Pancreatic islet transplantation has now become the procedure of choice for the treatment of insulin-dependent diabetes mellitus (IDDM) since the report in 2000 by Shapiro et al,1 in which they showed that insulin independence could be successfully achieved in patients with IDDM after islet transplantation. Currently, the outcome of islet transplantation has been improved, such that more than 50% of IDDM recipients are insulin-free 5 years later,2,3 and the serious complication of severe hypoglycemic events is resolved after islet transplantation.4,5 However, several important issues remain to be resolved, including the inability to access and retrieve transplanted islets if necessary, such as at the time of rejection. This is because the islets are transplanted via the portal vein of recipients,1 and they become diffusely localized in the periphery of the portal vein in the liver. More importantly, the efficiency of islet transplantation is low, requiring sequential transplantation of islets with the use of 2 to 3 donors for the treatment of a single recipient. This is mainly due to early loss of the islets caused by innate immune rejection of islets transplanted into the liver.6 These problems appear to be specific to the liver as a transplantation site, suggesting that an alternative functional site for islet transplantation may provide a way to overcome the current obstacles facing clinical islet transplantation.
Many sites other than the liver have been reported for experimental islet transplantation.7-9 Among them, we focused on the subcutaneous space as a clinically applicable site because the transplantation procedure itself is simple although its efficiency has previously been reported to be extremely low, in which 5 to 10 donors are required to reverse diabetes of a single recipient in rats.10,11 For transplanted islets to function, not only new vessel formation for supply of arterial blood but also the presence of latent feeding vessels adjacent to the transplanted islets to communicate with new vessels are essential, because otherwise, the transplanted islets eventually die due to hypoxia. Thus, we reasoned that the extremely low efficiency of islet transplantation into conventional sites in the subcutaneous space is caused by hypoxic cell death after transplantation due to the lack of feeding vessels. Therefore, we searched for a site in the subcutaneous space of mice with potential feeding vessels. As a result, we found that the inguinal subcutaneous white adipose tissue (ISWAT), with feeding vessels from the inferior epigastric artery and vein, fulfills this criterion.
In the present study, we describe a novel procedure for transplantation of islets to the ISWAT, where transplanted islets are engrafted to form clusters with neovascularization, allowing visualization of transplanted islets as enhanced mass by computed tomography (CT) and their easy retrieval. More importantly, hyperglycemia of streptozotocin (STZ)-induced diabetic mice is ameliorated after transplantation of only 200 syngeneic islets, which is the number of islets that can be isolated from a single donor. Of note, islet allograft rejection in this site can be prevented by posttransplant short-term treatment with costimulatory blocking biologics. Furthermore, human islet xenotransplants can reverse STZ-induced diabetes in NOD/scid mice when grafted in this site.
MATERIALS AND METHODS
All experiments were in accordance with protocols approved by the Animal Care and Use Committee at Fukuoka University (approval number: 1703020).
Male C57BL/6, BALB/c and NOD/scid mice were purchased from Charles River Japan (Kanagawa, Japan). Human islets were supplied by Prodo Laboratories (Aliso Viejo, CA). Mice were kept under specific pathogen-free conditions and used at 6 to 15 weeks of age.
Mouse Islet Isolation, Transplantation, and Retrieval
Islets were isolated from the mouse pancreas12,13 and those with a diameter of 150 to 250 μm were hand-picked and were transplanted into the left ISWAT (Figure S1, SDC,http://links.lww.com/TP/B546)14 of STZ-induced diabetic C57BL/6 recipient mice (180 mg/kg) (Sigma) at 3 days after the injection of STZ according to the tube method described by Ito et al,15 as follows. First, donor islets were allowed to settle in a 1.5-mL microfuge tube (Trefflab, Degersheim, Switzerland) and then were loaded into a PE50 polyethylene tube (Beckton Dickinson, Sparks, MD), approximately 20 cm in length, with the aid of a Hamilton syringe (Hamilton Company, Reno, NV). The PE50 tubing was then bent and placed inside a 15-mL conical tube so that the center of the tubing was placed in the bottom of the conical tube, which was centrifuged at 190g for 1 minute, thus forming an islet pellet in the center of the PE50 tubing. After measuring the length of the islet pellet using an Electronic Digital Caliper (General, New York, NY) as an objective index of donor islet mass (Figure S2, SDC, http://links.lww.com/TP/B546), the PE50 tubing was cut with scissors approximately 1 mm distant from the edge of the islet pellet and then, a Hamilton syringe was connected to the other side of the PE50 tubing.
