Yijun Zeng

Islet isolation assessment in man and large animals

SummaryRecent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity andin vitro andin vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 µ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason,in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers.

Pancreatic islet transplantation after upper abdominal exenteration and liver replacement

Nine patients who became diabetic after upper-abdominal exenteration and liver transplantation were given pancreatic islet-cell grafts obtained from the liver donor (eight cases), a third-party donor (one), or both (four). Two patients were diabetic when they died of infections after 48 and 109 days, as was a third patient who died of tumour recurrence after 178 days. The other 6 are alive 101-186 days postoperatively, and five are insulin-free or on insulin only during night-time parenteral alimentation. C-peptide increased 1.7 to 3.3 fold in response to intravenous glucose in these five patients who have had glycosylated haemoglobin in the high normal range. However, the kinetics of the C-peptide responses to intravenous glucose in all eight patients tested revealed an absent first-phase release and a delayed peak response consistent with transplantation and/or engraftment of a suboptimal islet cell mass. The longest survivor, who requires neither parenteral alimentation nor insulin, is the first unequivocal example of successful clinical islet-cell transplantation.

Human islet isolation and allotransplantation in 22 consecutive cases

This report provides our initial experience in islet isolation and intrahepatic allotransplantation in 21 patients. In group 1, 10 patients underwent combined liver-islet allotransplantation following upper-abdominal exenteration for cancer. In group 2, 4 patients received a combined liver-islet allograft for cirrhosis and diabetes. One patients had plasma C-peptide greater than 3 pM and was therefore excluded from analysis. In group 3, 7 patients received 8 combined cadaveric kidney-islet grafts (one retransplant) for end-stage renal disease secondary to type 1 diabetes mellitus. The islets were separated by a modification of the automated method for human islet isolation and the preparation were infused into the portal vein. Immunosuppression was with FK506 (group 1) plus steroids (groups 2 and 3). Six patients in group 1 did not require insulin treatment for 5 to greater than 16 months. In groups 2 and 3 none of the patients became insulin-independent, although decreased insulin requirement and stabilization of diabetes were observed. Our results indicate that rejection is still a major factor limiting the clinical application of islet transplantation in patients with type 1 diabetes mellitus, although other factors such as steroid treatment may contribute to deteriorate islet engraftment and/or function.

In vivo effect of FK506 on human pancreatic islets.

The purpose of this study was to evaluate the in vivo effect of FK506 on human pancreatic islets. Twenty-five nude mice were made diabetic by one intravenous injection of streptozotocin. Approximately 600 islets were administered in the renal subcapsular space 3-5 days following streptozotocin administration. One week after transplantation, the mice were divided in four groups. In group 1, the animals received 1 injection of 0.5 ml of diluent i.p. daily for one week. In groups 2, 3, and 4 the treatments were daily i.p. injection of 0.3, 1, and 3 mg/kg FK506, respectively. After treatment, the functional integrity of the transplanted human islets was tested by measuring the plasma glucose and human C-peptide response to intraperitoneal glucose injection in groups 1 and 4. IPGTT alone was assessed in groups 2 and 3. The results indicate that i.p. administration of FK506 for one week at a dose 0.3 mg/kg/day did not result in any significant alteration of glucose disappearance and C-peptide response to IPGTT. Higher doses of FK506 produced a significant delay in glucose disappearance in groups 3 and 4, and a significant inhibition of glucose-mediated C-peptide response in group 4.

