Islet transplantation at the Diabetes Research Institute Japan

Since the Edmonton Protocol was announced, more than 600 patients with type 1 diabetes at more than 50 institutions have received islet transplantation to treat their disease. We recently established a new islet isolation protocol, called the Kyoto Islet Isolation Method, based on the Ricordi method. It includes an in-situ cooling system for pancreas procurement, pancreatic ductal protection, a modified two-layer (M-Kyoto /perfluorochemical [PFC]) method of pancreas preservation, and a new islet purification solution (Iodixanol-based solution). Using this islet isolation method, we isolated islets from 19 human pancreata of non-heart-beating donors and transplanted 16 preparations into seven patients with type 1 diabetes between April 7, 2004 and November 18, 2005. The percentage of those meeting the release criteria of the Edmonton Protocol was more than 80%. We also performed living-donor transplantation of islets for unstable diabetes on January 19, 2005. Establishment of this method enables us to make diabetic patients insulin-independent, using islets not only from two or three pancreata of non-heart-beating donors but also using islets from half a pancreas from a living donor.     

Hematopoietic stem cell transplantation prevents diabetes in NOD mice but does not contribute to significant islet cell regeneration once disease is established

The treatment of type I diabetes by islet cell transplantation, while promising, remains restricted due to the incomplete efficacy and toxicity associated with current immunosuppression, and by limited organ availability. Given reports suggesting bone marrow derived stem cell plasticity, we sought to determine whether such cells could give rise to pancreatic islet cells in vivo. In the context of autoimmune diabetes, we transplanted unfractionated bone marrow from beta-gal trangenic donor mice into NOD mice prior to, at, and two weeks beyond the onset of disease. Successful bone marrow engraftment before diabetes onset prevented disease in all mice and for 1 year after transplant. However, despite obtaining full hematopoietic engraftment in over 50 transplanted mice, only one mouse became insulin independent, and no beta-Gal positive islets were detected in any of the mice. To test whether tolerance to islets was achieved, we injected islets obtained from the same allogeneic donor strain as the hematopoietic cells into 4 transplant recipients, and 2 had a reversion of their diabetes. Thus allogeneic bone marrow transplantation prevents autoimmune diabetes and tolerizes the recipient to donor islet grants, even in diabetic animals, yet the capacity of bone marrow derived cells to differentiate into functional islet cells, at least without additional manipulation, is limited in our model.     

Pancreatic islet cell transplantation using non-heart-beating donors (NHBDs)

Recent dramatic improvements in clinical islet cell transplantation demonstrated by the Edmonton group have increased the demand for this treatment, and donor shortage could become a major problem. Utilization of marginal donors could alleviate the donor shortage, and non-heart-beating donors (NHBDs) might be good resources. The University of Pennsylvania group demonstrated that it was possible to isolate islets from NHBDs, and the group actually transplanted islets from NHBDs, for the first time. The patient became insulin-independent; however, there had been no more cases using NHBDs until our group initiated islet transplantations from NHBDs in Japan. In order to utilize NHBDs effectively, we modified the standard islet isolation method. These modifications included minimizing the warm ischemic time, the use of trypsin inhibition during isolation, carrying out density measurement before purification and the use of a less toxic islet purification solution. With these modifications we were able to transplant nine of ten islet preparations from ten NHBDs (90%), into five type-1 diabetic patients. The first transplantation was performed on April 7, 2004 (the first time in Japan), and this patient became insulin-independent after the second islet transplantation (first time in Japan). All patients showed improved glycemic control and reduced insulin requirements, without hypoglycemic events. We also performed living-donor islet transplantation, with our modified islet isolation protocol, on January 19, 2005. The improved islet isolation protocol enabled us to perform effective islet transplantations from NHBDs, and it also enabled us to perform the living-donor islet transplantation.     

First human trial of pancreatic islet allo-transplantation in Korea--focus on re-transplantation

Over the past 20 years, allo-transplantation of islet or whole pancreas for reaching and sustaining near-normoglycemia, as close as possible to the physiological model, have been undertaken. As previously known, even though islet transplantation is possible as a safe re-transplant, it is not well known whether re-transplantation of islets is suitable for patients who have lost the grafted islet function. We have performed a human islet allo-transplantation and re-transplantation on an IDDM patient for the first time in Asia and Korea. The recipient was a 32-year-old male and his insulin requirement was 75-85 U per day. After islet transplantation, the basal C-peptide increased from 0.6 to 2.1 ng/ml and insulin requirement decreased from 80 to 36 U per day, indicating that the grafted islets were functional. However, the grafted islets lost function 70 days after the transplantation. So, we performed re-transplantation of the islets. After the re-transplantation, the glucose profile became more stable and frequent episodes of severe hypoglycemia completely disappeared. His severe neuropathic pain improved dramatically and he could engage his ordinary daily life without any antineuropathic drugs. The success of this re-transplantation is one step closer to becoming a viable alternative for the millions of individuals who are suffering from diabetes.     

