Yuanjun Gu

Reversal of diabetes in mice by xenotransplantation of a bioartificial pancreas in a prevascularized subcutaneous site.

BACKGROUND The subcutaneous site has been regarded as a potential site for a bioartificial pancreas. Transplantation of islets, encapsulated by the development of diverse biocompatible materials and structural designs, can reverse hyperglycemia in diabetic recipients. METHODS Approximately 750 Sprague-Dawley rat islets macroencapsulated in an agarose/poly (styrene sulfonic acid) mixed gel were implanted into a prevascularized subcutaneous site. The site was constructed by subcutaneous injection of basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres in streptozotocin-induced C57BL/6 diabetic mice. Diabetic mice treated with bFGF-free gelatin microspheres and diabetic mice without any treatment undergoing the same subcutaneous transplantation were used as controls. After transplantation, non-fasting blood glucose, body weight, intraperitoneal glucose tolerance test, and histologic evaluations were processed. RESULTS All the recipients undergoing the subcutaneous xenograft returned to normoglycemia within 1 week after transplantation. Eight of 10 recipients in the bFGF+ group maintained normoglycemia for a period of 38-101 days and gradually gained increase of body weight. Two of 10 recipients became hyperglycemic again when the grafts were respectively retrieved at days 31 and 63. Intraperitoneal glucose tolerance tests at month 1 and 2 revealed significant ameliorated glucose tolerance but a tendency to reduced glucose tolerance when compared respectively with those of the streptozotocin-induced diabetic mice and normal mice. Histologic examination revealed that islets within the retrieved grafts at days 31 and 63 were viable and intact; no fibrotic overgrowth was present around the surface of grafts. CONCLUSIONS A successfully prevascularized subcutaneous site could be constructed by a tissue bioengineering approach. Xenotransplantation of the agarose/poly (styrene sulfonic acid) mixed gel-based bioartificial pancreas in the prevascularized subcutaneous site could reverse diabetes in mice.

In vivo functioning and transplantable mature pancreatic islet-like cell clusters differentiated from embryonic stem cells.

Introduction Although the differentiation of embryonic stem (ES) cells to islet like clusters using differentiation method without employing gene transfer technique has been recently reported, neither endocrine granules in the cytoplasm nor in vivo function of differentiated islet like clusters has been demonstrated. Aims To investigate whether ES cells could be differentiated to mature islet like clusters which show in vivo function after transplantation as well as retain endocrine granules in the cytoplasm by electron microscopic observation. Methodology In this experiment, using mouse embryonic stem (mES) cells as a model system for lineage specific differentiation, we tried to differentiate mES cells to pancreatic islet-like cell clusters (PICCs) through a series of treatments (4-step procedure). Differentiated PICCs were analyzed and characterized by various techniques, such as RT-PCR, immunohistochemistry, electron microscopic observation, in vitro static incubation test, and in vivo transplantation to diabetic animals. Results Differentiated islet-like cell clusters from ES cells using our newly developed method (four-step procedure) showed strong expression of essential specific genes to the endocrine pancreas and also specific genes to the exocrine pancreas demonstrating that these islet-like clusters were mature from the developmental biologic point of view. These differentiated cells clearly revealed many mature insulin secretory granules of pleomorphic shape in the cytoplasm as well as well-developed rough endoplasmic reticulum. In vitro study indicated that differentiated cells retain a potent insulin secretory responsiveness to glucose stimulation. Furthermore, the islet-like cell clusters significantly decreased high blood glucose levels almost to normal levels when grafted to streptozotocin-induced diabetic mice without induction of any teratoma formation after transplantation. Conclusion Our results provide evidence that ES cells could differentiate to functioning and transplantable mature pancreatic islet-like cell clusters using our newly developed differentiation method without employing gene transfer technique. This study may lead to a basis for production of indefinite sources of islets that could be applicable for future clinical trial.

Evaluation of insulin secretion of isolated rat islets cultured in extracellular matrix.

