Adult Pancreata Research Articles

Heterogeneity of Islet Cells during Embryogenesis and Differentiation.

Diabetes is caused by insufficient insulin secretion due to β-cell dysfunction and/or β-cell loss. Therefore, the restoration of functional β-cells by the induction of β-cell differentiation from embryonic stem (ES) and induced-pluripotent stem (iPS) cells, or from somatic non-β-cells, may be a promising curative therapy. To establish an efficient and feasible method for generating functional insulin-producing cells, comprehensive knowledge of pancreas development and β-cell differentiation, including the mechanisms driving cell fate decisions and endocrine cell maturation is crucial. Recent advances in single-cell RNA sequencing (scRNA-seq) technologies have opened a new era in pancreas development and diabetes research, leading to clarification of the detailed transcriptomes of individual insulin-producing cells. Such extensive high-resolution data enables the inference of developmental trajectories during cell transitions and gene regulatory networks. Additionally, advancements in stem cell research have not only enabled their immediate clinical application, but also has made it possible to observe the genetic dynamics of human cell development and maturation in a dish. In this review, we provide an overview of the heterogeneity of islet cells during embryogenesis and differentiation as demonstrated by scRNA-seq studies on the developing and adult pancreata, with implications for the future application of regenerative medicine for diabetes.

Generation and characterization of a human iPSC line HECi001-A from a healthy adult donor pancreata

Human induced pluripotent stem cells (hiPSC) are considered promising sources of pancreatic islet organoids to treat diabetes. Due to that epigenetic memory predisposes iPSCs to enhanced differentiation into the parental cell type, we sought to generate iPSC lines from islet cells isolated from healthy adult pancreata. Islet cells were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vivoteratoma formation. This cell line will be a valuable resource for drug screening, and potential source for high-efficient generation of insulin-producing islets for treating diabetes.

Immunohistochemical characterization of pancreatic duodenal homeobox protein-1, neurogenin-3, and insulin protein expressions in islet-mesenchymal cell in vitro: a morphochronological evaluation.

Objective(s): The use of a co-culture of islets with mesenchymal stromal cells (MSCs) is a promising therapy in islet transplantation to revert hyperglycaemia, but the resulting insulin-producing cells (IPCs) express low levels of pancreas endocrine developmental genes. This study aims to investigate the morphochronology of a co-culture of islets with MSCs from injured adult pancreata, and characterize pancreatic duodenal homeobox protein-1 (Pdx1), neurogenin-3 (Ngn3) and insulin protein expressions to establish the fate of their interaction. Materials and Methods: Islets and MSCs were isolated from sham operated control (SOC) and duct-ligated (PPDL) pancreata. Islets from SOC or PPDL tissues were cultured with or without MSCs in RPMI1640, supplemented by 1% Penicillin-Streptomycin, and maintained at 37 °C±1 °C at 95% relative humidity and 95% /5% air/CO2. Pdx1, Ngn3 and insulin expressions were determined by immunohistochemistry and islet morphochronological changes were assessed. Results: Pdx1 was expressed in all islet-cell cultures with or without MSCs. Pdx1+ islet cells were significantly increased in the presence of MSCs compared to the islet culture without MSCs. Similarly, Ngn3 was highly expressed in all cultures with MSCs from both SOC and PPDL tissues and the expression was prolonged in cultures using PPDL tissues before it was down-regulated, thereby, extending the period of Ngn3+ cell expansion and differentiation into mature functional islets. Conclusion: In vitro, MSCs maintain a pool of Ngn3+ that contributes to insulin production from mature beta cells but the activation of insulin production from non-beta cells may not be induced by direct signals from MSCs.

