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Subcapsular Implantation of Pancreatic Islets in Syngeneic, Allogeneic, and Xenogeneic Mice
Subcapsular Implantation of Pancreatic Islets in Syngeneic, Allogeneic, and Xenogeneic Mice S.-N. Ma, Y.-H. Yuan, X.-R. Guo, and D.-S. Li* Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China
ABSTRACT Background. Reliable and reproducible transplantation is essential to the success of a number of particular investigations. Renal subcapsule is the most selected site for islet transplantation mainly owing to its easy access, readiness for retrieval, and possibility of reimplantation. Methods. Syngeneic, allogeneic, and xenogeneic islets were transplanted into kidney capsules of Balb/C and C57BL/6J mice, and the blood glucose levels of the experimental animals were periodically monitored. Detailed procedures on mouse diabetic model and islet implantation are described. Results. The values of blood glucose measured under varied transplant circumstances are presented, covering syngeneic, allogeneic, and xenogeneic islet transplantations. The methodology is straightforward and has been proven to be practicable and reproducible. Conclusions. The parallel observations in different transplant situations provide a valuable contribution to and useful information for diabetes-related studies.
I
SLET transplantation has become a very progressive diabetes research field in the recent few decades. It took almost 30 years from the first islet transplantation in rats to the first successful islet allotransplantation in patients with type 1 diabetes [1,2]. The transplantation of islets is widely used as an important method in almost all diabetes-related investigations, including diabetic therapy, antirejection drug selection, xeno-islet searching, and stem cellebased islet replacement. An ideal islet transplantation protocol is the prerequisite base for reliable outcomes and meaningful and accurate results of diabetic studies. As a concomitant methodology of the protocol for islet isolation from mouse pancreas [3], we present a practicable and reliable methodology for kidney subcapsular islet transplantation in mice. METHODS Animals Balb/C and C57BL/6J mice (8e12 weeks old) were provided by the animal care units of the Hubei University of Medicine and the University of British Columbia. All animal experiment protocols were conducted with appropriate permission from the Animal Rights Committee of China. Streptozotocin (STZ) ª 2016 Published by Elsevier Inc. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 48, 2821e2825 (2016)
and Nembutal solutions (50 mg/mL) were purchased from Sigma.
Preparation of Diabetic Mice Appropriate amounts of STZ were dissolved with STZ-acetate buffer (pH 4.5) and injected into each mouse under investigation (175e250 mg/kg of body weight, intraperitoneally [IP]). The blood glucose levels of the experimental mice were monitored with the use of a glucose meter on days 2, 3, and 4. When the animals were hyperglycemic (blood glucose, >18 mmol/L) on 2 consecutive days they were considered to be ready for islet transplantation.
Funding: National Natural Science Fund of China (81070614), Provincial Natural Science Fund of Hubei (2008CDA044), Canadian Institute of Health Research (mop-79414), and Juvenile Diabetes Research Foundation (1-2008-474). *Address correspondence to Dong-Sheng Li, Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, People’s Republic of China. E-mail: [email protected] 0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.06.045
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MA, YUAN, GUO ET AL
Fig 1. Packaging islets into transplantation tubing. A given amount of islets for each mouse were kept in an Eppendorf tube. After the islets settled down, they were collected into PE-50 tubing with the aid of a microsyringe. The islet-containing tubing was folded and inserted into a 200-mL pipette tip, then put into a 15-mL conical centrifuge tube. An islet pellet was formed after centrifugation. The islet pelletecontaining tubing was mounted onto the microsyringe for transplantation.
Packaging Islets for Transplantation A predetermined amount of islets for each recipient was individually transferred into 1.5-mL Eppendorf tubes, and the islets were allowed to settle down to the bottom. Then the islets were collected into PE-50 tubing at about one- half of the way of the full length of the tubing (15e20 cm) with the use of a microsyringe (Hamilten threaded plunger syringe; Fisher), and then the PE-50 tubing was folded at about the halfway point, leaving all of the islets in one end (Fig 1). Next, the folded PE-50 tubing was inserted into a 200-mL pipette tip, put into a 15-mL centrifuge tube, and centrifuged at 2,500 rpm for 10 minutes at 4 C (Fig 1).
After centrifugation, the PE-50 tubing was attached to a microsyringe again, the pipette tip was removed, and a cant was cut at about 1 cm from the islet pellet.
