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Y Mullen

Publications and source records attributed to Y Mullen.

At least 19 recordsLinked to original sources
PubMed2005

Multipotent progenitor cells isolated from adult human pancreatic tissue.

The supply of islet cells is a limiting factor for the widespread application of islet transplantation of type-1 diabetes. Islets constitute 1% to 2% of pancreatic tissue, leaving approximately 98% as discard after islet isolation and purification. In this report we present our data on the isolation of multipotent progenitor cells from discarded adult human pancreatic tissue. The collected cells from discarded nonislet fractions, after enzymatic digestion and gradient purification of islets, were dissociated for suspension culture in a serum-free medium. The cell clusters grown to a size of 100 to 150 mum contained cells staining for stage-specific embryonic antigens, but not insulin or C-peptide. To direct cell differentiation toward islets, clusters were recultured in a pancreatic differentiation medium. Insulin and C-peptide-positive cells by immunocytochemistry appeared within a week, reaching over 10% of the cell population. Glucagon and somatostatin-positive cells were also detected. The cell clusters were found to secrete insulin in response to glucose stimulation. Cells from the same clusters also had the capacity for differentiation into neural cells, as documented by staining for neural and glial cell markers when cultured as monolayers in media containing neurotrophic factors. These data suggest that multipotent pancreatic progenitor cells exist within the human pancreatic tissue that is typically discarded during islet isolation procedures. These adult progenitor cells can be successfully differentiated into insulin-producing cells, and thus they have the potential for treatment of type-1 diabetes mellitus.

Adult↗
PubMed2005

Inhibition of p38 mitogen-activated protein kinase protects human islets from cryoinjury and improves the yield, viability, and quality of frozen-thawed islets.

The development of an optimal islet cryopreservation method will permit transplantation of islets from multiple donors in a single procedure and contribute to alleviation of the islet shortage. In this study, we have improved human islet cryopreservation methods under serum-free conditions using an intracellular-based islet cryopreservation solution (ICS), especially supplemented with a p38 pathway inhibitor (p38IH) to suppress p38 mitogen-activated protein kinase (MAPK) activation. Three different solutions were compared for freezing and thawing of human islets (1) conventional RPMI1640 medium, (2) ICS, and (3) ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Islet cryopreservation with ICS-p38IH significantly improved islet recovery, viability, and quality after thawing of cryopreserved islets. This improvement may allow the use of cryopreserved islets in clinical islet transplantation.

Cell Culture Techniques↗
PubMed2001

Increased islet viability by addition of beraprost sodium to collagenase solution.

The digestion of pancreatic tissue with collagenase is an essential part of the islet isolation procedure. However, the process exposes islets to various types of harmful factors, including collagenase contaminants, enzymes released from the acinar cells, warm ischemia, and mechanical agitation. Nitrogen oxide production and cytokine release may also contribute to islet cell damage. Protection of islets from such damage would improve the islet yield, survival, and function. Beraprost sodium (BPS) is a prostaglandin I2 analogue, is stable in aqueous solution, and has a cytoprotective effect on various types of cells. BPS has been shown to improve the yield and function of cryopreserved and/or cultured islets. These findings prompted us to examine its cytoprotective effect on islets during the islet isolation process. Canine islets were isolated by means of a two-step digestion method and purified on Euro-Ficoll density gradient solutions (the procedure used for human islets). BPS at a concentration of 100 nM was added to the collagenase solution. After purification, the islet yield was 434,561 +/- 35.691 islet number expressed as 150 microm equivalent size (IEQ)/pancreas or 8,799 +/- 345 IEQ/g of pancreas in the BPS group and 349,987 +/- 52,887 IEQ/pancreas or 7,998 +/-1610 IEQ/g of pancreas in the control group (n = 8, each). The percent viability was 88.5 +/- 0.7% in the BPS group and 82.0 +/-0.9% in the control group (P < 0.01). Therefore, the recovery of viable islets (calculated by islet number x % viability) was 384,586 +/- 46,804 IEQ/pancreas (7,743 IEQ/g) in the BPS group and 286,989 +/- 43,367 IEQ/pancreas (6,558 IEQ/g) in the control group (P < 0.02). After culture, significantly higher numbers of islets were also recovered in the BPS group than in the control group. The islet insulin content was significantly higher in the BPS group than controls (237.8 +/- 38.5 versus 92.3 +/- 25.6 microU/IEQ; P < 0.02), although islets of both groups responded with high stimulation indices (>6). These results indicate that the addition of BPS to the collagenase solution increases the recovery of viable islets, and improves beta cell function.