Early islets and mesenchyme from an injured adult pancreas improve syngeneic engraftments and islet graft function in diabetic rats

A decrease in mass of isografts and a decline in islet function are major challenges in islet transplantations. Despite this, transplantation of 84 h harvested pancreatic duct ligation (PDL) tissues have been shown to have the same functional ability to foetal pancreata, but there was only 40% success in reverting hyperglycaemia. We tested the potential of early islets with mesenchymal stromal cells (MSCs) to promote isogeneic grafts survival and to restore normoglycemia in diabetic rats, in comparison with late islets. Islets were isolated from injured adult pancreata of donor rats at 24 h post ligation either with MSCs (24 h islet/MSC+) or without MSCs (24 h islet/MSC-), and at 84 h without MSCs (84 h islet/MSC-). These cells were transplanted under the renal capsule of syngeneic STZ-diabetic recipient rats. The islet grafts were monitored using the BGLs of recipients and the immunohistomorphology of the grafts were analysed using anti-insulin and anti-Ki67 antibodies. The mean BGL in 24 h islet/MSC+ recipients was reduced over time toward the control value. The curves of the mean BGLs in the control islet/MSC- and the 24 h islet/MSC- recipients dropped significantly below the control normal glucose group’s levels to reach their nadirs on weeks 4 and 6, respectively. Both curves had a peak overshoot on week 9, with no statistical significant difference between them. Engrafted islets were evident in these recipients, lasted for 5 and 6 weeks and correspondingly survived failure. However, insulin+ cells were present in the isografts of all recipients; but, only isografts in the 24 h islet/MSC+ presented with a homogenous subcapsular beta cell mass. In addition, the tendency of 24 h islet/MSC- to restore normoglycaemia with its survival capacity was statistically highly significant compared to the 84 islet/MSC- recipients (80%; 20%; p = 0.001). Transplantation of early islets with MSCs from injured adult pancreata prolongs islet graft survival and improves isograft function in diabetic rats. This novel observation requires much further exploration for its clinical application, but this model already provides hope for new sources of donor islets for transplantation.

Mesenchymal cells are required for epithelial duct cell-to-beta cell maturation and function in an injured adult pancreas in the rat

The islet, the endocrine portion of the pancreas − develops from an invagination of the pancreatic duct epithelial cells (PDECs) into the surrounding tissue. The contact of the PDECs with mesenchymal cells (MSCs) may be an essential drive for endocrine cell fate. During pancreatic development, cells that express Neurogenin-3 (Ngn3) biomarker are precursors of insulin- producing beta cells. These precursors have been reported in the neogenesis of islets from adult tissues following the surgical ligation of the main pancreatic duct (PDL). But the capacity of these precursors to induce the appropriate signals to complete the entire neogenesis program has been questioned. We studied the fate of co-culture of PDECs and MSCs from the ligated adult pancreas and established the exact location of adult stem- or progenitor-like cells that give rise to beta cells. PDECs were cultured in direct contact with or without MSCs in serum-containing culture media. The cytomorphology of the cells in co-cultures was determined and the immunocytochemical study of the cells was carried out using anti-Ngn3, anti-insulin and anti-cytokeratin-7 (CK7) antibodies. Both the PDEC/MSC- and PDEC/MSC+ cultures showed out- pocketing from duct epithelium by the end of the second week, which are distinct as cell clusters only in PDEC/MSC+ cells later in week four, exhibiting numerous branching ducts. Co-expression of Ngn3 with insulin was observed in both cultures from the second week. However, characterizations of these Ngn3+ cells in the PDEC/MSC+ culture revealed that these cells also co-expressed a CK7 biomarker. This study provides new evidence of the ductal epithelial nature of beta cells in injured adult pancreata; and that the mesenchymal stromal cells are required to sustain Ngn3 expression for beta cell maturation and function.

Novel Observations From Next-Generation RNA Sequencing of Highly Purified Human Adult and Fetal Islet Cell Subsets.

Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic α- and β-cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase β-cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify α-, β-, and δ-cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the subpopulations by flow cytometry, and, using next-generation RNA sequencing, we report the detailed transcriptomes of fetal and adult α- and β-cells. We observed that human islet composition was not influenced by age, sex, or BMI, and transcripts for inflammatory gene products were noted in fetal β-cells. In addition, within highly purified adult glucagon-expressing α-cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet α- and β-cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes.

