Donor Pancreata Research Articles

Embryonic stem cell-based diabetes therapy—a long road to travel

Embryonic stem (ES) cells have been widely heralded as a source of differentiated cells to be used in cell-replacement therapy (CRT), which has potential as a treatment for a number of degenerative diseases, including Parkinson’s disease, Alzheimer’s disease and diabetes [1–3]. The potential impact of CRT on human life is huge. Not only will healthcare systems be transformed, but a number of societal issues will emerge if cell-based therapy is fully developed. There are, however, widely diverging views as to when we can expect to see cell-based treatment for major diseases (see e.g. [4] in this issue), and over-optimistic views have been expressed in the press and elsewhere. Here I present a more pessimistic view on the amount of time that will be needed to develop CRT for the treatment of diabetes. Patients with type 1 diabetes are considered to be candidates for cell-replacement therapy (CRT), but those with advanced stages of type 2 diabetes might also benefit. Many obstacles, however, must be overcome before transplantation becomes the treatment of choice for patients newly diagnosed with diabetes. At present, only patients with poor metabolic control and a history of hypoglycaemic episodes are eligible for islet transplantation [5]. This selection is based on an unfavourable risk–benefit analysis in patients who are responding well to their current therapy of multiple daily insulin injections (i.e. patients with metabolic control sufficient to confer a low risk of secondary complications). Indeed, concerns over whether the immunosuppression needed to prevent graft rejection will be associated with serious side effects also limits the widespread use of CRT for diabetes [6]. Unless new forms of immunosuppression or encapsulation technologies with minimal side effects are developed, CRT faces a bleak future in the treatment of diabetes. The risks associated with the immune suppression must be lower than the risks of secondary complications associated with the disease itself before it will be ethically responsible to treat diabetes with CRT. It should not be forgotten that new generations of insulin analogues, and the development of closed-loop insulin delivery systems, may provide the means to improve the clinical outcome of conventional insulin therapy [7], raising further concerns as to whether the safety of future strategies to prevent graft rejection will allow a favourable risk–benefit analysis. Assuming that safe and efficient methods to prevent graft rejection are developed, a second and equally important obstacle emerges, namely the shortage of organ donors. The number of available donor pancreata per year is currently sufficient to treat only about 1 in 20 of the newly diagnosed type 1 diabetes patients in the USA each year [8] and it is obvious that an increase in donor pancreata sufficient to meet the demand is neither likely nor desirable. An alternative source of islet beta cells is therefore required before islet transplantation can be more Diabetologia (2006) 49:2537–2540 DOI 10.1007/s00125-006-0434-x

Successful Islet Transplantation from Nonheartbeating Donor Pancreata Using Modified Ricordi Islet Isolation Method

Current success of islet transplantation has led to donor shortage and the need for marginal donor utilization to alleviate this shortage. The goal of this study was to improve the efficacy of islet transplantation using nonheartbeating donors (NHBDs). First, we used porcine pancreata for the implementation of several strategies and applied to human pancreata. These strategies included ductal injection with trypsin inhibitor for protection of pancreatic ducts, ET-Kyoto solution for pancreas preservation, and Iodixanol for islet purification. These strategies significantly improved both porcine and human islet isolation efficacy. Average 399,469+/-36,411 IE human islets were obtained from NHBDs (n=13). All islet preparations met transplantation criteria and 11 out of 13 cases (85%) were transplanted into six type 1 diabetic patients for the first time in Japan. All islets started to secrete insulin and all patients showed better blood glucose control without hypoglycemic loss of consciousness. The average HbA1c levels of the six recipients significantly improved from 7.5+/-0.4% at transplant to 5.1+/-0.2% currently (P<0.0003). The average insulin amounts of the six recipients significantly reduced from 49.2+/-3.3 units at transplant to 11+/-4.4 units (P<0.0005) and five out of six patients reduced to less than half dose. The first patient is now insulin free, the first such case in Japan. This demonstrates that our current protocol makes it feasible to use NHBDs for islet transplant into type 1 diabetic patients efficiently.