Islet isolation involves enzymatic digestion of the interstitial matrix and mechanical disruption of the tissue. It is possible that a fundamental change of islet biology resulting from the loss of critical factors required for islet function or survival will occur. Extracellular matrix (ECM) is one of the most important components of the islet microenvironment. Reconstruction of the cell–matrix relationship seems to be effective for improving the loss of differentiated islet structure and function. The purpose of this study was to characterize and compare the effects of collagen gel mixture or Matrigel on β-cell function and islet cell survival. After isolation by the collagenase digestion technique, rat islets were divided and cultured with various types of collagen gel mixture. They were assessed for their glucose-stimulated insulin secretion and cell viability. Glucose-induced insulin secretion of islets cultured with collagen type I gel or a mixture of collagen type I and IV was improved after 11 days in culture. In conclusion, a type of gel composed of collagen type I and/ or type IV as an islet microenvironment is sufficient to maintain glucose responsiveness and may be useful for islet transplantation.

Bioartificial pancreas transplantation at prevascularized intermuscular space: effect of angiogenesis induction on islet survival.

Introduction Bioartificial pancreas (BAP) transplantation offers a potential treatment of diabetes mellitus. The optimal site for BAP transplantation has not yet been established. Aim To monitor the effect of induction of neovascularization at the intermuscular space on islet survival after allogenic transplantation of BAP. Methodology Angiogenesis was induced at the intermuscular space of diabetic Lewis rats by implanting a polyethylene terephthalate (PET) mesh bag, which enclosed a collagen sponge and biodegradable gelatin microspheres containing basic fibroblast growth factor. After confirmation of angiogenesis, BAP was prepared by mixing of 5% agarose with ≈2,800 isolated rat (Sprague–Dawley) islets and transplanted into the prevascularized PET mesh bag. Results Neovascularization was observed in and around the PET mesh bag within 10 days after implantation as confirmed by macroscopic and microscopic examinations. In the presence of a collagen sponge, new blood vessels penetrated into the PET mesh bag and formed a vascular bed. After transplantation, normoglycemia was achieved in the rats within 3 days and maintained for >35 days. The rats gradually gained body weight, and the results of intravenous glucose tolerance test showed normal patterns of blood glucose clearance 1 month after transplantation. Conclusion It can be concluded that the prevascularized PET mesh bag enabled transplanted BAP to survive and maintain function, thus indicating a potential site for BAP transplantation.

A newly developed three-layer agarose microcapsule for a promising biohybrid artificial pancreas : rat to mouse xenotransplantation

We examined the effectiveness of an improved version of a three-layer agarose microcapsule in islet xenotransplantation. The microcapsule is composed of a mixture of 5% agarose and 5% polystyrene sulfonic acid. The other two outer layers are polybrene and carboxymethyl cellulose. The agarose/polystyrene sulfonic acid membrane is for the purpose of immunoisolation, suppression of complement activity and reinforcement of the microcapsule. The polybrene layer suppresses the polystyrene sulfonic acid leakage by forming a polyionic complex at the surface of the agarose/polystyrene sulfonic acid membrane. The outermost layer, a carboxymethyl cellulose coating, improves the biocompatibility of the microcapsule. In vitro static incubation study showed that the insulin secretion from rat islets in microcapsules in response to 16.7 mM glucose stimulation was more than four times higher than that on 3.3 mM glucose stimulation (n = 8). In an in vivo study, 500 rat islets in microcapsules were xenogenically implanted in the abdominal cavity of mice with streptozotocin-induced diabetes. The graft survival times ranged from 2 to 5 mo, the average being 75 days (n = 5). Our results demonstrate that the improved version of the three-layer agarose microcapsule can effectively prolong the xenograft survival time without employing immunosuppressants, suggesting that this microcapsule could provide a promising biohybrid artificial pancreas for future clinical applications.

Experimental hybrid islet transplantation: application of polyvinyl alcohol membrane for entrapment of islets.

