Yoshiko Tamura

Seven consecutive successful clinical islet isolations with pancreatic ductal injection.

Inconsistent islet isolation is one of the issues of clinical islet transplantation. In the current study, we applied ductal injection to improve the consistency of islet isolation. Seven islet isolations were performed with the ductal injection of ET-Kyoto solution (DI group) and eight islet isolations were performed without the ductal injection (standard group) using brain-dead donor pancreata. Isolated islets were evaluated based on the Edmonton protocol for transplantation. The DI group had significantly higher islet yields (588,566 ± 64,319 vs. 354,836 ± 89,649 IE, p < 0.01) and viability (97.3 ± 1.2% vs. 92.6 ± 1.2%, p < 0.02) compared with the standard group. All seven isolated islet preparations in the DI group (100%), versus only three out of eight isolated islet preparations (38%) in the standard group met transplantation criteria. The islets from the DI group were transplanted into three type 1 diabetic patients and all three patients became insulin independent. Ductal injection significantly improved quantity and quality of isolated islets and resulted in high success rate of clinical islet transplantation. This simple modification will reduce the risk of failure of clinical islet isolation.

Elevation of high-mobility group box 1 after clinical autologous islet transplantation and its inverse correlation with outcomes.

A major problem after clinical autologous islet transplantation (AIT) is the difficulty in achieving insulin independence. To follow up on our demonstration in a murine model that high-mobility group box 1 (HMGB1) was released from islets and involved in early loss of transplanted islets, we tested the role of HMGB1 in clinical AIT. Serum HMGB1 levels from 15 AIT patients were significantly elevated during islet infusion (7.6 ± 1.2 ng/ml) and 24 h after infusion (8.0 ± 1.4 ng/ml) compared to admission levels (2.4 ± 0.6 ng/ml). The first elevation of HMGB1 was associated with islet damage, but the later elevation was not. The change in the HMGB1 level from admission to first peak (ΔHMGB1) was significantly higher in the AIT group (8.1 ± 1.1 ng/ml) than in the pancreatectomy-only control (2.2 ± 0.5 ng/ml) (p < 0.05). Circulating serum levels of soluble receptor for advanced glycation end products (sRAGE) were also elevated during islet infusion. In vitro studies demonstrated that damaged human islets released HMGB1 but not sRAGE. In terms of outcomes, the insulin-free group showed significantly lower ΔHMGB1 (5.2 ± 0.6 ng/ml) and higher ΔsRAGE (2.3 ± 0.6 ng/ml) than the insulin-dependent group (10.6 ± 1.9 ng/ml and 0.7 ± 0.2 ng/ml, respectively). The ΔHMGB1 correlated with the number of white blood cell, IP-10, EGF, and eotaxin. In conclusion, serum HMGB1 was elevated in AIT and could be associated with inflammatory reactions that deteriorate islet engraftment. Therefore, anti-HMGB1 therapy might be a candidate for further improving the outcomes of clinical AIT.

Islet cell transplantation for the treatment of type 1 diabetes in the USA

Islet cell transplantation (ICTx) is one of the most effective treatments for type 1 diabetes and is less invasive compared to whole organ transplantation. The US has been the leader in the research and clinical applications of ICTx for the last 40 years. ICTx requires complex procedures, including pancreas procurement and preservation; pancreas digestion; islet purification; and transplantation. Even with the dramatic progresses in each of the procedures listed above, there are still challenges to make ICTx the standard therapy. These challenges are: (1) obtaining enough islets from a single donor and (2) preventing graft loss due to allogenic rejection and recurrence of autoimmune islet destruction. A new preservation strategy for pancreata and pancreatic ducts using ET-Kyoto solution as well as a new islet purification method using iodixanol has substantially improved islet yields. Continuous research to improve the efficacy of islet isolation will solve the issue of obtaining enough islets from a single donor. Immunological tolerance is an ideal solution for the issue of rejection and autoimmune recurrence and a regulatory T cell strategy seems promising. Moreover, the SUITO index is a simple and powerful tool to assess engrafted islet mass and is, therefore, useful for evaluating the efficacy of new immunosuppressant strategies. Once ICTx becomes a standard treatment, the donor shortage will become the next challenge. Marginal or living donor islet transplantations could help alleviate this issue; however, bio-artificial islet transplantation with animal islets could be the ultimate solution.

Assessment of Islet Quality Following International Shipping of More Than 10,000 km

Islet transplantation is an attractive therapy for type 1 diabetes, although some issues remain. One of them is the severe donor shortage in some countries. In this study, we investigated the possibility of international islet shipping beyond 10,000 km to supply islets to countries with donor shortages. Human islets were isolated from six cadaver donors and cultured until shipment. Islets were packed in either gas-permeable bags or in non-gas-permeable bags and shipped from Baylor Research Institute (Dallas, TX, USA) to Fukuoka University (Fukuoka, Japan). Pre- and postshipment islet number, purity, viability, and stimulation index (by glucose stimulation test) were assessed. Shipped 1,500 IE islets were transplanted into streptozotocin-induced diabetic nude mice for in vivo assay. The distance of our shipment was 11,148.4 km, and the mean duration of the shipments was 48.2 ± 8.2 h. The islet number recovery rate (postshipment/preshipment) was significantly higher in gas-permeable bags (56.4 ± 10.1% vs. 20.5 ± 20.6%, p < 0.01). Islet purity was significantly reduced during shipment in non-gas-permeable bags (from 47.7 ± 18.6% to 40.2 ± 28.2 in gas-permeable bags vs. from 50.4 ± 6.4% to 25.9 ± 15.6% in non-gas-permeable bags, p < 0.05). Islet viability and stimulation index did not change significantly between pre- and postshipping, in either gas-permeable bags or in non-gas-permeable bags. One of three diabetic nude mice (33.3%) converted to normoglycemia. It is feasible to ship human islet cells internationally in gas-permeable bags. This strategy would promote basic and preclinical research for countries with donor shortages, even though the research centers are remote (over 10,000 km from the islet isolation center).

Impact of tissue volume and purification on clinical autologous islet transplantation for the treatment of chronic pancreatitis.

Autologous islet transplantation after total pancreatectomy is an excellent treatment for painful chronic pancreatitis. Traditionally, islets have been isolated without purification; however, purification is applied when the tissue volume is large. Nevertheless, the impact of tissue volume and islet purification on clinical outcomes of autologous islet transplantation has not been well examined. We analyzed 27 cases of autologous islet transplantation performed from October 2006 to January 2011. After examining the relationship between tissue volume and portal pressure at various time points, we compared islet characteristics and clinical outcomes between cases with complications (complication group) and without (noncomplication group), as well as cases with purification (purification group) and without (nonpurification group). Tissue volume significantly correlated with maximum (R = 0.61), final (R = 0.53), and delta (i.e., difference between base and maximum; R = 0.71) portal pressure. The complication group had a significantly higher body mass index, tissue volume, islet yield, and portal pressure (maximum, final, delta), suggesting that complications were associated with high tissue volume and high portal pressure. Only one of four patients (25%) in the complication group became insulin free, whereas 11 of 23 patients (49%) in the noncomplication group became insulin free with smaller islet yields. The purification group had a higher islet yield and insulin independence rate but had similar final tissue volume, portal pressure, and complication rates compared with the nonpurification group. In conclusion, high tissue volume was associated with high portal pressure and complications in autologous islet transplantation. Islet purification effectively reduced tissue volume and had no negative impact on islet characteristics. Therefore, islet purification can reduce the risk of complications and may improve clinical outcome for autologous islet transplantation when tissue volume is large.

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.