Organ utilization among deceased donors in Canada, 1993–2002

Optimizing organ utilization from consented donors is a critical need, given a static organ donation rate. We report changes in the characteristics of donors and organ utilization patterns in Canada over a ten-year period. For the decade spanning the years 1993-2002, data were extracted from the Canadian Organ Replacement Register (CORR), the national transplant registry. A donor was defined as a deceased person from whom at least one vital organ was retrieved and transplanted. The donor pool is aging (median age of donors increased eight years over the decade), with proportionately fewer donors dying from head trauma (motor vehicle collisions) and proportionately more from cerebrovascular accidents. At least four organs were utilized from approximately half the donors. These donors were significantly younger every year over the sampling period when compared with donors where < or = three organs were utilized. In 2002, utilization rates were: 87.0% (kidneys), 85.0% (livers), 42.2% (hearts), 30.6% (pancreata), 28.3% (lungs), and < or = 1% (intestines). There was increased utilization of donor pancreata, lungs and liver over the decade, but a flat utilization pattern for hearts, and a small decline in kidney utilization. Utilization rates vary from province to province. Trends in the Canadian organ donor pool are characterized by an increasing age and a shift towards cerebrovascular diseases as primary causes of death. In order to improve organ utilization and understand regional variability, the scope of data provided to the national registry requires enhanced detail to address the factors that lead to non-utilization. Addressing the low utilization rates for hearts and lungs is especially critical, given the need for thoracic transplantation in Canada.

Pancreas Versus Islet Transplantation in Diabetes Mellitus: How to Allocate Deceased Donor Pancreata?

Transplant options for the diabetic recipient include pancreas and islet transplantation. Pancreas transplantation has been increasingly performed in the last 3 decades with increasing success rates. Nevertheless, islet transplantation offers the advantage of being less invasive with fewer complications. However, current experience shows that multiple transplants are required to achieve and maintain insulin-independence in the intermediate term, and long-term function remains a problem even with multiple transplants. Early successes with single-donor islet transplants are encouraging and if maintained will largely substitute pancreas transplants. Currently, single-donor islet transplants have been shown to work in recipients with low insulin requirements who receive a pancreas from a donor with high body mass index. However, pancreas transplants from obese donors are associated with increased surgical risk. Therefore, it is logical to preferentially allocate obese donor pancreata to islet recipients. In addition, older donor (50 to 65 years) pancreata could be preferentially allocated to islets since their islet yield is still good, whereas they are associated with decreased survival in whole-organ pancreas transplants. With increasing efficiency and success of islet transplants the criteria for pancreas allocation for islets will need to be periodically reviewed.

Modified Two-Layer Preservation Method (M-Kyoto/PFC) Improves Islet Yields in Islet Isolation

Islet allotransplantation can achieve insulin independence in patients with type I diabetes. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and UW solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. However, UW solution has several disadvantages, including the inhibition of Liberase activity. In this study, we investigated the features of a new solution, designated M-Kyoto solution. M-Kyoto solution contains trehalose and ulinastatin as distinct components. Trehalose has a cytoprotective effect against stress, and ulinastatin inhibits trypsin. In porcine islet isolation, islet yield was significantly higher in the M-Kyoto/PFC group compared with the UW/PFC group. There was no significant difference in ATP content in the pancreas between the two groups, suggesting that different islet yields are not due to their differences as energy sources. Compared with UW solution, M-Kyoto solution significantly inhibited trypsin activity in the digestion step; moreover, M-Kyoto solution inhibited collagenase digestion less than UW solution. In conclusion, the advantages of M-Kyoto solution are trypsin inhibition and less collagenase inhibition. Based on these data, we now use M-Kyoto solution for clinical islet transplantation from nonheart-beating donor pancreata.

Islet transplantation: steady progress and current challenges

Purpose of review Clinical islet transplantation has evolved rapidly in recent years and current clinical trials are showing the benefits of this therapeutic approach for patients with diabetes. New challenges are emerging and must be overcome in order to extend this therapeutic option to larger cohorts of patients who may benefit. This review summarizes the current state of clinical islet transplantation and progress made in translational research to address limitations. Recent findings Transplantation of pancreatic islets has proven efficacy in improving metabolic control and quality of life, and in preventing severe hypoglycemia in patients with diabetes. New clinical islet transplant programs are being established worldwide and the annual number of transplants continues to increase. Recent improvements in pancreas procurement, islet isolation, and purification techniques have led to more consistent islet yields and better utilization of cadaveric donor pancreata. Improved methods for islet assessment are being developed to predict islet potency. A better understanding of the causes of islet dysfunction will be helpful in designing targeted interventions for islet cytoprotection and reducing the number of islets required to achieve insulin independence. Implementation of safer immunosuppressive/tolerogenic regimens will permit widespread application of islet transplantation to restore β-cell function in the future. Summary The benefits of islet transplantation in managing patients with diabetes are tangible. Very encouraging data are emerging from the numerous clinical trials and from translational research ongoing worldwide that justify cautious optimism for the near future.