Islet Transplantation Each diabetic mouse was anesthetized with the use of Nembutal (w0.05 mL IP per mouse), and its left flank was shaved. Then the shaved area was swabbed with iodophors, and a small incision was made through the skin and muscle of the left back side of the animal. The kidney was exposed outside the body with the use of 2 saline solutionewetted cotton-tipped applicators. A slight pressure
Fig 2. Illustration of renal subcapsule pouch making and islet transplantation. The stepwise flow chart is described as follows: Punch a hole on the top pole of the kidney / Insert a glass rod and make a subcapsular pouch / Insert the islet-containing tubing with the aid of the glass rod / Move the tubing back and forth as indicated by arrows / Release the islets into the bottom of the pouch with the aid of microsyringe.
ISLET TRANSPLANTATION IN MICE
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Fig 3. Blood glucose monitoring in diabetic mice. STZ-induced diabetic mice were maintained in the institutional animal center with free access to water and chow. Blood glucose concentration was continuously assessed and recorded in the control animals (n ¼ 4; red lines) and the islet-transplanted animals (n ¼ 7; green lines), respectively. Female C57BL/6J mice were used as recipients and male C57BL/6J mice as donors. In each animal, 400 islets were subcapsularly transplanted. Abbreviations: Tx, islet transplantation; Nc, nephrectomy.
Fig 4. Blood glucose monitoring in diabetic mice with 2 time points of islet transplantation. Each STZ-induced diabetic C57BL/6J mouse (n ¼ 7) were transplanted in the left kidney with 400 syngeneic islets on day 3. The left kidney was removed on day 65, and the 2nd islet transplantation was performed on the right kidney on day 70. Abbreviations: Tx, islet transplantation; Nc, nephrectomy.
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MA, YUAN, GUO ET AL
Fig 5. Blood glucose monitoring in diabetic mice with different types of islet transplantation. The left panels display the distributions of individual blood glucose detection. The right panels represent the trends of blood glucose changes as mean SE. Abbreviation: Tx, islet transplantation. (A) Syngeneic islet transplantation (n ¼ 11). Simultaneously with the other groups, 400 syngeneic islets were transplanted into each C57Bl/6J. However, the actual observation time was up to 120 days in this group. (B) Allogeneic islet transplantation (n ¼ 28). Each C57Bl/6J mouse were transplanted with 400 islets isolated from Balb/C mice. (C) Xenogeneic islet transplantation (n ¼ 15). C57Bl/6J mice were each transplanted with 3,000 IEQ human islets provided from Ike Barber Human Islet Transplant Laboratory at the University of British Columbia.
was applied to both sides of the incision, to raise or pop the kidney out of the abdominal cavity. The kidney was kept moist by applying saline solution with the use of a cotton-tipped swab. Through the use of a 25-G syringe needle, a small scratch was made on the upper pole of the kidney, creating a nick in the kidney capsule (Fig 2). Next, an “L”-type glass rod was inserted into the hole in the capsule and was carefully moved under the capsule to make a small pouch (Fig 2). The capsule was lifted slightly by the glass rod, and the islet-containing PE-50 tubing was carefully inserted into the pouch. Then, the isletpellet was released with the aid of a microsyringe. Once all of the islets were inside the pouch, the tubing was removed slowly, and the pouch was quickly sealed by cauterization. The muscle layer was sewn with a 4e0 absorbable silk suture, and the skin was closed with 2e3 staples.
Post-transplantation Recovery and Follow-up Each mouse under investigation was placed on a heating blanket and was injected with 1 mL of saline solution (IP). The animals were kept individually until completely recovered. During the
experimental period, the mice were allowed free access to tap water and chow. Blood glucose was measured every other day after transplantation.