Animals↗
PubMed2001

Prolongation of islet allograft survival in mice by combined treatment with pravastatin and low-dose cyclosporine.

Pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, is known to have suppressive effects on immune and inflammatory cells. We have previously shown in mice and dogs that this agent prevents primary nonfunction of islet iso- and autografts by reducing inflammation at the graft site. The present study was designed to further investigate whether pravastatin has a synergistic effect with cyclosporine (Cs) to prolong islet allograft survival in mice. Unpurified 3000 BALB/c newborn islets were transplanted under the renal capsule of a streptozotocin-diabetic C57BL/6 mouse. Pravastatin and Cs were administered for 10 days starting on the day of grafting (day 0). Five groups were set up based on the treatment protocol: group 1, treatment with 40 mg/kg pravastatin; group 2, 30 mg/kg Cs; group 3, 50 mg/kg Cs; group 4, 40 mg/kg pravastatin and 30 mg/kg Cs; group 5, vehicle alone. Graft survival was indicated by blood glucose levels sustained at <200 mg/dl, and graft rejection by >250 mg/dl for 2 consecutive days. Hyperglycemia persisted in six of the eight (75%) mice and grafts were rejected in 3.6 +/- 0.5 days (mean +/- SD) in group 5. In group 1, grafts were also rejected in 3.8 +/- 0.8 days, but blood glucose was transiently <200 mg/dl in three of the five mice. Despite Cs, grafts were rejected between 7 and 15 days (10.3 +/- 2.4 days) in group 2. Among six mice in group 3, one maintained euglycemia for >60 days, the other rejected the graft on day 15, and the remaining four died with functioning grafts between 9 and 13 days due to Cs toxicity. A combination of a low dose of Cs and pravastatin (group 4) prolonged graft survival for >19 days in five of the eight mice, and for 7-13 days in the remaining three mice. Histological examination of the grafts in this group showed significantly reduced local inflammation. Results indicate a synergistic effect of pravastatin and Cs on prevention of islet allograft rejection.

Animals↗
PubMed2000

Current progress and perspectives in immunoisolated islet transplantation.

This review summarizes the recent results of immunoioslated islet allo- and xenotransplants, especially in large animal species, and presents certain issues that would be important for the advancement of this technology toward clinical application. To date, the best results have been obtained with alginate microcapsules in both allo- and xenogeneic (porcine) islet transplantation in the spontaneously diabetic dogs and monkeys. Reversal of diabetes was also achieved in mice with cryopreserved, microencapsulated rat islets. However, results with these microcapsules have been highly variable and inconsistent from one laboratory to another. The causes of these discrepancies are multiple, but not totally understood. Immunoisolation devices have been investigated as an alternate approach to house a large number of islets in a space constructed by membranes of a defined pore size. Vascularized bioartificial devices were among the few cases that obtained long-term allo- and xenogeneic islet survival in totally pancreatectomized dogs. Thrombosis and associated problems were the major cause of failure with these devices. Diffusion chambers that have no vascular connection have been tested successfully in rodents, but no report has been available in large animal species. We have learned many important lessons and made considerable progress in islet immunoisolation. However, immunoisolated islet transplants have been only minimally successful in large animal models, and such technologies must be improved to achieve consistent success in large animal models prior to clinical trials. This will requires strict quality control of the islets, the membrane material, and the device construction. The membrane pore size that allows the permeation of molecules necessary for islet survival and function also permits the entry of cytokines, oxygen-radicals, and other small-size inflammatory products. Thus, the key to success appears to be to minimize inflammatory reactions generated by immuneisolation devices/capsules.