In this study, we first examined in vitro a polyvinyl alcohol membrane to be used to contain hybrid islet cells, and second we tested a bioartificial pancreas with entrapment of pancreatic islets in polyvinyl alcohol membrane in rats with experimentally induced diabetes. The permeability of the polyvinyl alcohol membrane to different substances was studied in a two-cell chamber system. Glucose, insulin, and nutrients passed through the membrane easily, whereas the passage of immunoglobulin G was completely prevented, indicating that this membrane could be effective in protecting the bioartificial pancreas from immunorejection. Approximately 2,000 islets collected from three Sprague-Dawley rats were enclosed in a mesh-reinforced polyvinyl alcohol tube and transplanted into the peritoneal cavity of six Wistar rats with streptozotocin-induced diabetes. Their nonfasting serum glucose levels were significantly decreased for at least 12 days. Six diabetic rats receiving intra-peritoneal transplantation of free islets without the tube showed a slight but significant decrease in nonfasting serum glucose levels for only 3 days. One diabetic rat with transplantation of the bioartificial pancreas had a significant and sustained decrease in nonfasting glucose levels from pretransplanted levels of 440–500 mg/dl to a mean value of 162 ± 13 mg/dl for over 3 months without immunosuppression. The bioartificial pancreas was then removed, and glucose levels gradually increased to over 500 mg/dl. The results of the present study suggest that a bioartificial pancreas with entrapment of islets in a polyvinyl alcohol membrane could be a promising therapeutic approach to diabetes mellitus.

Subcutaneous transplantation of macroencapsulated porcine pancreatic endocrine cells normalizes hyperglycemia in diabetic mice.

Background. The ultimate goal of islet transplantation is the unlimited availability of insulin-secreting cells to be transplanted in a simple procedure that requires no use of immunosuppressive drugs. Immunoisolation of xenogeneic pig islets for transplantation has great potential therapeutic benefits for treatment of diabetes. Methods. Approximately 4×106 porcine pancreatic endocrine cells (PEC) isolated from 6-month-old pigs were macroencapsulated in agarose-poly(styrene sulfonic acid) mixed gel and implanted into a prevascularized subcutaneous site in streptozotocin-induced C57BL/6 diabetic mice. Animals receiving an equal number of free porcine PEC were used as controls. After transplantation, nonfasting blood glucose, body weight, intraperitoneal glucose tolerance test, and immunohistologic evaluations were processed. Results. All 10 animals receiving the subcutaneous xenografts of the macroencapsulated porcine PEC normalized hyperglycemia within 5 days after transplantation, maintained the duration of normoglycemia for 24 to 76 days, and gradually gained weight. The subcutaneous xenografts of free porcine PEC could not reverse hyperglycemia. The recipient became hyperglycemic again when the implanted graft was retrieved at day 45 after transplantation. The glucose clearances were significantly ameliorated at day 21 and day 45 after transplantation when compared with those in diabetic mice. The immunohistochemical results revealed an inherent intact structure of the macroencapsulated porcine PEC and positive double-immunofluorescence staining for insulin and glucagon. Conclusions. Subcutaneous transplantation of macroencapsulated porcine PEC normalized hyperglycemia in diabetic mice. Our results identified a potential for a favorable development of subcutaneous transplantation of porcine PEC as a cure for diabetes.

Effectiveness of acidic oxidative potential water in preventing bacterial infection in islet transplantation.

