G.S. Olsen

Induction of Insulin-Producing Cells From Human Pancreatic Progenitor Cells

INTRODUCTION We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions. MATERIALS AND METHOD Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein. RESULTS The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes. CONCLUSION Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells.

Assessment of Human Islet Isolation With Four Different Collagenases

BACKGROUND The isolation of islets from the human pancreas critically depends on the efficiency of the digestive enzymes. Liberase HI had been used as a standard preparation until the issues concerning bovine spongiform encephalopathy. Thus, we must now use other collagenases for clinical islet transplantation, four of which we have evaluated herein. METHODS The digestion of each of 17 pancreata from brain-dead donors was performed using the following collagenases: Liberase HI (HI; Roche, n = 9); Liberase MTF C/T (MTF; Roche, n = 4); Collagenase NB1 Premium Grade (NB1; Serva, n = 7); or Clzyme Collagenase HA (CI, VitaCyte, n = 4). Islet isolations were based on the Edmonton protocol for HI, whereas our modified islet isolation method was used for the three new enzymes (MTF, NB1, and CI). RESULTS There were no significant differences in donor age, body mass index, pancreas size, and cold ischemic time among the four groups. The phase I time in the NB1 group was significantly shorter than in the CI group (P = .0014). The prepurification IEQ/g in the HI group was significantly lower than the others (P = .0003 vs MTF, .0007 vs NB1, and .0009 vs CI, respectively). The postpurification IEQ/g in the MTF group was significantly higher than in the HI group (P = .006). The viability in the NB1 group was significantly greater than the HI group (P = .003). CONCLUSION Three new enzymes (MTF, NB1, and CI) may enable us to obtain higher islet yields than with HI.

Body Mass Index Reflects Islet Isolation Outcome in Islet Autotransplantation for Patients with Chronic Pancreatitis

Total pancreatectomy with autologous islet cell transplantation (TP with AIT) is an effective treatment for chronic pancreatitis patients with severe abdominal pain. Body mass index (BMI) of the pancreatic donor is proven to be a useful predictor for islet isolation and transplantation outcomes in allogenic islet transplantation. However, the association between BMI and islet isolation outcome and/or metabolism after AIT was previously unclear. Twelve patients who received TP with AIT at our hospital were included in this study. All pancreata were preserved with both pancreatic ductal injection and oxygen-charged static two-layer method using ET-Kyoto solution. The cohort was divided into two groups: low BMI group (BMI < 23 kg/m2, n = 5) and high BMI group (BMI ≥ 23, n = 7). The high BMI group had a significantly higher islet yield per gram than the low BMI group both in pancreas postdigestion and in final product (postdigestion: 7330 ± 539 vs. 3509 ± 563 IE/g; p < 0.001; final product: 6555 ± 585 vs. 3476 ± 546 IE/g; p = 0.004). For islet yield in final product per patient body weight, the high BMI group also had significantly higher islet yield than the low BMI group (7997 ± 779 vs. 4175 ± 750 IE/kg, p = 0.007). Insulin independence rate in the high BMI group (71%) was also higher than that low BMI group (40%), but it did not reach statistical significance. Pancreata from patients with higher BMI could obtain higher islet yield in the setting of autologous islet cell transplantation for chronic pancreatitis.

Comparison of Fresh and Cultured Islets From Human and Porcine Pancreata

INTRODUCTION For clinical islet transplantation, many centers have recently introduced of human islet cultures prior to transplantation. They provide flexibility to evaluate isolated islets and pretreat patients. However, isolated islets deteriorate rapidly in culture. In the present study, we compared fresh human and porcine islets with cultured islets for c-Jun NH(2)-terminal kinase (JNK) activity. MATERIALS AND METHODS Islet isolations from human and porcine pancreata were performed using the standard Ricordi technique with a modified Edmonton protocol. Isolated islets cultured for 24 hours at 37 degrees C with 5% CO(2) in culture medium were evaluated for counts and JNK activity. RESULTS After 24 hours of culture, the percentages of surviving islets were 86.9% for human and 47.3% for porcine sources. JNK activity in isolated islets declined to a low baseline level after 24-hour culture. CONCLUSION Both human and porcine islets deteriorated rapidly in 24-hour cultures, although the in vitro conditions did not induce JNK activation.