The fractal spatial distribution of pancreatic islets in three dimensions: a self-avoiding growth model

The islets of Langerhans, responsible for controlling blood glucose levels, are dispersed within the pancreas. A universal power law governing the fractal spatial distribution of islets in two-dimensional pancreatic sections has been reported. However, the fractal geometry in the actual three-dimensional pancreas volume, and the developmental process that gives rise to such a self-similar structure, has not been investigated. Here, we examined the three-dimensional spatial distribution of islets in intact mouse pancreata using optical projection tomography and found a power law with a fractal dimension of 2.1. Furthermore, based on two-dimensional pancreatic sections of human autopsies, we found that the distribution of human islets also follows a universal power law with a fractal dimension of 1.5 in adult pancreata, which agrees with the value previously reported in smaller mammalian pancreas sections. Finally, we developed a self-avoiding growth model for the development of the islet distribution and found that the fractal nature of the spatial islet distribution may be associated with the self-avoidance in the branching process of vascularization in the pancreas.

PERK Is Required in the Adult Pancreas and Is Essential for Maintenance of Glucose Homeostasis

Germ line PERK mutations are associated with diabetes mellitus and growth retardation in both rodents and humans. In contrast, late embryonic excision of PERK permits islet development and was found to prevent onset of diabetes, suggesting that PERK may be dispensable in the adult pancreas. To definitively establish the functional role of PERK in adult pancreata, we generated mice harboring a conditional PERK allele in which excision is regulated by tamoxifen administration. Deletion of PERK in either young adult or mature adult mice resulted in hyperglycemia associated with loss of islet and β cell architecture. PERK excision triggered intracellular accumulation of proinsulin and Glut2, massive endoplasmic reticulum (ER) expansion, and compensatory activation of the remaining unfolded-protein response (UPR) signaling pathways specifically in pancreatic tissue. Although PERK excision increased β cell death, this was not a result of decreased proliferation as previously reported. In contrast, a significant and specific increase in β cell proliferation was observed, a result reflecting increased cyclin D1 accumulation. This work demonstrates that contrary to expectations, PERK is required for secretory homeostasis and β cell survival in adult mice.

Superiority of Visipaque (Iodixanol)-Controlled Density Gradient Over Ficoll-400 in Adult Porcine Islet Purification

Objective Sufficient and favorable biological functions of islets are major problems hindering xenotransplantation. The aim of the present study was to evaluate the effects on harvesting, purity, viability, and function of using improved Visipaque (iodixanol) and Ficoll-400 for adult porcine islet purification. Methods Twelve adult porcine pancreata were randomly divided into an Iodixanol-University of Wisconsin (UW) group and a Ficoll-400-UW group according to the purification method. Porcine pancreata were isolated by collagenase digestion. After isolation and purification, the islet yield and purity were evaluated by dithizone staining, and islet function assessed by in vitro insulin release assays and in vivo islet xenotransplantation. Results There were no marked differences in the islet yield before purification (5254.67 ± 189.44 IEQ/g vs 5092.67 ± 178.94 IEQ/g, P > .05). After purification, there were significantly more islets harvested in Iodixanol-UW group than in the Ficoll-400-UW group: 4222.00 ± 228.84 IEQ/g vs 3036.83 ± 79.60 IEQ/g ( P < .05). Islets from the two groups showed satisfactory insulin secretory ability. There were no significant differences in islet survival times between the two groups in diabetic rats: 8.2 ± 1.619 days vs 6.9 ± 1.197 days ( P > .05). Conclusion The improved iodixanol-UW density gradient method was superior to Ficoll-400 method to improve the number, viability, and insulin secret of purified adult porcine islets although the benefits did not improve in vivo survival.

High Levels of Thyrotropin-Releasing Hormone Receptors Activate Programmed Cell Death in Human Pancreatic Precursors

Thyrotropin-releasing hormone (TRH) is expressed in rodent and human adult pancreata and in mouse pancreas during embryonic development. However, expression of TRH receptors (TRHRs) in the pancreas is controversial. We sought to provide evidence that the TRH/TRHR system might play a role in fetal development. We used quantitative reverse transcription-polymerase chain reaction to measure TRH and TRHR messenger RNA (mRNA). To study the effects of TRHR expression in a pancreatic progenitor population, we expressed TRHRs in human islet-derived precursor cells (hIPCs) by infection with adenoviral vector AdCMVmTRHR. Thyrotropin-releasing hormone receptor signaling was measured as inositol phosphate production and intracellular calcium transients. Thyrotropin-releasing hormone receptor expression was measured by [H]methyl-TRH binding. Apoptosis was monitored by release of cytochrome c from mitochondria. We show that TRH mRNA is expressed in human fetal and adult pancreata, and that TRHR mRNA is expressed in fetal human pancreas but not in adult human pancreas. Thyrotropin-releasing hormone receptors expressed in hIPCs were shown to signal normally. Most importantly, TRH treatment for several days stimulated apoptosis in hIPCs expressing approximately 400,000 TRHRs per cell. These findings suggest a possible role for TRH/TRHR signaling in pancreatic precursors to promote programmed cell death, a normal constituent of morphogenesis during embryonic development in humans.