RESULTS AND DISCUSSION
Blood glucose changes of diabetic mice are presented in Fig 3. The STZ-induced hyperglycemia was promptly corrected by syngeneic islet transplantation. The corrected euglycemia was reversed to hyperglycemia when the islet graftebearing kidney was removed, thereby confirming the function of the transplanted islets. A short-term rebound of blood glucose levels after islet transplantation is not a rare phenomenon. It might be due to operation-related stress and/or manipulation-induced islet instability. The hyperglycemia that developed after nephrectomy in the STZ-induced diabetic mice was corrected again by the 2nd transplantation on the other kidney (Fig 4). The 2nd islet transplantation may be a useful option for some investigations. The
ISLET TRANSPLANTATION IN MICE
immunohistochemistry characteristics of the islet grafts are not given in the present protocol. However, interested investigators may refer to our previous report [4]. The period of time until the rejection of islet grafts is determined mainly by the histologic conformity between donor and recipient. Figure 5 displays the varied patterns under different transplant combinations. The syngeneic islet transplantation showed no rejection during the observation period (Fig 5A), whereas evident rejection started on days 10e14 after the transplantation of allogeneic islets (Fig 5B). Rejection after the xenogeneic islet transplantation, however, took place almost instantly after the transplantation procedures (Fig 5C). CONCLUSION
Currently, the hepatic portal vein is widely used in clinical trials as the site for islet transplantation. However, the long-term outcomes of islet transplantation are far from satisfactory [5,6], which is mainly due to the extremely high concentration of glucose and antirejection drugs in the hepatic portal environment [7]. It is therefore reasonable to propose alternate potential implantation sites [8]. Various other targets are considered or are under experimental studies, including pancreas [9], spleen [10,11], bone marrow [12], omental pouch [13], muscle [14], and kidney [15]. A number of comparative studies have revealed that the kidney subcapsule is the best site for islet implantation in mice regarding operative feasibility, implantation efficiency, and glycemic control efficiency [10,16]. Among the potential sites for transplantation, mouse renal subcapsular islet transplantation holds some unique advantages: 1) feasible graft removal for the confirmation of graft function; 2) potential to use both kidneys for some paralleled and controlled studies; and 3) possibility to perform graft retransplantation. Renal subcapsular islet transplantation has been the technique most frequently used in mouse models. For experimental reference purposes, the fluctuations in blood glucose levels during the experimental period were shown, representing the changes in blood glucose rates after the islet transplantation and nephrectomy, respectively. Furthermore, 3 types of islet transplantations were performed: syngeneic, allogeneic, and xenogeneic. This information may be referenced during the implementation of certain investigations. In summary, a practical and reliable methodology for kidney subcapsular islet transplantation in mice was introduced. The representative values of blood glucose
2825
measurements were presented after syngeneic, allogeneic, and xenogeneic islet transplantation in mice, which can serve as references for further diabetes-related research. ACKNOWLEDGMENTS The authors are grateful to Crystal Robertson for her assistance in the manuscript preparation.
REFERENCES [1] Ballinger WF, Lacy PE. Transplantation of intact pancreatic islets in rats. Surgery 1972;72:175e86. [2] Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000;343:230e8. [3] Li DS, Yuan YH, Tu HJ, Liang QL, Dai LJ. A protocol for islet isolation from mouse pancreas. Nat Protoc 2009;4: 1649e52. [4] Wang X, Hao J, Metzger DL, et al. Endogenous expression of B7-H4 improves long-term murine islet allograft survival. Transplantation 2013;95:94e9. [5] Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006;355:1318e30. [6] Robertson RP. Islet transplantation a decade later and strategies for filling a half-full glass. Diabetes 2010;59:1285e91. [7] Vardanyan M, Parkin E, Gruessner C, Rodriguez Rilo HL. Pancreas vs islet transplantation: a call on the future. Curr Opin Organ Transplant 2010;15:124e30. [8] Rajab A. Islet transplantation: alternative sites. Curr Diab Rep 2010;10:332e7. [9] Rilo HL, Fontes PA, Nussler AK, et al. Intrapancreatic islet transplantation as a potential solution to chronic failure of intraportal islet grafts. Transplant Proc 1993;25:988. [10] Mellgren A, Schnell Landstrom AH, Petersson B, Andersson A. The renal subcapsular site offers better growth conditions for transplanted mouse pancreatic islet cells than the liver or spleen. Diabetologia 1986;29:670e2. [11] Gray DW. Islet isolation and transplantation techniques in the primate. Surg Gynecol Obstet 1990;170:225e32. [12] Salazar-Banuelos A, Wright J, Sigalet D, BenitezBribiesca L. The bone marrow as a potential receptor site for pancreatic islet grafts. Arch Med Res 2008;39:139e41. [13] Stagner JI, Samols E. Induction of angiogenesis by growth factors: relevance to pancreatic islet transplantation. Exs 1992;61: 381e5. [14] Weber CJ, Hardy MA, Pi-Sunyer F, Zimmerman E, Reemtsma K. Tissue culture preservation and intramuscular transplantation of pancreatic islets. Surgery 1978;84:166e74. [15] Leow CK, Shimizu S, Gray DW, Morris PJ. Successful pancreatic islet autotransplantation to the renal subcapsule in the cynomolgus monkey. Transplantation 1994;57:161e4. [16] Jaeger C, Wohrle M, Federlin K, Bretzel RG. Pancreatic islet xenografts at two different transplantation sites (renal subcapsular versus intraportal): comparison of graft survival and morphology. Exp Clin Endocrinol Diabetes 1995;103(Suppl 2): 123e8.