Utility of co-transplanting mesenchymal stem cells in islet transplantation

Islet transplantation is characterized by the transplantation of isolated islets from donor pancreata into a diabetic recipient. Although it is a viable choice in the treatment of insulin dependent diabetes mellitus, most patients (approximately 90%) require insulin five years after transplantation. Recently, the co-transplantation of mesenchymal stem cells (MSCs) and islets in animal studies has revealed the effectiveness of MSCs co-transplantation for improving islet function. Themechanisms underlying the...     

Islet cell transplantation for Type 1 diabetes

Islet transplantation is an attractive concept for the treatment of Type 1 diabetes because of its potential high efficacy and minimal invasion to patients. The treatment may effectively control blood glucose for brittle Type 1 diabetes, resulting in a marked reduction in hypoglycemic episodes and improvements in HbA 1 c. In addition, approximately 70% of transplanted Type 1 diabetic patients have achieved insulin independence. However, there are still important issues to be addressed before this treatment is widely applicable, including difficulty in maintaining insulin independence, low islet isolation success rate, multiple donor requirements, and side effects associated with the use of immunosuppressants. Donor shortage is another dilemma. To address the issue of donor shortage, living donor islet transplantation and bioartificial islet transplantation using pig islets are being evaluated. Bioartificial islet transplantation could be the ultimate solution of the donor shortage. Currently, overcoming immunological hurdles, establishing reliable islet isolation methods, and controlling porcine endogenous retrovirus are the primary obstacles to the implementation of this treatment. If bioartificial islet transplant becomes a clinical reality, it may even be applicable in the treatment of select Type 2 diabetic patients. β‐Cell regeneration from naïve pancreas and β‐cell generation from embryonic stem cells or induced pluripotent stem cells are the next‐generation treatments for Type 1 diabetes.     

Transplantation of insulin-producing tissue

A series of new findings have shown that it is possible to prevent rejection of islet allografts and islet xenografts in animals without the continued use of immunosuppressive agents. The survival of allografts of rat islets has been prolonged for more than 100 days by in vitro culture of the islets at 24 °C for seven days prior to transplantation in conjunction with a single injection of rat antilymphocyte serum at the time of transplantation. Xenograft survival of rat islets transplanted into diabetic mice has been prolonged for more than 100 days by the use of culture of rat islets at low temperature, with a single injection of antiserums to mouse and rat lymphocytes at the time of transplantation. Rejection of established islet allografts in rats was induced by the injection of donor peritoneal exudate cells and donor T lymphocytes, whereas the injection of B lymphocytes did not induce rejection. The pretreatment regimens used for prolonging islet allograft and xenograft survival apparently destroy or alter passenger leukocytes in the grafts, and it would appear that these cells are needed for the induction of immune recognition by the recipient. Islet cells in mice express products of the H-2K and H-2D loci, but the cells do not express the I region (Ia) antigens. These new developments in the prevention of immune rejection of the islets raise the question as to whether these approaches may be applicable to the transplantation of islets into human subjects with diabetes.     

Islet transplantation outcomes in mice are better with fresh islets and exendin-4 treatment

Aims/hypothesis Although islet transplantation in diabetes holds great promise, two or three donor pancreases are usually required to achieve normoglycaemia in human or rodent recipients. We investigated whether there were differences between fresh and cultured islets in terms of transplantation outcome. We also investigated the effects of normoglycaemia during engraftment and the effects of exendin-4, a glucagon-like peptide-1 receptor agonist, on islet transplantation.Materials and methods Seventy-five fresh islets were transplanted to the right kidney of diabetic mice and 425 fresh islets were transplanted to the left kidney. The mice were treated with exendin-4 or vehicle for 14 days, after which the large graft was removed by left nephrectomy. In a separate set of experiments, islets cultured in the presence or absence of exendin-4 for 72 h, or fresh islets, were transplanted to diabetic mice. In both sets of experiments, blood glucose levels were monitored.Results Compared with cultured islets, fresh islets were more effective at reversing hyperglycaemia in mice. The treatment of the recipient mice with exendin-4 did not have beneficial effects on glucose homeostasis. However, when islets are cultured, exendin-4 treatment increases the rate of reversal of hyperglycaemia, but not to the degree of fresh islets.Conclusions/interpretation Fresh islets are more effective than cultured islets at reversing hyperglycaemia. Exendin-4 has beneficial effects on islet transplantation.     