Alginates↗
PubMed2000

Improved quality and yield of islets isolated from human pancreata using a two-step digestion method.

A new approach, involving a two-step digestion process and Los Angeles preservation solution #1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty-six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age, cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5% (43 of 46 cases) in group 1, and 56.5% (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 +/- 36,244 islets equivalent to 150 microm (IEQ) and 6,233 +/- 681 IEQ/g of pancreas with 83 +/- 2.5% purity, whereas the old method yielded a total of 195,587 +/- 25,242 IEQ and 3,763 +/- 5,509 IEQ/g with 69.2 +/- 4.7% purity. Isolated islets in group 1 maintained a good three-dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transplantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a single pancreas.

Adolescent↗
PubMed1999

Improved human islet isolation by a tube method for collagenase infusion.

BACKGROUND: Collagenase infusion into the pancreatic duct is an essential step in human islet isolation. We developed a new method for ductal canulation and collagenese infusion. METHODS: A total of 53 pancreata were divided into two groups: group 1 (n=23), the new tube method, and group 2 (n=30), the standard angiocatheter method. In group 1, a polyethylene tube was inserted into the duct and pushed to the tail. The tail was first expanded, followed by expansion of the body and then the head, by pulling out the tube. RESULTS: Total islet number and number/g pancreas (mean+/-SE) were significantly higher in group 1 (481,123+/-43,218 and 8,010+/-722) (mean+/-SE) than in group 2 (300,974+/-35,122 and 5,090+/-515, P<0.01). Total islet equivalent number and islet equivalent number per gram pancreas were also significantly higher in group 1 (319,176+/-39,354 and 5,455+/-652) than in group 2 (202,022+/-23,331 and 3,722+/-468, P<0.04). Islet purity and fragmentation showed no differences between the groups. CONCLUSIONS: The tube method improved islet yields. We recommended this method for human islet isolation.

Catheterization↗
PubMed1999

Improved recovery of cryopreserved canine islets by use of beraprost sodium.

Cryopreservation of pancreatic islets provides many advantages for clinical transplantation. Unfortunately, the freezing and thawing processes lead to a significant loss of islets. In this study, an attempt was made to increase the yield and viability of islets after cryopreservation and thawing. By using canine islets, we evaluated whether beraprost sodium (BPS), a stable prostacyclin analog, protects islets during the freeze-thaw processes and improves the recovery of frozen-thawed islets. Canine islets were frozen and thawed by the procedures used routinely for storage of human islets. In this study, we deliberately used islets of lower purity (60+/-3.6%), which is undesirable for cryopreservation. The recovery of viable islets after thawing is poorer with islets of lower purity than with highly purified islets. BPS was added to both the cryopreservation solutions containing dimethyl sulfoxide (DMSO) and the thawing solution containing sucrose. After thawing, the islet recovery (islet number after thawing divided by islet number before freezing) was 71.1+/-12.7% with 1 nM BPS, 77.8+/-5.6% with 10 nM BPS, 79.3+/-6.7% with 100 nM BPS, and 69.2+/-7.2% in control preparations without BPS. Islet viability assessed by supravital staining was 57.5+/-5.6%, 64.7+/-7.0%, 67.5+/-6.5%, and 57.7+/-4.9% with 1 nM, 10 nM, and 100 nM BPS and controls, respectively. Both islet recovery and viability were significantly better with 10 nM and 100 nM BPS than with the controls (p<0.03). After 3 days in culture, islet numbers in the 10 nM and 100 nM BPS groups were significantly higher and showed better insulin-release responses than those from the 1 nM BPS and control groups. Histologically, islet structure was well preserved in the 10 nM and 100 nM BPS groups, whereas many islets of the control group were smaller and fragmented. Electron microscopic examination revealed that 10 nM and 100 nM BPS preserved the microstructure of islet cells, and signs of apoptosis or necrosis were rare. It was concluded that BPS improved the recovery and viability of canine islets after cryopreservation and thawing. BPS would be a useful agent for improving the recovery of cryopreserved human islets for clinical transplantation.

Animals↗
PubMed1998

Prevention of primary islet isograft nonfunction in mice with pravastatin.