LONG-TERM SURVIVAL OF DONOR-SPECIFIC PANCREATIC ISLET XENOGRAFTS IN FULLY XENOGENEIC CHIMERAS (WF RAT → B10 MOUSE)

We recently reported that reconstitution of lethally irradiated B10 mouse recipients with 40 x 10(6) untreated WF rat bone marrow cells resulted in stable fully xenogeneic chimerism (WF rat----B10 mouse). In these animals, the tolerance induced for skin xenografts was highly MHC specific in that donor-specific WF rat skin grafts were significantly prolonged while MHC-disparate third-party xenografts were rapidly rejected (median survival time [MST] = 9 days). We have now examined whether islet cell xenografts placed under the renal capsule of chimeras rendered diabetic with streptozotocin would be accepted and remain functional to maintain euglycemia. Animals were prepared, typed for chimerism at 6 weeks, and diabetes induced with streptozotocin. Donor-specific WF (Rt1Au) islet cell xenografts were significantly prolonged (MST greater than 180 days) in WF----B10 chimeras, while MHC-disparate third-party F344 rat (Rt1A1) grafts were rejected with a time course similar to unmanipulated B10 mice (MST = 8 days). The transplanted donor-specific islet cells were functional to maintain euglycemia, since removal of the grafts at from 100 to 180 days in selected individual chimeras uniformly resulted in return of the diabetic state. These data suggest that donor-specific islet cell xenografts are accepted and remain functional in mice rendered tolerant to rat xenoantigens following bone marrow transplantation.

Long-term (>3-Year) insulin independence in a patient with pancreatic islet cell transplantation following upper abdominal exenteration and liver replacement for fibrolamellar hepatocellular carcinoma

In the University of Pittsburgh experience, the most successful setting for human islet allografts is in patients undergoing upper abdominal exenteration with total pancreatectomy and liver transplantation for the indication of malignancy (cluster). In this group of patients 6/11 were insulin-independent for long periods. We report herein the metabolic course or the longest survivor (> 3 years). This patient has been free of exogenous insulin since the third postoperative month and has sustained her body weight without total parenteral nutrition since the 4th postoperative month. The patient has some postprandial hyperglycemia but average capillary glucoses are near-normal to normal as are glycosylated hemoglobin values. The clearance of glucose during the administration of an intravenous glucose load has been well preserved and is currently normal. C-peptide stimulates significantly in response to intravenously injected glucose. The absolute levels of stimulation during the test have declined possibly related to improvements in renal function, decreased immunosuppression or the natural history of cells transplanted into the portal site. The kinetics of the C-peptide response to intravenously injected glucose shows a persistent abnormality of first-phase insulin release and a prolonged second phase release. Basal glucagon levels are low but stimulate to a mixed meal. This patients results demonstrate long-term function of islet cells from a single donor transplanted into the portal vein using FK506 as an immunosuppressant agent.

Acceleration of chronic failure of intrahepatic canine islet autografts by a short course of prednisone

A topic of current interest in islet transplantation is the selection of an optimal site for long-term graft survival since the intrahepatic site may be characterized by long-term failure. Additionally, the use of immunosuppressive agents such as prednisone may adversely affect long-term graft function. In this study, we examined the long-term outcome of intrahepatic canine islet autografts and compared this with results obtained in animals treated with a short-term course of steroids or steroids plus insulin. Islets were isolated using the automated method and were purified on discontinuous Euro-Collins Ficoll gradients (densities: 1.108, 1.096, 1.037). Prednisone-treated dogs were hyperglycemic during treatment but returned to normoglycemia after steroid withdrawal. Control and insulin-treated animals were normoglycemic following autotransplant, with no difference in plasma glucose levels between controls and the insulin-treated animals. All control dogs became diabetic at 11, 14, 17, and 19 months following islet autograft. Prednisone-treated dogs had more rapid onset of diabetes at 7, 11, and 12 months following ITx. Prednisone-treated dogs given insulin became hyperglycemic at 10, 14, 18, and 19 months post ITx. Graft failure was preceded by a decline in IVGTT Kg values and diminished insulin secretion. At the time of graft failure islets showed no lymphocytic infiltration and islets stained positive for glucagon but few insulin-containing cells were seen. Thus, even when an initially adequate B cell mass was transplanted, the intrahepatic site was characterized by long-term canine autograft failure. A short course of prednisone accelerated the time to graft failure and insulin treatment reversed this acceleration.

Detection of pancreatic islet tissue following islet allotransplantation in man.