Regarding the transplantation procedures, a vertical skin incision is made in the left inguinal area of the recipient mouse (Figure 1A, left), and the inferior epigastric artery and vein (Figure 1A, arrows) are identified in the ISWAT, and a small pocket is created superiorly to the vessels (Figure 1A, middle, left), where islets loaded in P50 tubing are deposited with the aid of a Hamilton syringe (Figure 1A, middle, right). Then, the opening is closed with an Anastoclip (Lemaitre, Burlington, MA) (Figure 1A, right).
Transplantation of islets into the liver via the portal vein was performed as reported previously.16 Transplantation of islets beneath the kidney capsule was performed as reported previously17 with minor modification as follows. Briefly, after the exposure of the left kidney, a small incision was made on the kidney capsule, and the subcapsular space was created with the use of a glass spatula where islets loaded in the P50 tube were placed as described above.
The retrieval of transplanted islets in the ISWAT was performed as follows. After a vertical skin incision, the ISWAT containing transplanted islets was dissected free and cut with a Bovie coagulator (Clearwater, FL) at a line approximately 3 mm distant from the edge of transplanted islets cluster bilaterally.
The nonfasting blood glucose levels of recipient mice were measured before and twice after the injection of STZ and once a week after syngeneic islet transplantation.
In the allogeneic islet transplants, the nonfasting blood glucose levels of recipient mice were measured 3 times a week until 30 days after transplantation and once a week thereafter. Rejection was considered to have occurred when the blood glucose levels of recipient mice exceeded the pretransplant levels of 400 mg/dL after transplantation.
The blood glucose levels were measured using a GlucoCard DIA meter (Arkray, Kyoto, Japan).
Macro Photographs and Histology
Transplanted islets in the ISWAT of recipient mice were exposed periodically after transplantation and observed under a dissecting microscope (Olympus, Tokyo, Japan). The excised ISWAT of recipient mice bearing islet grafts were fixed in 10% formalin, processed, embedded in paraffin, and sectioned for histological analysis. Sections were stained with hematoxylin and eosin or for immunofluorescent microscopic analysis. Primary and secondary antibodies used were the following: guinea pig anti-insulin (DAKO, 1:200), rabbit anti-glucagon (Thermo, 1:200). Secondary antibodies used were the following: 488-Alexa antirabbit or antiguinea pig IgG (Molecular Probes), Cy3 antirabbit IgG (Jackson). Nuclei of the cells were costained with DAPI (Sigma-Aldrich, 1 mg/mL). Images were acquired by fluorescent microscopy (Keyence BZ-9000) and by confocal microscopy (Zeiss LSM710).
Intraperitoneal Glucose Tolerance Test
The intraperitoneal glucose tolerance test (IPGTT) was performed in recipient mice at 120 days after islet transplantation. The mice were fasted for 15 hours before the start of the test. Blood samples were obtained from the tail vein of recipient mice at 0, 30, and 120 minutes after the IP injection of glucose (1 g/kg body weight).
Forty minutes after the injection of contrast material (ExiTron Nano12000; Miltenyi Biotec, Bergisch Gladbach, Germany), 0.1 mL in volume via the left jugular vein of recipient mice, a whole-body CT (Skyscan 1178; Bruker, Kontich, Belgium) was performed, and the CT images were acquired by NRecon (Bruker microCT), and the data were analyzed with DataViewer ver184.108.40.206 (Bruker microCT).