BACKGROUND: Nonspecific inflammatory damage in the early stages of transplantation is the major cause of primary islet graft nonfunction. Using murine isografts, we attempted to prevent this islet graft damage by treating recipients with pravastatin (Pravacol), a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor. Nicotinamide was also tested to determine the synergistic effect of both agents. METHODS: Unpurified newborn BALB/c islets, ranging in number from 1800 to 2500, were transplanted into the left renal subcapsular space of a syngeneic adult mouse made diabetic with streptozotocin. Recipient mice were divided into the following four groups, based on treatment protocols: treatment with 40 mg/kg pravastatin (group 1), 500 mg/kg nicotinamide (group 2), 40 mg/kg pravastatin and 500 mg/kg nicotinamide (group 3), and vehicle alone (group 4). Pravastatin and nicotinamide were administered orally every day for 14 days, starting on the day of transplantation (day 0). Nonfasting blood glucose levels, urine glucose levels, and the intravenous glucose tolerance test were used to monitor the diabetic state. The reversal of diabetes was defined by normoglycemia and negative urine glucose maintained for more than 7 days. RESULTS: After islet transplantation, levels of blood and urine glucose were significantly lower in groups 1 and 3, compared with those in group 4. K-values of an intravenous glucose tolerance test performed on day 14 were significantly higher in groups 1 and 3 than those of group 4. Reversal of diabetes had occurred in 63% of mice in group 1 and 67% in group 3, levels that were higher than those in group 2 (17%) and group 4 (0%) (P<0.02, groups 1 and 3 vs. group 4). Histological examination of grafts, biopsied on day 21, revealed well preserved islets with little sign of inflammation in groups 1 and 3, whereas grafts in groups 2 and 4 contained broken, smaller islets surrounded by severe fibrosis and mononuclear cell infiltration. CONCLUSION: Our results in mice have shown the effectiveness of pravastatin for protecting islets from nonspecific inflammatory damage. Nicotinamide did not show a synergistic effect with pravastatin at the dosage used in this study. These results indicate that pravastatin may be a useful agent for clinical islet transplantation.

Animals↗
PubMed1998

LAP-1 cold preservation solution for isolation of high-quality human pancreatic islets.

The most critical factors that affect the outcome of clinical pancreatic islet transplantation are the number and quality of donor islets available for transplantation. Toward this goal, we attempted to obtain islets that are both of better quality and higher number than are obtainable by the islet-isolation process that is now widely used. We paid special attention to two critical components of the isolation procedure: minimizing the exposure of pancreatic tissue and freed islets to warm enzyme solution, and development of a preservation solution suitable for islets during cold storage of digested pancreatic tissue-free islets. For this purpose, we developed both a two-step procedure for pancreas digestion and a new cold preservation solution, the LAP-1 solution (Los Angeles preservation solution 1). In this study, we evaluated the effect of four preservation solutions by storing digested pancreatic tissues on ice for 90 min. After the cold storage, islets were purified on three layers of Euro-Ficoll solutions in a 50-ml tube, and the islet yield, viability, and function were determined. These experiments were performed by using samples from 10 consecutive islet isolations. Results with LAP-1, original University of Wisconsin solution (oUW), and modified UW solution (mUW;UW without hydroxyethyl starch) were compared with those obtained with Hank's balanced salt solution (HBSS). The islet yield was significantly higher in the LAP-1 and mUW groups as compared with the HBSS group (p < 0.01). The islet purity was significantly better in the LAP-1, oUW, and mUW groups than the HBSS (p < 0.001). The islet viability was lowest in the HBSS group immediately after purification (vs. LAP-1, oUW, and mUW, p < 0.05) and further decreased during culture (p < 0.01). Both the number and viability of cultured islets were the highest with LAP-1 solution but without statistical significance between mUW and oUW. Electron microscopic examination showed only slight damage to cell membranes immediately after purification of islets stored in LAP-1 solution and their complete recovery within 1-2 days of culture. These islets also exhibited normal insulin responses to high glucose by static incubation and perifusion assays.

Adolescent↗