Hyperemic Islets: A Possible Explanation for Poor Yields in Human and Porcine Islet Isolation

When studying histological characteristics of human and porcine pancreata in relation to islet isolation, we encountered a remarkably high number of hyperemic islets. The abnormalities observed in these islets ranged from a single dilated vessel through multiple widely dilated vessels to hemorrhages extending into the surrounding exocrine tissue. We determined their possible relevance for outcomes of islet isolation. This study involved a histological examination of 143 porcine pancreata (72 juvenile and 71 adult) and islet isolation from 48 adult pancreata. Human pancreata obtained from 71 multiple organ donors yielded islet isolation in 24 cases. To determine their endocrine content, tissue samples were stained with Aldehyde Fuchsin. The presence of hyperemic islets was scored semiquantitatively with pancreata allotted to categories based on the severity. In humans and pigs we observed hyperemic islets in 48% of pancreata, but only 4.0 ± 2.4% of the islets were hyperemic. In both humans and pigs, significantly higher endocrine content was found in the most severely affected pancreata. When the higher endocrine content was taken into account and isolation results were expressed as ratios of yield and content, we observed significantly lower yields in the most affected pancreata in pigs with a trend toward lower yields in humans. A substantial proportion of human and porcine pancreata contain hyperemic islets. Although the results in humans are preliminary, our data suggest that this phenomenon may contribute to the unpredictable, highly variable islet yields in pigs and humans.

Notch Signaling Is Required for Exocrine Regeneration After Acute Pancreatitis

The mechanisms for tissue regeneration and renewal after acute pancreatitis are not well understood but may involve activation of Notch signaling. To study the effect of Notch signaling ablation during acute experimental pancreatitis, we used a chemical and genetic approach to ablate Notch signaling in cerulein-induced pancreatitis in mice. Acute pancreatitis was induced by cerulein treatment in mice treated with the gamma-secretase inhibitor dibenzazepine or in conditional Notch1 knockout mice. Mice were characterized using immunohistologic, biochemical, and molecular methods. To investigate Notch and beta-catenin interaction, acinar 266-6 cells were analyzed using transfection and biochemical assays. Loss of Notch signaling results in impaired regeneration after acute pancreatitis with fewer mature acinar cells in dibenzazepine-treated and Notch1-deficient mice in the regenerative phase 3 days after induction. beta-catenin expression was increased and prolonged during exocrine regeneration. Crosstalk between Notch and beta-catenin-mediated signaling was identified, with Notch1-IC inhibiting beta-catenin-mediated transcriptional activity. This inhibition was dependent on a functional RAM domain. Inhibition of Notch signaling in vivo leads to impaired regeneration of the exocrine pancreas after acute pancreatitis. Our results suggest an interaction of Notch and Wnt signaling in pancreatic acinar cells, providing evidence for a role of these pathways in the regulation of the maturation process of acinar cells.