Prevention of Islet Allograft Rejection in the ISWAT
BALB/c islets were grafted in the left ISWAT of STZ-induced diabetic C57BL/6 mice. CTLA4-Ig (500 μg/injection per mouse, Orencia, Bristol-Myers Squibb) and/or anti-mouse CD40L antibody (200 μg/injection per mouse, BioXcell, West Lebanon, NH) were administered IP at days 0, 2, 4, 6, and 8 after transplantation and recombinant human Fc-G1 (BioXcell) and Armernian hamster IgG (BioXcell) were used as controls, respectively.
Human Islet Experiments
Human islets were cultured at 37°C for additional 2 to 3 days in CMRL1066 medium (Mediatech, Corning, Tewksbury, MA) containing 2% human albumin (CSL Behring, King of Prussia, PA) before the following experiments. For islet transplantation, human islets, 2000 to 2500 islet equivalent (IEQ)/transplant/mouse, were grafted in the ISWAT of STZ (160 mg/kg, iv injection)-induced diabetic male NOD/scid mice. Functional and histological studies of transplanted human islets were performed similarly to the mouse study. Human C-peptide was measured with an ELISA Kit (Mercodia, Uppsala, Sweden).
The statistical significance of IPGTT data was determined by 2-way analysis of variance (ANOVA). Values were expressed as mean ± SD from independent experiments. The statistical significance of Kaplan-Meier curve was determined by log-rank test. Any difference with a P value less than 0.05 was considered significant.
The ISWAT Is a Novel Functional Site of Islet Transplantation
To analyze the function of the ISWAT transplanted islets, we first used 400 syngeneic islets from 2 donors per transplant because this is the number of islets needed to ameliorate hyperglycemia in STZ-induced diabetic mice after transplantation into the liver (Figure S3, SDC,http://links.lww.com/TP/B546).6,18 We found that the blood glucose levels of diabetic mice receiving 400 syngeneic islets in the ISWAT became less than 300 mg/dL after transplantation (Figure 1B, right), whereas those of nontransplanted mice remained the pretransplant levels of more than 400 mg/dL (Figure 1B, left). If the ISWAT containing transplanted islets were removed after transplantation, the mice promptly became hyperglycemic again (Figure 1B, * in the right panel), indicating that amelioration of hyperglycemia after transplantation is dependent on the transplanted islets and that they remain within the ISWAT.
Intraperitoneal glucose tolerance test revealed that the glucose intolerance of STZ-induced diabetic mice was improved at 120 days after transplantation of islets into the ISWAT, and that this improvement was abolished after retrieval of the transplanted islets (Figure 1C).
Macroscopically, islets grafted in the ISWAT were found to form clusters approximately 1 to 2 mm in diameter with vascularization and located close to the epigastric vessels (Figure 1D). Histologically, transplanted islets were surrounded by adipose tissue in which individual pancreatic endocrine cells were clearly visible (Figure 1E).
Visualization of Transplanted Islets in the ISWAT by μCT
The macroscopic appearance of transplanted islets, forming clusters surrounded by subcutaneous adipose tissue, prompted us to determine whether they could be visualized by CT. To test this, recipient mice bearing functional islet grafts in the ISWAT were analyzed at day 120. First, transplanted islets were exposed under anesthesia and a pencil rod, 0.5 mm in diameter was placed close by as an index of the location as well as the size of transplanted islets (Figure 2A). CT was performed after injection of contrast material via the jugular vein (Figure 2B, and a cluster of transplanted islets could be visualized as an enhanced mass close to the pencil rod (Figure 2C).