The effect of transgenic expression of TGF-beta1 on transplanted islet graft survival

BACKGROUND/AIMS: Transgenic mice expressing the active form of porcine TGF-beta1 (NOD- TGF-beta1 Tg) were completely protected from autoimmune diabetes in the NOD genetic background in our previous study. Here, we attempted to determine whether transgenic expression of TGF-beta1 in transplanted islets prevents autoimmune destruction in NOD mice. METHODOLOGY: We transplanted islets to the subcapsular region of the kidney using NOD-TGF-beta1 Tg and NOD mice as donor and recipient or vice versa. Cyclophosphamide was administered twice to streptozocin-induced diabetic females NOD-TGF-beta1 Tg or their female littermates after islet transplantation. RESULTS: All islets grafts of NOD-TGF-beta1 Tg in spontaneously diabetic NOD mice were rejected earlier than those of their littermates. Hyperglycemia was induced in all littermates, but three out of four NOD-TGF-beta1 Tg, which were STZ-induced diabetic female mice, remained normoglycemic in response to the administration of cyclophosphamide after islet transplantation. CONCLUSIONS: Our results lack direct evidence for the local paracrine TGF-beta1 to protect the transplanted islet grafts. We observed, however, prolonged survival of NOD islets grafts in diabetic NOD-TGF-beta1 Tg suggesting the protective role of transgenic TGF-beta1 to suppress the autoimmune process in our syngenic transplantation model. We are convinced that this data could help resolve many problems regarding islet transplantation for type 1 diabetes.     

Insulin gene transfer enhances the function of human islet grafts

AIMS/HYPOTHESIS: Recent success in islet transplantation renews the hope for the complete cure of patients afflicted with Type 1 diabetes. However, in the Edmonton series, two to four pancreas donors were required to obtain a sufficient islet mass to reverse the diabetes of each patient. In view of the donor shortage, this represents a major obstacle preventing greater application of islet transplantation to diabetic patients. We hypothesised that increasing the expression of the insulin gene in transplanted islets would augment their capacity for insulin production, thereby allowing reversal of diabetes with a reduced islet mass. METHODS: We used a replication defective adenovirus to deliver the human proinsulin gene (Ad-Ins) to isolated human islets. The function of Ad-Ins-transduced human islets was compared to islets transduced with a control vector (Ad-lacz). RESULTS: Ad-Ins-transduced islets produced two to three times more insulin than normal islets or those infected with Ad-lacz, as assessed by in vitro perifusion tests of glucose stimulated insulin release. When transplanted, Ad-Ins-transduced islets normalised the blood glucose of diabetic immunodeficient NOD-Scid mice, and less than half as many Ad-Ins islets were required for reversal of diabetes than when normal islets were transplanted. CONCLUSION/INTERPRETATION: Our results suggest a simple and effective approach that could enhance the efficiency of islet transplantation for treatment of diabetes in humans.     

Veno-venous extracorporeal membrane oxygenation bridged living-donor lung transplantation for rapid progressive respiratory failure with pleuroparenchymal fibroelastosis after allogeneic hematopoietic stem cell transplantation

Cases of extracorporeal membrane oxygenation (ECMO) bridged lung transplantation (LTx) are rare in Japan because an allocation system to prioritize patients based on urgency remains to be established. For critically ill patients who cannot wait for a brain-dead donor LTx, ECMO bridge to living-donor LTx may be the only practical option. A 21-year-old woman with pleuroparenchymal fibroelastosis after hematopoietic stem cell transplantation was admitted to our hospital with rapidly progressive respiratory failure. She was waitlisted for 6 months before admission, but veno-venous ECMO was initiated. She was transported under ECMO support via a jet plane and underwent successful living-donor LTx.     