Protein Transduction: A Novel Approach to Induce In Vitro Pancreatic Differentiation

It is widely believed that human embryonic stem (huES) cells may represent a valid alternative to donor pancreata as a source of islets for transplantation. Much is known about the transcription factors whose sequential activation results in the generation of islets during pancreatic development. This knowledge has been used to articulate the theoretical possibility that such process might be recapitulated in vitro from stem cells. However, our understanding of the extracellular signals that prompt the developing pancreas to follow this sequence of molecular events is very limited. Also, the simplicity of in vitro systems makes it difficult, if not impossible, to mimic the complex signaling pattern observed in living embryos. Protein transduction (PT) technology may provide researchers with a new powerful tool to sequentially induce stem cell differentiation, entirely bypassing the need for unraveling the signaling pattern that drives the process in vivo. Here we discuss this novel application of the flourishing PT technology, which may revolutionize the way we direct stem cells along any specific lineage.

Small rat islets are superior to large islets in in vitro function and in transplantation outcomes

Barriers to the use of islet transplantation as a practical treatment for diabetes include the limited number of available donor pancreata. This project was designed to determine whether the size of the islet could influence the success rate of islet transplantations in rats. Islets from adult rats were divided into two groups containing small (diameter <125 microm) or large (diameter >150 microm) islets. An average pancreas yielded three times more small islets than large. Smaller islets were approximately 20% more viable, with large islets containing a scattered pattern of necrotic and apoptotic cells or central core cell death. Small islets in culture consumed twice as much oxygen as large islets when normalized for the same islet equivalents. In static incubation, small islets released three times more insulin under basal conditions than did large islets. During exposure to high glucose conditions, the small islets released four times more insulin than the same islet equivalencies of large islets, and five times more insulin was released by the small islets in response to glucose and depolarization with K+. Most importantly, the small islets were far superior to large islets when transplanted into diabetic animals. When marginal islet equivalencies were used for renal subcapsular transplantation, large islets failed to produce euglycemia in any recipient rats, whereas small islets were successful 80% of the time. The results indicate that small islets are superior to large islets in in vitro testing and for transplantation into the kidney capsule of diabetic rats.

Comparison of the Collagen VI Content Within the Islet-Exocrine Interface of the Head, Body, and Tail Regions of the Human Pancreas

Efficient islet isolation depends on the use of collagenase to digest the extracellular matrix within the islet-exocrine interface, the molecular structure of which is poorly understood. Recently it has been reported that transplantable yields of islets can be isolated from the tail segment of the pancreas alone. This study aimed to quantify and compare the amount of collagenase-resistant collagen VI within the islet-exocrine interface of the head, body, and tail of the human pancreas. Human adult pancreata (n = 5) were retrieved from heart-beating donors (age range, 40–62 years; cold ischemia times <10 hours). Tissue blocks from the head, body, and tail region of each pancreas were fixed in formalin and processed for immuno-labelling of collagen VI, which was quantified in the islet-exocrine interface using a Zeiss KS-400 image analysis system. Data were expressed as area of collagen at the interface relative to the islet area. Statistical analysis was done using paired t test. The mean islet areas in the head, body, and tail regions were not significantly different. Collagen VI was uniformly present within the islet-exocrine interface of all regions of the pancreas and was 0.326 ± 0.064, 0.324 ± 0.060, and 0.334 ± 0.052 μm 2/islet area ( P = .441) in the head, body, and tail, respectively. The content of collagen VI within the islet-exocrine interface was uniform throughout all parts of the adult pancreas. Targeting this collagen subtype with novel collagenase blends may result in consistently improved islet yields and enable a wider number of available donor pancreata to be used.