ABSTRACT Background. Reliable and reproducible transplantation is essential to the success of a number of particular investigations. Renal subcapsule is the most selected site for islet transplantation mainly owing to its easy access, readiness for retrieval, and possibility of reimplantation. Methods. Syngeneic, allogeneic, and xenogeneic islets were transplanted into kidney capsules of Balb/C and C57BL/6J mice, and the blood glucose levels of the experimental animals were periodically monitored. Detailed procedures on mouse diabetic model and islet implantation are described. Results. The values of blood glucose measured under varied transplant circumstances are presented, covering syngeneic, allogeneic, and xenogeneic islet transplantations. The methodology is straightforward and has been proven to be practicable and reproducible. Conclusions. The parallel observations in different transplant situations provide a valuable contribution to and useful information for diabetes-related studies.
I
SLET transplantation has become a very progressive diabetes research field in the recent few decades. It took almost 30 years from the first islet transplantation in rats to the first successful islet allotransplantation in patients with type 1 diabetes [1,2]. The transplantation of islets is widely used as an important method in almost all diabetes-related investigations, including diabetic therapy, antirejection drug selection, xeno-islet searching, and stem cellebased islet replacement. An ideal islet transplantation protocol is the prerequisite base for reliable outcomes and meaningful and accurate results of diabetic studies. As a concomitant methodology of the protocol for islet isolation from mouse pancreas [3], we present a practicable and reliable methodology for kidney subcapsular islet transplantation in mice. METHODS Animals Balb/C and C57BL/6J mice (8e12 weeks old) were provided by the animal care units of the Hubei University of Medicine and the University of British Columbia. All animal experiment protocols were conducted with appropriate permission from the Animal Rights Committee of China. Streptozotocin (STZ) ª 2016 Published by Elsevier Inc. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 48, 2821e2825 (2016)
and Nembutal solutions (50 mg/mL) were purchased from Sigma.
Preparation of Diabetic Mice Appropriate amounts of STZ were dissolved with STZ-acetate buffer (pH 4.5) and injected into each mouse under investigation (175e250 mg/kg of body weight, intraperitoneally [IP]). The blood glucose levels of the experimental mice were monitored with the use of a glucose meter on days 2, 3, and 4. When the animals were hyperglycemic (blood glucose, >18 mmol/L) on 2 consecutive days they were considered to be ready for islet transplantation.
Funding: National Natural Science Fund of China (81070614), Provincial Natural Science Fund of Hubei (2008CDA044), Canadian Institute of Health Research (mop-79414), and Juvenile Diabetes Research Foundation (1-2008-474). *Address correspondence to Dong-Sheng Li, Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, People’s Republic of China. E-mail: [email protected] 0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.06.045
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MA, YUAN, GUO ET AL
Fig 1. Packaging islets into transplantation tubing. A given amount of islets for each mouse were kept in an Eppendorf tube. After the islets settled down, they were collected into PE-50 tubing with the aid of a microsyringe. The islet-containing tubing was folded and inserted into a 200-mL pipette tip, then put into a 15-mL conical centrifuge tube. An islet pellet was formed after centrifugation. The islet pelletecontaining tubing was mounted onto the microsyringe for transplantation.
Packaging Islets for Transplantation A predetermined amount of islets for each recipient was individually transferred into 1.5-mL Eppendorf tubes, and the islets were allowed to settle down to the bottom. Then the islets were collected into PE-50 tubing at about one- half of the way of the full length of the tubing (15e20 cm) with the use of a microsyringe (Hamilten threaded plunger syringe; Fisher), and then the PE-50 tubing was folded at about the halfway point, leaving all of the islets in one end (Fig 1). Next, the folded PE-50 tubing was inserted into a 200-mL pipette tip, put into a 15-mL centrifuge tube, and centrifuged at 2,500 rpm for 10 minutes at 4 C (Fig 1).
After centrifugation, the PE-50 tubing was attached to a microsyringe again, the pipette tip was removed, and a cant was cut at about 1 cm from the islet pellet.