Inhibition of Inflammatory Cytokine-Induced Response in Human Islet Cells by Withaferin A

BACKGROUND After islet cell transplantation, a substantial mass of islets are lost owing to nonspecific inflammatory reactions. Cytokine exposure before or after transplantation can upregulate expression of proinflammatory genes via the nuclear factor-kappaB signaling pathway, eventually resulting in islet loss. OBJECTIVE To test the effects of a naturally occurring nuclear factor-kappaB inhibitor, withaferin A, on regulation of inflammatory genes in human islets. METHODS Human pancreatic islets were isolated using a modified Ricordi protocol. Purified islets were cultured for 2 days. The effect of withaferin A treatment on islet cell viability was examined using the fluorescein diacetate-propidium iodide dye exclusion test, and on function using a static glucose stimulation assay. Islet cells were treated with a cytokine mixture (50 U/mL of interleukin-1beta, 1000 U/mL of tumor necrosis factor-alpha, and 1000 U/mL of interferon-gamma) for 48 hours with or without withaferin A, 1 microg/mL. Treated islets were used for real-time polymerase chain reaction (PCR) array analysis for expression of inflammatory genes, and expression of other selected genes was analyzed using real-time PCR with single primers. RESULTS Glucose stimulation and viability assays demonstrated that withaferin A was not toxic to islet cells. Of 84 inflammation-related genes examined using real-time PCR array analysis, 9 were significantly upregulated by cytokine treatment compared with the control group. However, addition of withaferin A to the culture significantly inhibited expression of all genes. CONCLUSION Withaferin A significantly inhibits the inflammatory response of islet cells with cytokine exposure.

ET-Kyoto Ductal Injection and Density-Adjusted Purification Combined With Potent Anti-Inflammatory Strategy Facilitated Single-Donor Islet Transplantation: Case Reports

BACKGROUND The necessity to use multiple donors for achieving insulin independence in clinical islet transplantation is still a major issue. We have developed a modified islet isolation method for non-heart-beating donors (Kyoto method) to significantly increase islet yield. In this study, we further modified the method for brain-dead donors and in addition, introduced a potent anti-inflammatory strategy aiming for single-donor islet transplantation. MATERIALS AND METHODS Two islet isolations used pancreatic ductal preservation with the modified Kyoto solution and a density-adjusted purification method. Anti-interleukin-1-beta antibody (Anakinra) and anti-tumor necrosis factor-alpha (Eternacept) were administered during and after transplantation. The efficacy of the islet transplantation was assessed by the insulin requirement and SUITO (Secretory Unit of Islet Transplant Objects) index, wherein a value of more than 26.0 seems to be associated with insulin independence. RESULTS Both isolated islet preparations met the criteria for transplantation. They were transplanted into two type 1 diabetic patients, both of whom became insulin independent with stable glycemic control. The average SUITO index within 1 month was 29.2 and 45.3. CONCLUSION The islet isolation method combined with a potent anti-inflammation strategy made it possible to achieve single-donor islet transplantation achieving a high SUITO index.

Improved Method of Human Islet Isolation for Young Donors

BACKGROUND Although islet transplantation using young donors is more effective than older donors, islet isolation from young donor is notoriously difficult. This may relate to islet ontogeny and collagen composition in the young pancreas. Therefore, we examined whether a high concentration of collagenase could improve the separation of islets from exocrine tissues resulting in an high islet yield. METHODS We used six human pancreata from brain-dead donors of less than 30 years old. Islet isolation was performed based on the Edmonton protocol with modifications. All pancreata were digested with Collagenase NB1 Premium Grade (Serva). The pancreas was expanded by injecting either 200 mL of cold collagenase solution (2.5 mg/mL, standard group, n = 3) or 100 mL of solution (5 mg/mL, new group, n = 3) in a controlled manner under low pressure for 5 minutes. Then the pressure was raised for another 5 minutes. The following procedure and evaluation were performed based on the Edmonton protocol. RESULTS Phase II time in the new group was significantly shorter than the standard group. The ratio of embedded islets in the new group was significantly lower than the standard group. The postpurification islet equivalents per pancreas weight (IEQ/g) and the recovery rate in the new group were higher than the standard group, but not significantly. There was no significant difference in the postpurification purity, viability, and final tissue volume. CONCLUSION Our simple modification with an initially concentrated collagenase preparation using a syringe significantly improved the ratio of embedded islets, resulting in a higher yield from young donors.