Possibility of Islet Transplantation from a Nonheartbeating Donor Pancreas Resuscitated by the Two-Layer Method

The shortage of cadaveric donors is a problem in islet transplantation, and recent improvements in this field have led to renewed interest in the use of nonheartbeating (NHB) donors. NHB donor pancreata that could provide a significant source for islet transplantation are associated with warm ischemic injury. We tested whether the two-layer method (TL) could improve islet yield and function from damaged pancreata after warm ischemia (WI). Lewis rats were divided into six groups. In groups 1 to 3, rats were subjected to 0, 30, and 45 minutes of WI, respectively. Islets were isolated immediately (subgroup a) or after 3-hour preservation with TL (subgroup b). Isolated islets were assessed in terms of islet yield and in vivo function. We also assessed the pancreatic tissue ATP concentration before isolation and distended pancreata morphologically after chemical digestion by H&E staining. Islet yield decreased significantly after 30 minutes of WI in group 2a, whereas TL preservation doubled this decreased yield in group 2b. Forty-five minutes of WI resulted in nearly no islet yield in both groups 3a and 3b. The success rates of transplantation in groups 1a, 1b, 2a, and 2b were 100%, 100%, 0%, and 75%, respectively. Increased tissue ATP levels and alleviation of morphological islet damage were observed in group 2b. These results demonstrated that pancreata damaged from 30-minute WI were restored by 3-hour TL preservation. TL may allow the selective use of NHB donors as an alternative source for islet transplantation.

Effect of Oxygenated Perfluorocarbons on Isolated Rat Pancreatic Islets in Culture

One impediment for a wider application of islet transplantation is the limited number of donor pancreata for islet isolation. A more efficient utilization of available organs could in part alleviate this problem. Perfluorocarbons (PFCs) have a high oxygen solubility coefficient and maintain high oxygen partial pressures for extended time. They serve also as oxygen "reservoirs" for harvested organs in pancreas organ transplantation. The aim of this study was to test whether the use of PFCs could also be beneficial for the secretory activity and overall viability of cultured purified islets before transplantation. Purified rat islets were cultured in static conditions with or without oxygen-saturated PFCs for 1 or 7 days. Cell death and apoptosis were assessed by trypan blue staining, DNA strand breaks, and caspase 3/7 activity. mRNA levels of insulin and ICA512/IA-2, a membrane marker of secretory granules (SGs), were quantitated by real-time PCR, whereas insulin content and secretion were measured by RIA. Polypyrimidine tract binding protein (PTB), which promotes SG biogenesis, was assessed by Western blotting. The number of SGs and the ultrastructural appearance of beta5-cells were analyzed by cryoimmunoelectronmicroscopy for insulin. Various parameters, including caspase activity, insulin and ICA512/IA-2 mRNA levels, PTB expression, number of secretory granules, and ultrastructural appearance did not significantly differ between control and PFC-cultured islets. On the other hand, PFC culture islets showed significantly increased DNA fragmentation and a reduced insulin stimulation index at both time points compared to control islets. While advantageous for the transport of human harvested organs, the use of PFH in the culture may be comparable to and/or not provide advantage over conventional protocols for culture of islets for transplantation.

Regenerative medicine of the pancreatic β cells

Diabetes mellitus is a metabolic disorder that affects millions of people. The number of patients suffering from diabetes continues to increase all over the world. Both type 1 and type 2 diabetes result from an inadequate mass of functioning beta cells. To achieve the ultimate goal of curing diabetes in the future, the mechanism of the regenerative process of the adult human pancreas must be elucidated. In this review, we first summarize the regenerative processes of the pancreas observed in animal models in vivo, and approaches to promote the regeneration of the pancreas in vivo. Next we consider other new approaches, such as stem cell research and cell-based therapy, for the cure of diabetes in the future. Based on the innovative success of the Edmonton protocol, islet transplantation has been considered to be a new therapeutic option for the treatment of diabetes. However, a serious shortage of donor pancreata is a critical problem. We suggest that the following issues should be solved in order to realize cell-based therapy. The first is to establish a source of stem/progenitor cells that will multiply easily in vitro and maintain their property as progenitor cells. The probable use of adult stem cells will circumvent potential ethical problems, and autotransplantation will become possible. The most difficult and as yet unsolved issue is how to differentiate these cells and acquire fully functional islets. Further investigations to understand the regenerative process of the adult pancreas and the appropriate induction of stem cell differentiation will help to establish cell-based therapy in diabetes.