Islet Transplantation Each diabetic mouse was anesthetized with the use of Nembutal (w0.05 mL IP per mouse), and its left flank was shaved. Then the shaved area was swabbed with iodophors, and a small incision was made through the skin and muscle of the left back side of the animal. The kidney was exposed outside the body with the use of 2 saline solutionewetted cotton-tipped applicators. A slight pressure
Fig 2. Illustration of renal subcapsule pouch making and islet transplantation. The stepwise flow chart is described as follows: Punch a hole on the top pole of the kidney / Insert a glass rod and make a subcapsular pouch / Insert the islet-containing tubing with the aid of the glass rod / Move the tubing back and forth as indicated by arrows / Release the islets into the bottom of the pouch with the aid of microsyringe.
ISLET TRANSPLANTATION IN MICE
2823
Fig 3. Blood glucose monitoring in diabetic mice. STZ-induced diabetic mice were maintained in the institutional animal center with free access to water and chow. Blood glucose concentration was continuously assessed and recorded in the control animals (n ¼ 4; red lines) and the islet-transplanted animals (n ¼ 7; green lines), respectively. Female C57BL/6J mice were used as recipients and male C57BL/6J mice as donors. In each animal, 400 islets were subcapsularly transplanted. Abbreviations: Tx, islet transplantation; Nc, nephrectomy.
Fig 4. Blood glucose monitoring in diabetic mice with 2 time points of islet transplantation. Each STZ-induced diabetic C57BL/6J mouse (n ¼ 7) were transplanted in the left kidney with 400 syngeneic islets on day 3. The left kidney was removed on day 65, and the 2nd islet transplantation was performed on the right kidney on day 70. Abbreviations: Tx, islet transplantation; Nc, nephrectomy.
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Fig 5. Blood glucose monitoring in diabetic mice with different types of islet transplantation. The left panels display the distributions of individual blood glucose detection. The right panels represent the trends of blood glucose changes as mean SE. Abbreviation: Tx, islet transplantation. (A) Syngeneic islet transplantation (n ¼ 11). Simultaneously with the other groups, 400 syngeneic islets were transplanted into each C57Bl/6J. However, the actual observation time was up to 120 days in this group. (B) Allogeneic islet transplantation (n ¼ 28). Each C57Bl/6J mouse were transplanted with 400 islets isolated from Balb/C mice. (C) Xenogeneic islet transplantation (n ¼ 15). C57Bl/6J mice were each transplanted with 3,000 IEQ human islets provided from Ike Barber Human Islet Transplant Laboratory at the University of British Columbia.
was applied to both sides of the incision, to raise or pop the kidney out of the abdominal cavity. The kidney was kept moist by applying saline solution with the use of a cotton-tipped swab. Through the use of a 25-G syringe needle, a small scratch was made on the upper pole of the kidney, creating a nick in the kidney capsule (Fig 2). Next, an “L”-type glass rod was inserted into the hole in the capsule and was carefully moved under the capsule to make a small pouch (Fig 2). The capsule was lifted slightly by the glass rod, and the islet-containing PE-50 tubing was carefully inserted into the pouch. Then, the isletpellet was released with the aid of a microsyringe. Once all of the islets were inside the pouch, the tubing was removed slowly, and the pouch was quickly sealed by cauterization. The muscle layer was sewn with a 4e0 absorbable silk suture, and the skin was closed with 2e3 staples.
Post-transplantation Recovery and Follow-up Each mouse under investigation was placed on a heating blanket and was injected with 1 mL of saline solution (IP). The animals were kept individually until completely recovered. During the
experimental period, the mice were allowed free access to tap water and chow. Blood glucose was measured every other day after transplantation.