Qualitative and quantitative standards have been recently proposed for islet isolation assessment in humans and large animals (1). The ultimate test of viability is functional activity after transplantation into a diabetic recipient. Morphologic identification of insulin-containing transplanted islets could provide important correlative data and eventually confirm the functional results. Recently, several centers reported prolonged insulin independence after human islet allotransplantation (2–5). The present report describes the first identification of islet tissue in a liver biopsy after intrahepatic islet allotransplantation in man. Human islets were obtained from 2 multiorgan donors by a modification (6) of the automated method for human islet isolation (7). The islets were purified on Eurocollins-Ficoll gradients (8) using a cell separator (9, 10) (COBE 2991, Lakewood, CO). On March 16, 1990 258,000 islets of an average diameter of 150 μ (1) were transplanted via portal vein injection following an upper abdominal exenteration and liver replacement (2). The procedure was performed in a 52-year-old woman for adenocarcinoma of the ampulla of Vater infiltrating the duodenal wall, with liver metastasis. Immunosuppression was with FK506 as described previously (2).

Detection of Intrahepatic Human Islets Following Combined Liver-Islet Allotransplantation

This article describes the localization of intact insulin-containing intrahepatic islets after combined liver-islet allotransplantation. The patient was a 36-year-old woman who underwent upper abdominal exenteration for neuroendocrine carcinoma; 289,000 islets were transplanted via portal vein infusion immediately after complete revascularization of the liver. Immunosuppression was with low-dose FK-506. OKT3 and steroids were used to treat one rejection episode 2 weeks after transplantation, but the patient subsequently developed multiple infections and died 109 days after transplantation. At autopsy, the transplanted liver did not show any sign of rejection and well-preserved islets were present in portal triads sampled from the anterior inferior edge of the right lobe. Immunohistochemical labeling confirmed the presence of insulin-containing cells. This finding indicated that human islets can survive after intrahepatic allotransplantation, despite positive cross-match with no HLA antigen match, suggesting that upper abdominal exenteration and liver transplantation may constitute a protective factor for the survival of allogeneic human islets.

Evidence that canine pancreatic islets promote the survival of human hepatocytes in nude mice.