Pancreas Divisum: A Study of the Cadaveric Donor Pancreas for Islet Isolation

Pancreas divisum, the most frequent congenital variant of pancreatic ductal configuration, might impair intraductal delivery of enzyme for islet isolation. We investigated the frequency of pancreas divisum and its effect on the digestion process in islet isolation. Two catheters were placed into the main duct in opposite directions following dissection of the duct in the mid-body of cadaveric donor pancreata. When the majority of outflow was observed through the Santorini orifice by injecting solution through the catheter toward head, a diagnosis of pancreas divisum was made. Twenty-eight cases of a total of 127 pancreata evaluated were described as having pancreas divisum. There were no significant differences in donor demographics, donor instability, pancreas weight, or undigested tissue weight between donors with and without pancreas divisum. In the 28 cases of pancreas divisum, there was moreover no observable difference in the quality of distention between the ventral and dorsal pancreas. Islet yield after digestion was comparable between cases with and without pancreas divisum. We found that Santorini's duct was the major drainage route of the pancreas in 22% of individuals. Pancreas divisum did not have a negative impact on the digestion process and outcome of islet isolation.

Saline Contamination of University of Wisconsin Solution Preserved Human Pancreata

The use of University of Wisconsin (UW) solution is widely accepted for the cold storage and transport of human donor pancreata destined for islet isolation. While several variables exist in islet isolation, proper procurement and packaging is essential for optimizing preprocessing conditions. This study examined the effect of normal saline (NS) contamination/dilution of transport UW solution in 89 human pancreata processed by our islet isolation program. Samples of UW were taken from the primary pancreas container and examined for the presence of blood and or hemolysis. The cold ischemia time (CIT) was calculated, and processing by cold collagenase perfusion commenced. Islets were then isolated by collagenase digestion, purified by continuous gradient Ficoll centrifugation, and islet equivalent (IE) yield determined by dithizone staining. Viability of isolated islets was assayed by using the fluorescent SYTO/ethidium bromide (SYTO/EB) method. Na+ and K+ values were assayed using ion-specific electrode methodologies. Isolation outcomes were grouped by successful versus unsuccessful categories, and statistically compared using an unpaired t-test, where p < 0.05 was deemed to be statistically significant. Values are expressed as mean ± SEM. There was no significant relationship between increased Na+ and an unsuccessful isolation as a single variable. The study did suggest that the prolonged ischemic pancreas is more vulnerable to isolation failure if significant Na+ is present. In either case, a shorter CIT in UW was beneficial to increased IE yield and viability in human islet isolation.

Transdifferentiation molecular pathways of neonatal pig pancreatic duct cells into endocrine cell phenotypes

Restrictions in availability of cadaveric human donor pancreata have intensified the search for alternate sources of pancreatic endocrine tissue. We have undertaken to assess whether nonendocrine pancreatic tissue, with special regard to ducts, including epithelial cells, and retrieved from neonatal pig pancreata that are used for islet isolation, may under special in vitro culture conditions generate endocrine cell phenotypes. Special care was taken to identify the time-related appearance of molecular and biochemical markers associated with β-cell specificity, in terms of glucose-sensing apparatus and insulin secretion. For this purpose, established ductal origin monolayer cell cultures were incubated with a battery of mono- or polyvalent growth factors. Morphological, immunocytochemical, molecular, and functional assays indicated that under special culture conditions ductal origin cells acquired an endocrine identity, based upon expression of key gene transcripts that govern the stimulus-coupled insulin secretory activity. Among factors eliciting transdifferentiation of ductal epithelial into endocrine cells, Sertoli cell (SC)-conditioned medium seemed to be the most powerful inducer of this process. In fact, the resulting cultures not only expressed β-cell–oriented metabolic markers but also were associated with insulin and C-peptide output at equimolar ratios. This finding indicates that SC coincubation, more than other conditions, caused originally ductal cell cultures to gradually differentiate and mature into β-cell–like elements. In vivo studies with this early cell differentiation product will test whether our approach may be suitable for correction of hyperglycemia in diabetic animal models.

Can the density of native pancreatic tissue slices predict human islet isolation and purification outcome?