Efficiency of Islet Transplantation Into the ISWAT Is Superior to the Liver
It was then critically important to evaluate the efficiency of islet transplantation in a new site of the ISWAT compared with the liver, the currently favored site for clinical islet transplantation. To evaluate the efficiency of islet transplantation into the ISWAT, we reduced the number of syngeneic donor islets from 400 to 200 and further to 100 (half the islet number yielded from 1 mouse pancreas). For these experiments, the mass of donor islets was evaluated objectively by the volume (length) of donor islets occupied in the PE50 tubing after centrifugation at 190G for 1 minute in addition to islet count as expressed by the islet equivalent (IEQ), and found that the number of 100 islets is equal to the islet volume of 1 mm in length in the PE50 tubing and of 250 IEQ by the count (Figure S2, SDC,http://links.lww.com/TP/B546). Then, it was found that hyperglycemia of STZ-diabetic mice was ameliorated after transplantation of 200 islets to the ISWAT (Figure 3A, upper left). When the number of islets was 100, the blood glucose levels of recipient mice became gradually decreased to less than 300 mg/dL in 50% of mice at 150 days after transplantation (Figure 3B, lower right; Kaplan-Meier curve). Removal of the fat tissue containing transplanted islets (* in Figure 3A, upper left and Figure 3B, upper left) in recipient mice bearing 200 or 100 islets, promptly resulted in blood glucose levels of more than 400 mg/dL, indicating that the glucose tolerance of recipient mice is dependent on the transplanted islets. In contrast, when 200 or 100 syngeneic islets were grafted into the liver of STZ-diabetic mice, recipient mice remained hyperglycemic after transplantation (Figure 3A, upper right and Figure 3B, upper right). To examine the glucose tolerance of recipient mice after transplantation of 100 islets in the ISWAT versus the liver, IPGTT was performed at 150 day after transplantation and showed that the glucose tolerance of recipient mice bearing 100 syngeneic islet grafts in the ISWAT was significantly superior to that of mice with 100 intrahepatic islet grafts (Figure 3C). When the efficiency of islet transplantation in the ISWAT was compared with that in the renal subcapsular space, it was found that the efficiency in the ISWAT was equal or inferior to that in the renal subcapsular space when the number of donor islets was 200 (Figure 3A, lower right) or 100 (Figure 3B, lower right), respectively, as shown by Kaplan-Meier curves.
Islet Allograft Rejection in the ISWAT Is Prevented by Blockade of Costimulatory Signals
We next determined whether islet allograft rejection in the ISWAT is preventable, because there have been no reports showing prevention of islet allograft rejection in the subcutaneous space by immunosuppressive agents. Because costimulatory blockade has been reported to be effective in prevention of islet allograft and xenograft rejections in other sites,19-21 we determined whether CTLA4Ig and/or anti-CD40L prevent islet allograft rejection in the ISWAT. We found that the simultaneous IP administration of CTLA4Ig and anti-CD40L antibody 5 times at days 0, 2, 4, 6, and 8 after transplantation to STZ-induced diabetic C57BL/6 mice receiving 400 BALB/c islets produced long-term acceptance of islet allografts, such that 6 of 8 recipient mice became normoglycemic for more than 120 days after transplantation (Figure 4A, upper left), whereas that of control antibodies did not (Figure 4B, lower left) (Figure 4C). Histologically, intact islet cells were seen in the ISWAT accepting islet allografts (Figure 4A, right), whereas transplanted islets infiltrated with mononuclear cells were observed in the ISWAT of control group of mice rejecting islet allografts at 10 days after transplantation (Figure 4B, right). When diabetic mice received 400 BALB/c islets and were treated with either CTLA4Ig or anti-CD40L antibody, only 1 of 6 or 1 of 5 mice was normoglycemic at 120 days after transplantation (Figure S4, SDC,http://links.lww.com/TP/B546).
Human Islets Can Reverse STZ-induced Diabetes in NOD/scid Mice After Transplantation Into the ISWAT
Finally, we determined whether human islets can function when grafted into the ISWAT. To test this, human islets were transplanted into the ISWAT of STZ-induced diabetic NOD/scid mice. Without islet transplantation, all mice remained hyperglycemic and died (Figure 5A, left). In marked contrast, hyperglycemia of the diabetic recipient mice was ameliorated by transplantation of 2000 to 2500 IEQ human islets into the ISWAT, and removal of the islet grafts promptly made recipient mice hyperglycemic again (* in Figure 5A, right). The IPGTT revealed that the blood glucose levels of recipient mice at 0, 30, and 120 minutes after glucose injection were similar to those of untreated naive mice, whereas, in contrast, the glucose tolerance of diabetic mice without islet transplantation was significantly worse (Figure 5B). Furthermore, human c-peptide was detected in the plasma of transplanted mice (Figure 5C). Histologically, the human islet cells were found to be maintained intact (Figure 5D, day 60).