RESULTS AND DISCUSSION
Blood glucose changes of diabetic mice are presented in Fig 3. The STZ-induced hyperglycemia was promptly corrected by syngeneic islet transplantation. The corrected euglycemia was reversed to hyperglycemia when the islet graftebearing kidney was removed, thereby confirming the function of the transplanted islets. A short-term rebound of blood glucose levels after islet transplantation is not a rare phenomenon. It might be due to operation-related stress and/or manipulation-induced islet instability. The hyperglycemia that developed after nephrectomy in the STZ-induced diabetic mice was corrected again by the 2nd transplantation on the other kidney (Fig 4). The 2nd islet transplantation may be a useful option for some investigations. The
ISLET TRANSPLANTATION IN MICE
immunohistochemistry characteristics of the islet grafts are not given in the present protocol. However, interested investigators may refer to our previous report [4]. The period of time until the rejection of islet grafts is determined mainly by the histologic conformity between donor and recipient. Figure 5 displays the varied patterns under different transplant combinations. The syngeneic islet transplantation showed no rejection during the observation period (Fig 5A), whereas evident rejection started on days 10e14 after the transplantation of allogeneic islets (Fig 5B). Rejection after the xenogeneic islet transplantation, however, took place almost instantly after the transplantation procedures (Fig 5C). CONCLUSION
Currently, the hepatic portal vein is widely used in clinical trials as the site for islet transplantation. However, the long-term outcomes of islet transplantation are far from satisfactory [5,6], which is mainly due to the extremely high concentration of glucose and antirejection drugs in the hepatic portal environment [7]. It is therefore reasonable to propose alternate potential implantation sites [8]. Various other targets are considered or are under experimental studies, including pancreas [9], spleen [10,11], bone marrow [12], omental pouch [13], muscle [14], and kidney [15]. A number of comparative studies have revealed that the kidney subcapsule is the best site for islet implantation in mice regarding operative feasibility, implantation efficiency, and glycemic control efficiency [10,16]. Among the potential sites for transplantation, mouse renal subcapsular islet transplantation holds some unique advantages: 1) feasible graft removal for the confirmation of graft function; 2) potential to use both kidneys for some paralleled and controlled studies; and 3) possibility to perform graft retransplantation. Renal subcapsular islet transplantation has been the technique most frequently used in mouse models. For experimental reference purposes, the fluctuations in blood glucose levels during the experimental period were shown, representing the changes in blood glucose rates after the islet transplantation and nephrectomy, respectively. Furthermore, 3 types of islet transplantations were performed: syngeneic, allogeneic, and xenogeneic. This information may be referenced during the implementation of certain investigations. In summary, a practical and reliable methodology for kidney subcapsular islet transplantation in mice was introduced. The representative values of blood glucose
2825
measurements were presented after syngeneic, allogeneic, and xenogeneic islet transplantation in mice, which can serve as references for further diabetes-related research. ACKNOWLEDGMENTS The authors are grateful to Crystal Robertson for her assistance in the manuscript preparation.
REFERENCES [1] Ballinger WF, Lacy PE. Transplantation of intact pancreatic islets in rats. Surgery 1972;72:175e86. [2] Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000;343:230e8. [3] Li DS, Yuan YH, Tu HJ, Liang QL, Dai LJ. A protocol for islet isolation from mouse pancreas. Nat Protoc 2009;4: 1649e52. [4] Wang X, Hao J, Metzger DL, et al. Endogenous expression of B7-H4 improves long-term murine islet allograft survival. Transplantation 2013;95:94e9. [5] Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006;355:1318e30. [6] Robertson RP. Islet transplantation a decade later and strategies for filling a half-full glass. Diabetes 2010;59:1285e91. [7] Vardanyan M, Parkin E, Gruessner C, Rodriguez Rilo HL. Pancreas vs islet transplantation: a call on the future. Curr Opin Organ Transplant 2010;15:124e30. [8] Rajab A. Islet transplantation: alternative sites. Curr Diab Rep 2010;10:332e7. [9] Rilo HL, Fontes PA, Nussler AK, et al. Intrapancreatic islet transplantation as a potential solution to chronic failure of intraportal islet grafts. Transplant Proc 1993;25:988. [10] Mellgren A, Schnell Landstrom AH, Petersson B, Andersson A. The renal subcapsular site offers better growth conditions for transplanted mouse pancreatic islet cells than the liver or spleen. Diabetologia 1986;29:670e2. [11] Gray DW. Islet isolation and transplantation techniques in the primate. Surg Gynecol Obstet 1990;170:225e32. [12] Salazar-Banuelos A, Wright J, Sigalet D, BenitezBribiesca L. The bone marrow as a potential receptor site for pancreatic islet grafts. Arch Med Res 2008;39:139e41. [13] Stagner JI, Samols E. Induction of angiogenesis by growth factors: relevance to pancreatic islet transplantation. Exs 1992;61: 381e5. [14] Weber CJ, Hardy MA, Pi-Sunyer F, Zimmerman E, Reemtsma K. Tissue culture preservation and intramuscular transplantation of pancreatic islets. Surgery 1978;84:166e74. [15] Leow CK, Shimizu S, Gray DW, Morris PJ. Successful pancreatic islet autotransplantation to the renal subcapsule in the cynomolgus monkey. Transplantation 1994;57:161e4. [16] Jaeger C, Wohrle M, Federlin K, Bretzel RG. Pancreatic islet xenografts at two different transplantation sites (renal subcapsular versus intraportal): comparison of graft survival and morphology. Exp Clin Endocrinol Diabetes 1995;103(Suppl 2): 123e8.