Introduction With currently available technology, the outcomes of human islet isolation and purification are still inconsistent, in part due to a lack of control of the pancreas donor and the procurement conditions. Using a single donor pancreas, the critical islet mass for establishing insulin independence of approximately 5000 engrafted islet equivalents (IEQ)/kg of recipient weight can only be retrieved from about one third of isolations. The purpose of this study was to analyze whether successful islet isolation and purification outcomes might be predicted from the density of native pancreatic tissue. Methods Tissue slices (TS) were obtained from the neck of 9 nondistended human donor pancreata. The density of the TS was determined using gravity sedimentation in continuous density gradients under either iso-osmolar or hyperosmolar conditions. Correlation coefficients were calculated with regard to the density of isolated exocrine and endocrine tissue, donor age, body mass index (BMI), cold ischemia time (CIT), IEQ prepurification and postpurification, IEQ recovery, and purity. Results (1) There was no change in density over time for TS in 300 mOsm/kg (mean, 1.079 ± 0.0019 g/cm 3) (2) In 500 mOsm/kg, there was a significant increase in density from 1.086 ± 0.0021 g/cm 3 to 1.092 ± 0.0021 g/cm 3 over time. (3) Density of isolated exocrine and endocrine became more distinct with lower density of TS (r = −0.776; P < .05). (4) Donor age, BMI, recovery of IEQ from gradients, and number of IEQ after purification did not correlate significantly with TS density. (5) In contrast, a significant inverse correlation existed betwen TS and CIT (r = −0.829; P < .05), and between TS versus IEQ number prior to purification (r = −0.867; P < .05). Conclusion No homogeneous distribution of pancreas tissue density was seen among 9 consecutive human organs. Taken together, the density of native pancreas TS is not a suitable sole predictor for successful islet isolation and purification.

Efficacy of human islet isolation from the tail section of the pancreas for the possibility of living donor islet transplantation.

Islet transplantation is on the rise for the treatment of type 1 diabetes. Apparent donor shortages could be alleviated through use of living donor pancreata. A critical issue for using a section of pancreas from living donors is whether islet yields would be sufficient for transplantation. After obtaining human pancreata, islets were isolated from the head section (n=20, head group), tail section (n=23, tail group) or whole pancreas (n=24, whole group). Islets were isolated by enzymatic digestion followed by purification, then assessed for yields, purity, morphology, functionality, and insulin content. Fifteen of twenty cases (75%) in the head group, all cases (100%) in the tail group, and 23 of 24 cases (96%) in the whole group were successfully completed for islet isolation. Islet yield per gram pancreas was significantly higher in the tail group compared with both the head and whole groups (head, 1,472+/-326 IE/g; tail, 4,256+/-574 IE/g; whole, 2,424+/-506 IE/g). Total islet yield from the head group was significantly lower compared with both tail and whole groups (head, 75,016+/-18,933 IE; tail, 197,469+/-28,236 IE; whole, 208,207+/-43,414 IE), and the tail group showed similar islet yield to the whole group. The whole group showed significantly lower purities and the head group showed significantly lower morphologic scores. There were no significant differences in viability, function, and insulin content among the three groups. The tail section of the human pancreas is suitable for islet isolation. The living donor islet transplantation may be feasible using only this section of the pancreas for the first transplantation to reduce hypoglycemic unawareness for small recipients, which might be followed by the second islet transplantation from cadaveric donor.

ANALYSIS OF FACTORS LEADING TO SUBOPTIMAL USE OF DECEASED DONOR PANCREATA IN THE ERA OF ISLET CELL TRANSPLANATION.

P131 Aims: In the USA only 30 % of consented deceased organ donors have their pancreata procured and transplanted (PTX), leaving about 4,500 organs unused. In an era of anticipated strong need of pancreatic islets for transplant (ICT), this low utilization pattern may be problematic. We undertook this study to try to understand the factors which led to non-use of deceased donor pancreata in a single, large Organ Procurement Organization (OPO). Methods: All records of a single large OPO over a consecutive 8 month period in 2003 were reviewed. Charts were analyzed for presence/absence of organ placement, discard, and reasons for no-use/turndown of pancreata. Published accepted criteria of organ placement for ICT were compared to outcome of attempted placement in these deceased donors. Results: From 173 consented donors 144 were deemed medically suitable and were used for organ recovery (81.4%). Organs recovered were: 235 kidneys (81.6%), 136 livers (94.4%), 51 hearts (35.4%), 14 heart valves (9.7%), 34 lungs (11.8%), 52 pancreata (36.1%) and 1 small bowel (0.69%). 4 pancreata were discarded after procurement. In all, 96 (55% of all donors, 67 % of medically suitable donors) pancreata were not procured or not accepted by centers for the following reasons:donor age below 16 and above 64 in 27 cases (28%), positive hepatitis B and/or C serology in 13 cases (14%), donor comorbidities, such as cardiovascular and renal, and donor instability in 12 cases (13%); another 12 pancreata were not procured because of intraoperative findings, such as chronic pancreatitis, blunt pancreatic trauma, and fatty organs (13%). Ten donors had diabetes mellitus- type I or type II on insulin (10%) and another seven had severe hyperglycemia and necessitated insulin for glucose control without previously known diabetes mellitus (7%). In six cases there was no consent for pancreas retrieval for transplant and research (6%); another five donors had concerning social history (5%). Four pancreata were not placed for logistical reasons (4%). All four were marginal organs that were not allocated for pancreas transplantation and were declined subsequently by islet isolation laboratories for long cold ischemia time or manpower or budget issues. Of the remaining 48 pancreata accepted, 28 were used for whole organ transplant (58.3%) and 20 for islet isolation (41.7%). All of the 20 pancreata used for islet isolation (13.9% of organ donors) were allocated only after being declined for whole organ transplant for the following reasons: age above 50 in six cases (30%), obesity with BMI>35 kg/m2 in four cases (20%), high serum glucose in four cases (20%), donor instability with renal dysfunction in four cases (20%), intraoperative surgical damage in one case (5%). One donor (5%) had a BMI of 20 kg/m2 and his pancreas not accepted for PTX, but was used for islet isolation. Conclusions: Since there are different acceptance criteria in place for PTX and ICT, a significant number of organs can be allocated for ICT without depleting the donor pool for PTX. Current practices of organ allocation fall far short of maximal utilization of pancreata for both PTX and ICT. Different patterns of use and allocation will have to be developed to promote the success of ICT.