Low revascularization of experimentally transplanted human pancreatic islets

Pancreatic islets are avascular immediately after transplantation. Although the islets are rapidly revascularized, it is uncertain whether the revascularization produces an adequate oxygenation of the transplanted islet tissue. We measured pO(2), blood flow and vascular density in mouse or human islets 1 month after transplantation to nude mice. Tissue pO(2) was measured with Clark microelectrodes. Blood perfusion was measured with laser-Doppler flow cytometry, whereas vascular density was determined in histological specimens stained for the lectin Bandeiraea simplicifolia (BS-1). Both the transplanted mouse and human islets had a pO(2) 15-20% of that in endogenous mouse islets. Moreover, the vascular density of the transplanted islets was decreased compared with that of endogenous mouse and human islets. Graft blood perfusion was approximately 50% of renal cortex blood flow. A negative correlation was found between donor age and blood perfusion of the human islet grafts. A similar correlation was seen between donor age and the total vascular density of these grafts. In conclusion, transplanted human islets had a markedly decreased vascular density and pO(2) compared with endogenous islets. This has potential implications for clinical islet transplantations, because poor vascular engraftment may significantly increase the number of islets needed to obtain insulin independence.     

Pankreas- und Inseltransplantation

Pancreas transplantation is a successful and effective procedure resulting in tight glucose control. Due to the postoperative morbidity and the need for immunosuppression pancreas transplantation should be considered in patients with type I diabetes at the time of kidney transplantation. Besides this pancreas transplantation alone can be taken into consideration for patients with very poor metabolic control and quality of life despite optimal medical treatment. Recently, islet transplantation became a less invasive alternative to pancreas transplantation. Due to the lack of long-term follow-up and due to the need of multiple donor grafts for one recipient, islet transplantation should be performed under experimental settings in experienced centers. New developments in protecting transplanted islets and in the induction of donor-specific tolerance could increase the indication to perform the procedure. Therefore alternative sources of β-cells have to be identified.     

Prolongation of islet allograft and xenograft in nonimmunosuppressed rat recipients

The temperature at which islets were cultured have been observed in this study to play a major role in the prolongation of islet survival when subsequently allotransplanted across a major histocompatibility barrier. Culturing of Lewis (Le) and Wistar rat islets for 7 days at 26° prior to transplantation prolonged their functional period significantly in $\text{4}\text{6}$ and $\text{3}\text{5}$ of the nonimmunosuppressed diabetic ACI recipient animals. Monkey islets when cultured for 7 days at 26°C had significantly prolonged the functional period in $\text{2}\text{5}$ of the diabetic recipient Le rats. In contrast, in vitro culture for 7 days at 32°C or for 7–14 days at 37° had failed to prolong Le islet survival in the ACI recipient rats. Rejection of successful allografts of cultured rat islets was subsequently induced with an intravenous (i.v.) injection of donor splenocytes, suggesting that the successful allografts maintain their antigenicity. The immunogenicity of the islet allograft in provoking humoral response by the recipient was found to decrease following in vitro culture for 7 or more days regardless of the variation in culturing temperature. This was demonstrated by the lowered antidonor antibody titer in the recipients of cultured islets as compared with those that received fresh islets. Data in this investigation demonstrates that culture of islets under a relative low temperature (26°C) can prolong the survival of islet allograft or xenograft in the nonimmunosuppressed recipient, and alteration of humoral immunity alone cannot account for prolongation of islet allograft survival.     

9a Techniques of pancreas and islet transplantation

Techniques for vascularized pancreas transplantation are relatively standardized, whereas those for human islet isolation and transplantation are rapidly changing and evolving. The commonest method for transplanting the vascularized pancreas is to use the entire pancreas together with a segment of donor duodenum, and to anastomose this to the recipient bladder. This technique offers the advantages of technical ease, the maximum β-cell mass is transplanted and graft function can be monitored by measuring urinary amylase levels. Human islet isolation requires that the pancreas is dissociated, the islets purified and then transplanted to a wellvascularized location. The pancreas is dispersed by a combination of the intraductal injection of collagenase and gentle mechanical agitation, and the islets separated from contaminating exocrine tissue by density-gradient centrifugation. Once purified, the islets can be placed into tissue culture or cryopreserved. The commonest site for human islet transplantation is intraportal injection so that the islets are embolized throughout the liver. Alternatives include transplantation to the renal subcapsular space or the spleen.     

Transmission of disseminated histoplasmosis via cadaveric renal transplantation: case report.

A 30-year-old woman received a cadaveric renal allograft from a donor who was subsequently found to have renal infection due to Histoplasma capsulatum. The recipient had funguria in the early postoperative period and later developed cutaneous manifestations of disseminated histoplasmosis. We report a case of histoplasmosis transmitted via renal transplantation and describe the subsequent 20-year clinical course.     