At a number of points in the current procedures of islet isolation and islet culture after the harvesting of donor pancreata, microorganisms could potentially infect the islet preparation. Furthermore, the use of islets from multiple donors can compound the risks of contamination of individual recipients. Acidic oxidative potential water (also termed electrolyzed strong acid solution, function water, or acqua oxidation water), which was developed in Japan, is a strong acid formed on the anode in the electrolysis of water containing a small amount of sodium chloride. It has these physical properties: pH, from 2.3 to 2.7; oxidative-reduction potential, from 1,000 to 1,100 mV; dissolved chlorine, from 30 to 40 ppm; and dissolved oxygen, from 10 to 30 ppm. Because of these properties, acidic oxidative potential water has strong bactericidal effects on all bacteria including methicillin-resistant Staphylococcus aureus (MRSA), viruses including HIV, HBV, HCV, CMV, and fungi as a result of the action of the active oxygen and active chlorine that it contains. We conducted this study to evaluate the effect of acidic oxidative potential water irrigation on bacterial contamination on the harvesting of porcine pancreata from slaughterhouses for islet xenotransplantation by counting the number of pancreatic surface bacteria using the Dip-slide method, and on the results of islet culture; and to evaluate the direct effect on isolated islets when it is used to prevent bacterial contamination by the static incubation test and by morphorogical examination. Direct irrigation of the pancreas by acidic oxidative potential water was found to be very effective in preventing bacterial contamination, but direct irrigation of isolated islets slightly decreased their viability and function.

Stem cells and regenerative medicine for diabetes mellitus.

A profound knowledge of the development and differentiation of pancreatic tissues, especially islets of Langerhans, is necessary for developing regenerative therapy for severe diabetes mellitus. A recent developmental study showed that PTF-1a is expressed in almost all parts of pancreatic tissues, in addition to PDX-1, a well-known transcription factor that is essential for pancreas development. Another study suggested that &agr; cells and β cells individually, but not sequentially, differentiated from neurogenin-3–expressing precursor cells. Under strong induction of pancreas regeneration, it is likely that pancreatic duct cells dedifferentiate to grow, express PDX-1, and re-differentiate toward other cell types including islet cells. Duct epithelium-like cells can be cultivated from crude pancreatic exocrine cells and can be induced to differentiate toward islet-like cell clusters under some culture conditions. These cell clusters made from murine pancreas have been shown to control hyperglycemia when transplanted into diabetic mice. Liver-derived oval cells and their putative precursor H-CFU-C have been shown to differentiate toward pancreatic cells. Furthermore, extrapancreatic cells contained in bone marrow and amniotic membrane are reported to become insulin-producing cells. However, their exact characterization and relationship between these cell types remain to be elucidated. Our recent study has shown that islet-like cell clusters can be differentiated from mouse embryonic stem cells. Transplantation of these clusters could ameliorate hyperglycemia of STZ-induced diabetic mice without forming teratomas. Interestingly, these cells expressed several genes specific to exocrine pancreatic tissue in addition to islet-related genes, suggesting that stable and efficient differentiation toward certain tissues can only be achieved through a process mimicking normal development of the tissue. Perhaps recent developments in these fields may rapidly lead to an established regenerative therapy for diabetes mellitus.

Development of a new method to induce angiogenesis at subcutaneous site of streptozotocin-induced diabetic rats for islet transplantation

The subcutaneous space is a potential site for clinical islet transplantation. Even though there are several advantages, poor blood supply at this site mainly causes failure of islet survival. In this study, angiogenesis was induced in advance at the diabetic rats subcutis for islet transplantation by implanting a polyethylene terephthalate (PET) mesh bag containing gelatin microspheres incorporating basic fibroblast growth factor (bFGF) (MS/bFGF) and a collagen sponge. The bFGF was incorporated into gelatin microspheres for controlled release of bFGF. As controls, a PET mesh bag with or without either collagen sponges or MS/bFGF was implanted at the subcutaneous site of diabetic rats. Macroscopic and microscopic examinations revealed the formation of capillary network in and around the PET mesh bag containing MS/bFGF and collagen sponges 7 days after implantation when compare with other control groups. When tissue hemoglobin level was also measured, a significantly high level of hemoglobin amount was observed compared with that of control groups. When allogeneic islets mixed with 5% agarose were transplanted into the prevascularized rat subcutis, normoglycemia was maintained for more than 40 days, while other control groups were ineffective. This study demonstrated that combination of gelatin microspheres incorporating bFGF and collagen sponges enabled the mesh to induce neovascularization even at the subcutaneous site of streptozotocin-induced diabetic rats, resulting in improved function of islet transplantation.