Presence of nonhematopoietic side population cells in the adult human and nonhuman primate pancreas

Side population (SP) cells defined by their ability to efflux Hoechst dye 33342 (Hst), demonstrate functional stem cell capabilities in adult murine tissues and may represent organ-specific stem cells. We examined adult human (Hu) and rhesus macaque (Rh) pancreatic tissue for the presence of SP cells. Methods Hu cadaver ( n = 4) and Rh donor ( n = 5) pancreata were dispersed with collagenase and separated by density gradient centrifugation to relatively enrich fractions for islet, ductal, and acinar tissue in human and islet and nonislet tissue in Rh. Single cell suspensions were incubated with varying Hst concentrations to determine optimal conditions for SP cell analysis. Cellular heterogeneity was assessed using a panel of monoclonal antibodies positive for hematopoietic and/or endothelial cells. Results Hu SP cells comprised approximately 0.12%, 0.08%, and 0.45% of the gated populations for Hu islet, ductal, and acinar fractions respectively. In Rh, 5.5% and 3.7% of the islet and nonislet fractions were identified as SP cells. FACS analysis of Hu pancreas-derived SP cells indicated that greater than 95% were CD45 −, and only 6% were CD34 +CD45 −. A similar phenotype was detected in Rh pancreas-derived SP cell populations: greater than 70% were CD45 − and less than 2% were endothelial lineage positive. Conclusions SP cells are found in both islet- and nonislet-enriched fractions of the adult Hu and Rh pancreas. The majority of pancreatic SPs are CD45 − and CD34 −, suggesting nonhematopoietic lineage. Further preclinical study is needed to establish the phenotype and functional role of adult tissue-specific versus tissue-resident stem cells.

Analysis of donor factors affecting human islet isolation with current isolation protocol

Background Appropriate donor selection is one of the keys for successful human islet isolation. Previous studies identified several critical donor factors; however, significant improvements in current human islet isolation protocols make reevaluation of donor factors necessary. Study design Review was performed on 31 human islet isolations. Islet isolations were conducted using the standard automated islet isolation method with three protocol revisions that included the two-layer method (TLM) of pancreas preservation prior to islet isolation, usage of purified collagenase mixture Liberase, and continuous density gradient for islet purification. Factors leading to successful isolations (islet yield > 100,000 IE and static incubation stimulation index greater than 2.0) were analyzed. The impacts of various risk factors were also examined. Results Donors in the successful islet isolation group had a significantly lower incidence of elevated peak transaminases and creatinine levels, lower usage of norepinephrine or cardiac arrest, less prolonged hospitalization (> 96 hours), and less prolonged preservation time of donor pancreata (>25 hours). The TLM extended acceptable preservation time of donor pancreata from 8 to 25 hours. When donors had no risk factor, the success rate was 14/16 (87.5%). In sharp contrast, when donors had two or more risk factors, the success rate was 0/7 (0%; P < .001). Conclusion Risk factors for human islet isolation with the current islet isolation protocol were identified. The decision to process pancreata based on review of donor factors should improve the consistency of human islet isolations and transplantation for curing type 1 diabetes.