Super-High-Dose Islet Transplantation Is Associated With High SUITO Index and Prolonged Insulin Independence: A Case Report

INTRODUCTION One of the current issues of clinical islet transplantation is the difficulty to achieve a prolonged insulin-free status. Functional islet mass gradually decreased after transplantation. We developed the SUITO index, which reflects engrafted islet mass. The SUITO (Secretory Unit of Islet Transplant Objects) index more than 26.0 is associated with an insulin-free status. In this study, we have experienced that super-high-dose islet transplantation maintained insulin-free status and a high SUITO index for a prolonged period. MATERIALS AND METHODS Two islet isolations were performed in February 2007 and January 2008. Ductal injections were performed at the procurement site using the ET-Kyoto solution and pancreata preserved by a two-layer method. Islets were isolated using the modified Ricordi method. Both isolated islets were transplanted into a type 1 diabetic patient. Efficacy of islet transplantation was assessed by the amount of insulin requirements and SUITO index. RESULTS Islet yields were 514,467 islet equivalents (IE) and 872,174 IE, with purities of 49% and 85% for the first and second islet transplantations, respectively. The patient received a total of 24,327 IE/kg body weight. The immunosuppression was based on the Edmonton protocol. After the second islet transplantation, the average SUITO index for the following 1 month was 48.5, and the patient became insulin-free. At postoperative day 1006, the SUITO index was 44.6 and the patient maintained an insulin-free status with excellent glycemic control. CONCLUSION Super-high-dose islet transplantation was associated with an high SUITO index and prolonged insulin independence.

Secretory Unit of Islet Transplant Objects (SUITO) Index Can Predict Outcome of Intravenous Glucose Tolerance Test

BACKGROUND Simple monitoring of engrafted islet function is important for follow-up of recipients after islet transplantation. We previously developed a simple assessment tool for islet graft function; the secretory unit of islet transplant objects (SUITO) index. The aim of this study was to clarify the relationship between the SUITO index and the outcomes of intravenous glucose tolerance tests (IVGTT). METHODS Fifteen series of blood samples from 6 islet recipients were collected before 3, 5, 10, 20, and 30 minutes after injection of 0.5 g/kg 50% dextrose. The SUITO index was calculated using plasma C-peptide and glucose level at fasting baseline. Samples were divided into the following 3 groups; low-SUITO (SUITO index <10; n = 3); middle-SUITO (SUITO index > or =10 to <26; n = 4); and high-SUITO (SUITO index > or =26; n = 8). RESULTS A threshold SUITO index of 26 showed good sensitivity (85.7%) and specificity (75.0%) to predict a blood glucose level of >10 mmol/L at 30 minutes. Blood glucose levels in the low-SUITO group were significantly higher than among the other 2 groups at baseline and 10, 20 and 30 minutes (P < .05). Glucose-level areas under the receiver-operating characteristic curve during IVGTT in the low-SUITO group were also significantly larger than among the other 2 groups (P < .05). CONCLUSION The SUITO index, using only a fasting blood sample, predicted IVGTT outcomes.

Adverse events in clinical islet transplantation: one institutional experience.

Islet transplantation is one of the most promising treatments for an unstable form of type 1 diabetes. However, islet transplantation still has some obstacles, such as low success rate of islet isolation, difficulty to obtain long-term insulin freedom, and adverse events related to transplant protocol. We describe the adverse events of current clinical islet transplantation at our institute in this report. Nine type 1 diabetic patients received 17 islet infusions from March 2005 to October 2008. The islet infusion procedure and immunosuppression regimen were based on a modified Edmonton protocol. Severe adverse events (SAEs) were defined as events that were more than grade 3 according to the Terminology Criteria for Adverse Events in Trials of Adult Pancreatic Islet Transplantation, version 4.1 (Collaborative Islet Transplant Registry, CITR). Sixteen events were reported as SAEs and among them 12 events were probably or definitely related to transplant protocols; all occurred within 1 year after infusion except for one. Five adverse events (31%) occurred within 10 days after transplantation and were related to infusion procedures. Seven events (44%) occurred after 50 days and were related to immunosuppressive therapy. SAEs related to the protocol included three events of elevated liver enzymes, two of hemorrhage into gall bladder or peritoneal cavity, two of neutropenia, two of infection, one of vomiting, one of diarrhea, and one of renal dysfunction. All events were grade 3, except for one case that was grade 4 of neutropenia. All SAEs resolved with no sequelae. Neoplasms and deaths were not observed in our study. The present study suggests need to improve both infusion procedure and immunosuppressive strategy from the view of preventing SAEs.