特有的效应。     

Combination of Mangiferin and Dipeptidyl Peptidase-4 Inhibitor Sitagliptin Improves Impaired Glucose Tolerance in Streptozotocin-Diabetic Rats

In this study, we combined the dipeptidyl peptidase-4 inhibitor sitagliptin with the antidiabetic drug mangiferin to examine the effects on active glucagon-like peptide-1 (GLP-1) and glucose tolerance in streptozotocin-diabetic rats. Active GLP-1 levels were measured by an ELISA kit. Insulin levels were measured by an RIA kit. Islet morphology was determined by double immunolabeling. Sitagliptin (1 mg/kg) or mangiferin (20 mg/kg) single treatment improved glucose tolerance during an oral glucose tolerance test. In addition, the combination therapy improved glucose tolerance with an increase in plasma insulin level and active GLP-1 levels. Islets from combination-treated diabetic rats had markedly increased β-cell/islet area ratio compared with islets from the diabetic or single-treatment rats. In conclusion, these results indicate that the combination therapy is useful as a therapeutic agent for impaired glucose tolerance and type 2 diabetes.     

Melatonin improves the therapeutic role of mesenchymal stem cells in diabetic rats

The present study aimed to investigate the role of bone marrow mesenchymal stem cells (MSCs) and/or melatonin (MT) for improvement of β-cell functions in STZ diabetic rats. Male albino rats (130–150?g) were divided into six groups. Control group: received phosphate-buffered saline (PBS); melatonin group received melatonin (10?mg/kg b.wt./day for 2 months by oral gavage); diabetic untreated group; diabetic group treated with melatonin; diabetic group treated with MSCs (a single intravenous injection of 3?×?10⁶ cell in PBS); and diabetic group co-treated with stem cells and melatonin. The results showed significant improvement in glucose, insulin, total antioxidant, and malondialdehyde level in diabetic rats treated with either MSCs alone or in combination with melatonin. The imumuno-histochemical analysis showed that MSCs and/or melatonin treatment reduced the rate of inflammation and apoptosis of the islet cells as well as increased the rate of pancreatic cell division. Such results were indicated by a significant improvement in the level of TNF-α, IL-10, PCNA, and caspase-3 to levels very close to the control. Co-treatment of MSCs and MT resulted in an improvement in the tissue of the pancreas and reduced number of damaged β-cells. It can be concluded that co-treatment of stem cells and melatonin has a significant role in restoring the structural and functional efficiency of β-cells in the pancreas more than stem cells alone. Such results may be due to the role of melatonin as an antioxidant in increasing the efficiency and vitality of stem cells.     

TFP5 attenuates cyclin-dependent kinase 5-mediated islet β-cell damage in diabetes

Islet β-cell damage and dysfunction represent the pathophysiological basis of diabetes. Excessive activation of cyclin-dependent kinase 5 (CDK5) is involved in the pathogenesis of type 2 diabetes mellitus (T2DM), although the exact mechanism remains unclear. Therefore, this study investigated the role of a CDK5 inhibitor (TFP5) in islet β-cell damage under diabetic conditions by regulating the expression of CDK5 in vitro and in vivo. CDK5 was upregulated under high glucose conditions in vivo and in vitro, which resulted in inflammation, oxidative stress, and apoptosis of islet β-cells, thereby decreasing insulin secretion. However, TFP5 treatment inhibited the overexpression of CDK5; reduced the inflammatory response, oxidative stress, and apoptosis of islet β cells; and restored insulin secretion. In conclusion, CDK5 is involved in islet β-cell damage under high glucose conditions, and TFP5 may represent a promising candidate for the development of treatments for T2DM.