XIAP inhibition of β-cell apoptosis reduces the number of islets required to restore euglycemia in a syngeneic islet transplantation model

Clinical pancreatic islet transplantation has great promise as a treatment for type 1 diabetes but despite recent advances, it is still limited by the need for lifelong immunosuppression, restricted availability of donor islets, and uncertainty regarding long-term graft survival. Using a syngeneic, suboptimal islet transplantation model, we asked whether adenoviral overexpression of an anti-apoptotic protein, the X-linked inhibitor of apoptosis protein (XIAP) would protect transplanted islet cells from death and reduce the number of islets required for successful transplantation. Transplantation of 100 XIAP-expressing islets into the kidney capsule of syngeneic Balb/c mice restored euglycemia in 86% of recipients, where transplantation of 100 islets transduced with a control adenovirus expressing LacZ restored euglycemia in only 27% of recipients. Analysis of islet grafts by insulin/TUNEL double immunostaining revealed fewer apoptotic beta-cells in recipients of XIAP- compared with LacZ-expressing grafts (0.8±0.5 vs. 2.4±0.8 double-positive cells/graft), suggesting that XIAP enhances graft success by inhibiting β-cell apoptosis in the immediate post-transplant period. In summary, XIAP overexpression inhibits beta cell apoptosis in syngeneic islet transplants, thereby reducing the number of islets and decreasing the number of days required to restore euglycemia. These data raise the possibility that ex vivo XIAP gene transfer in islets prior to transplantation has the potential to increase the number of donor islets available for transplantation and may enhance graft function and long-term transplant success.     

Maintenance of aerobic metabolism increases immunoisolated islet survival

The progress of immunoisolation as a treatment for diabetes has been hampered by the diminished long term viability of islets within the immunoisolation device. Chronic hypoxia is greatly responsible for islet cell death within an immunoisolation device and remains an obstacle to the success of this form of islet transplantation. In order to address this problem, isolated rat islets were transfected with a plasmid encoding cytoglobin, an intracellular oxygen binding protein. Untreated or transfected islets were placed in polyacrylonitrile-polyvinychloride hollow fiber and implanted beneath the hepatic capsule in streptozotocin-diabetic rats. Fasting blood glucose was used as an indicator of islet survival and function. Rats receiving fibers containing transfected islets remained normoglycemic through the 60 day trial. Untreated islets failed within two weeks after implantation resulting in elevated blood glucose in the recipient. The fibers were recovered and tested for insulin content. Cytoglobin promoted islet cell survival and insulin synthesis and secretion. The induction of cytoglobin in islets may reduce cell loss from chronic hypoxia and may be a useful method to improve the feasibility of immunoisolation as an islet transplantation modality.     

Maintenance of aerobic metabolism increases immunoisolated islet survival

The progress of immunoisolation as a treatment for diabetes has been hampered by the diminished long term viability of islets within the immunoisolation device. Chronic hypoxia is greatly responsible for islet cell death within an immunoisolation device and remains an obstacle to the success of this form of islet transplantation. In order to address this problem, isolated rat islets were transfected with a plasmid encoding cytoglobin, an intracellular oxygen binding protein. Untreated or transfected islets were placed in polyacrylonitrile-polyvinychloride hollow fiber and implanted beneath the hepatic capsule in streptozotocin-diabetic rats. Fasting blood glucose was used as an indicator of islet survival and function. Rats receiving fibers containing transfected islets remained normoglycemic through the 60 day trial. Untreated islets failed within two weeks after implantation resulting in elevated blood glucose in the recipient. The fibers were recovered and tested for insulin content. Cytoglobin promoted islet cell survival and insulin synthesis and secretion. The induction of cytoglobin in islets may reduce cell loss from chronic hypoxia and may be a useful method to improve the feasibility of immunoisolation as an islet transplantation modality.     

Biocompatibility of Implanted Diabetes Devices: Part 1: Thrombosis and Inflammation in Intraportal Islet Transplantation: A Review of Pathophysiology and Emerging Therapeutics

With the inception of the Edmonton Protocol, intraportal islet transplantation (IPIT) has re-emerged as a promising cell-based therapy for type 1 diabetes. However, current clinical islet transplantation remains limited, in part, by the need to transplant islets from 2–4 donor organs, often through several separate infusions, to reverse diabetes in a single patient. Results from clinical islet transplantation and experimental animal models now indicate that the majority of transplanted islets are destroyed in the immediate post-transplant period, a process largely facilitated by deleterious inflammatory responses triggered by islet-derived procoagulant and proinflammatory mediators. Herein, mechanisms that underlie the pathophysiology of thrombosis and inflammation in IPIT are reviewed, and emerging approaches to improve islet engraftment through attenuation of inflammatory responses are discussed.