These findings clearly demonstrate that the ISWAT is a novel functional site for islet transplantation. This transplantation therapy ameliorates diabetes in mice, the transplanted islets are easily accessed, visualized by CT and retrievable, and with the improved efficiency compared with the liver, currently favorable site of clinical islet transplantation. Furthermore, islet allograft rejection is preventable by costimulatory blockade.
Currently, the liver via the portal vein is the preferred site of clinical islet transplantation, and transplanted islets delivered by this method become lodge in the periphery of the liver. Because the average size of individual islets is 150 to 250 μm in diameter,22 transplanted islets in the liver are hardly detected by ultrasound, MRI or CT after transplantation, which makes it almost impossible to evaluate the transplanted islets when necessary, such as at the time of rejection. Thus, from the clinical perspective, the accessibility to transplanted islets is of great importance and significance to evaluate their morphology as well as function. In the present study, we found that transplanted islets in the ISWAT are engrafted together to form clusters 1 to 2 mm in diameter, which allows their visualization by CT.
The most important finding in the present study is that the efficiency of islet transplantation into the ISWAT is superior to that when the liver is the site of transplantation. Previously, we have shown that early loss of transplanted islets caused by innate immune rejection is a major factor responsible for the low efficiency of islet transplantation in the liver and that islet transplantation from 1 donor to 1 recipient in mice becomes feasible when innate immune rejection is prevented by targeting HMGB1-NKT cell-mediated pathway in the liver.6,18,23,24 In the present study, we show that the efficiency of islet transplantation in the ISWAT is superior to that in the liver because hyperglycemia of STZ-induced diabetic mice was ameliorated after transplantation of 200 syngeneic islets, which is the number of islets yielded from 1 mouse pancreas, without any treatment when the site of transplantation was the ISWAT but not when it was the liver (Figure 3A). The superiority in the efficiency of islet transplantation in the ISWAT to the liver may depend on the difference in innate immune response in the site after islet transplantation, although it remains undetermined and is a matter of future investigation.
In the current clinical setting, where the liver is the site of transplantation, sequential islet transplantations with the use of 2 to 3 donor pancreases are required for the treatment of a single IDDM patient.1-4 Thus, it will have a tremendous impact on clinical islet transplantation if the beneficial effect of islet transplantation into the ISWAT is recapitulated in humans. As to one of the potential reasons for the improved efficiency of islet transplantation into the ISWAT of mice, the present study may suggest that β-cell mass in the adipose tissue containing transplanted islets may increase after transplantation, based on the finding that the blood glucose levels of recipient mice declined gradually after transplantation. The issue is of interest and need to determine whether transplanted islets expand after transplantation in this particular site.
Recently, it has been reported that insulin-producing cells other than allogenic islets, including islets from other species, such as pig25 and those derived from embryonic stem cells26 and induced pluripotent stem cells,27,28 might be potential donor sources. This may afford a solution to an essential obstacle facing clinical islet transplantation, the shortage of donor organs for allogenic islet transplantation. For transplantation of these cells, subcutaneous site has now become of much attention with the strategy to induce new vascular formation by the treatment of the subcutaneous site before transplantation to improve engraftments of transplanted islets.11,29 In this regard, the ISWAT might have an advantage because it contains the feeding vessels of the epigastric artery and vein (Figures S1 and S5, SDC,http://links.lww.com/TP/B546) and, therefore, there is no need for such pretreatment of transplant site to induce neovascularization. Thus, the ISWAT might be an ideal site for transplantation of these insulin-producing cells, where the transplanted cells can be easily evaluated and retrieved when necessary in case of serious side effects, such as infection or tumor formation after transplantation.
Taken collectively, the findings reported here should have a significant impact on islet transplantation by demonstrating that the ISWAT is a novel functional site, providing a potential solution to current obstacles facing clinical islet transplantation if the finding obtained in mice is also the case in humans.
The authors are grateful to Dr. Peter Burrows for helpful comments and constructive criticisms in the preparation of the manuscript. Technical supports by Yuriko Hamaguchi, Nozomi Okuyama and Yuri Otsu, Islet Institute Fukuoka University, are greatly appreciated.
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