In the last 15 years there have been numerous reports demonstrating that transplanted hepatocytes can retain hepatocellular functions (1), correct congenital enzyme deficiencies (2–4), and improve survival rates in animal models of acute hepatic failure (5–7). Recently, Moscioni et al. (8) showed that human liver cells attached to collagen-coated microcarriers (9, 10) were able to correct metabolic defects following transplantation into mutant rat recipients that were genetically deficient in either bilirubin-uridine diphosphate glucuronosyltransferase activity or albumin synthesis. Interestingly, the intraperitoneally injected hepatocyte-microcarriers that were well vascularized were almost exclusively found on the surface of the pancreas. This apparent cellular tropism raises questions about the presence and mechanism of action of pancreas-derived hepatotrophic factors and their role in the maintenance of hepatocyte integrity (11–14). The aim of this study was to determine the effect of pancreatic islets on the survival of human hepatocytes in an ectopic site in nude mice. Human hepatocytes were obtained from segments of preserved cadaver donor livers that had been discarded during size reduction procedures before transplantation of the residual fragments to pediatric recipients. A modification of the automated procedure for isolation of human pancreatic islets (15) was used for the isolation of hepatocyte aggregates. After cannulation of its portal branch, the liver segment was perfused with 200 ml of a solution of 1 mg/ml collagenase (Boehringer-Mannheim, type P) in Hanks solution (28°C) and placed into a digestion chamber. Separation of hepatocellular clusters was accomplished by the same separation procedure used previously for isolation of human pancreatic islets (15). Between 80% and 95% of the harvested hepatocytes were viable, as assessed by trypan blue exclusion. Collagen-coated microcarriers (Cytodex 3, Pharmacia Fine Chemicals, Upsala, Sweden) were prepared as described by Demetriou et al. (9). Pancreatic islets were obtained from the pancreata of mongrel dogs (18–25 kg) by a modification (16) of the automated method for human islet isolation (15). Male BALB/c nude mice (20 g) were used as recipients in three groups (n=6 in each group). In group 1 (control) approximately 4000 hepatocyte clusters of an average diameter of 150 µ were placed beneath the left renal capsule as previously described (17). In group 2 (secondary control), hepatocyte clusters were transplanted in combination with 400 collagen-coated microcarriers. In group 3 the same number of hepatocyte clusters was transplanted without microcarriers but in combination with 400 pancreatic islets of 150 µm average diameter. Thirty days after transplantation the nude mice were sacrificed and the morphologic integrity of the renal subcapsular grafts was determined histopathologically. Hematoxylin-eosin stain was used in all sections. In addition, immunostain for insulin (immunoperoxidase) was used in sections of combined islet-hepatocyte transplants to distinguish the islets from the hepatocellular implants. The pathologist, who was blinded to the experimental groups from which the specimens came, correctly and without exception matched the tissues to the groups as follows. No hepatocytes were detectable in the renal subcapsular implants 1 month after transplantation of hepatocyte clusters alone (group 1). In group 2, the addition of collagen-coated microcarriers to the hepatocellular aggregates resulted in the survival of a thin rim of epithelioid cells attached to the microcarriers, while the remaining hepatocytes degenerated, leaving only fibrosis and a nonspecific mononuclear cell infiltrate around the microcarriers in the transplant site (Fig. 1). This suggested a minor augmentation of hepatocellular survival by the microcarrier. In contrast, when islets were included at the transplant site (group 3) multilayers of human hepatocytes were found. The surviving hepatocytes were healthy, in large clumps, and glycogen-containing, PAS-positive, and diastase-sensitive. These results are consistent with previous demonstrations that pancreatic-derived hepatotrophic factors are essential for maintenance of hepatocyte integrity (11–14). Scattered pancreatic islets were detected (Fig. 2). FIGURE 1 Photomicrograph of combined human hepatocytes and collagen-coated microcarriers 4 weeks after renal subcapsular transplantation. Degeneration of the hepatocytes occurred, leaving only a thin rim of epithelioid cells attached to the microcarriers (M) (H&E ... FIGURE 2 Photomicrograph of combined human hepatocytes and dog pancreatic islets 4 weeks after renal subcapsular transplantation in nude mice. Insulin immunoperoxidase (dark) stain distinguished islets (I) from the human hepatocytes (H), which appeared well preserved ... In past experience, when hepatocytes were transplanted by themselves in the presence of an intact host liver, no evidence of hepatocyte proliferation could be observed at any time after transplantation (18). Whenever cells without nuclei and multinucleated cells were found at the transplant site (19), hepatic degeneration had occurred. Similarly, it was not possible to detect the hepatocytes at either 2 or 4 days after intrasplenic transplantation (20), except when there was a proliferative stimulus provided by 70% recipient hepatectomy. Using the model of Demetriou et al. (9, 10), the collagen-coated hepatocyte microcarriers, the microcarriers may have promoted the survival of a small number of hepatocytes. In the earlier experiments of Moscioni et al. (8), the selective survival of the intraperitoneally injected hepatocyte-microcarrier aggregates on the surface of the pancreas was congruent with the possible role of factors produced from the pancreas in the maintenance of normal hepatocytes. Direct evidence supporting this concept (11–14) has come from rodent experiments in which combined hepatocyte-islet clusters were placed in ectopic sites (21, 22). The present study has established that human hepatocytes are subject to the hepatotrophic influence of islet cell factors, and because dog islets were used with human hepatocytes in the nude mouse recipient, it could be shown that the hepatotrophic effect was not species-specific. These findings will be important in defining effective procedures for the maintenance of human hepatocytes both in vitro and in vivo, and for their use in hepatocellular transplantation, artificial liver, and gene therapy applications.