SPONTANEOUS DEVELOPMENT OF MURINE PANCREATIC INTRAEPITHELIAL NEOPLASIA (MPANIN) IN ELA-CREERT2; LSL-KRASG12D MICE

Background: Pancreatic ductal adenocarcinoma (PDAC) is a uniformly lethal neoplasm. PDAC arises in association with microscopic precursor lesions known as pancreatic intraepithelial neoplasia (PanIN). The cellular origins of PDAC remain an enigma, and despite the "ductal" moniker, studies in genetically engineered mice (GEM) have failed to definitively establish an ontogenic relationship with mature ductal epithelium. Recent GEM models of PDAC recapitulating the cognate human disease were created by developmental targeting of mutant KrasG12D to the entire pancreatic anlage, using Pdx1-regulated Cre recombination. However these elegant studies did not specifically address the cellular origins of murine PanINs (mPanINs) in the context of adult pancreata, which is the biologically relevant soil for PDAC. Methods: In order to determine the phenotypic effects of oncogenic KrasG12D in the mature pancreas, we generated Pdx1-CreERT2; LSL-KrasG12D and Elastase (Ela)-CreERT2; LSL-KrasG12D mice. Post-natally, Pdx1 expression is restricted to endocrine cells, while Ela expression is restricted to the exocrine acinar / centro-acinar population. Tamoxifen-mediated CreERT2 activation in these GEM permits targeting mutant KrasG12D to the respective Pdx1 and Ela-expressing cell types. Therefore, at 6 weeks of age, tamoxifen was administered as once daily intraperitoneal injection for a period of 5 days. Thirty mice were generated from each cohort (Pdx1-CreERT2; LSL-KrasG12D and Ela-CreERT2; LSL-KrasG12D), and groups of five mice were harvested at two-monthly intervals, beginning at 2 months post-tamoxifen injection, and culminating at 1 year. Non-induced littermates were used as control. Results: No pancreatic phenotype was observed in Pdx1-CreERT2; LSL-KrasG12D mice even at 1 year post-induction. In contrast, Ela-CreERT2; LSL-KrasG12D mice spontaneously developed mPanIN at 4 months, and by 1 year, had developed the entire histologic spectrum of pre-invasive lesions, including high-grade mPanIN-3. The advanced mPanINs typically arose in association with lobular atrophy, acinar to ductal metaplasia, and presence of intermediate acinar-ductal cell types. Conclusions: While developmental targeting of mutant KrasG12D to Pdx1-expressing cells consistently results in a pre-invasive ductal phenotype, Pdx1-expressing cells in the post-natal pancreas appear to be largely resistant to effects of the oncogenic allele. In contrast, the mature Ela-expressing acinar / centroacinar population retains its susceptibility for spontaneous mPanIN formation. A recent report by Guerra and colleagues had suggested the absolute requirement for concomitant exocrine injury (chronic pancreatitis) in order for mPanINs to develop in the context of Kras misexpression in mature exocrine tissues. However, as the Ela-CreERT2; LSL-KrasG12D mice demonstrate, the Ela-expressing acinar / centroacinar cells can be spontaneously induced along a path towards pancreatic neoplasia by a single initiating genetic event.

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.

Adaption of neutral protease activity for islet isolation from the long-term two-layer method–stored pig pancreas

Background Islet release from the pancreas is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. To prove the hypothesis that adjustment of NP reduces islet damage after prolonged ischemia, adult pig pancreata were digested after 7-hour preservation by the two-layer method (TLM) using a 2-component enzyme blend consisting of collagenase NB-8 and NP. Methods After intraductal University of Wisconsin (UW) flush resected pancreata were distended with 4.4 PZ-U/g of UW-dissolved Serva collagenase either before (TLM-preloaded, n = 7) or after (TLM-postloaded, n = 10) cold storage, or for immediate processing (n = 6). NP was adjusted after preliminary experiments to respectively 1.1, 0.2, or 0.8 DMC-U/g for unstored, TLM-preloaded, or postloaded organs. Results Purified islet yield decreased from 3670 ± 730 islet equivalents (IEQ)/g in unstored pancreata to 1800 ± 180 and 2080 ± 290 IEQ/g in TLM-preloaded or postloaded organs, respectively ( P < .05). Although purity was always >90%, IEQ recovery was significantly decreased in TLM-preloaded pancreata. Quality control revealed consistently high viability as determined using trypan-blue exclusion (>95%) or formazan production. Compared with unstored organs (2.47 ± 0.36; P < .05), glucose stimulation index was reduced in TLM-preloaded (1.48 ± 0.15) and TLM-postloaded pancreata (1.81 ± 0.20). Normoglycemia in diabetic nude mice transplanted with islets from TLM-preloaded pancreata was transient in contrast to sustained function in the other groups. Conclusions Significant amounts of viable pig islets can be isolated after prolonged TLM preservation by reducing NP activity. Nevertheless, early enzyme administration prior to long-term storage deteriorates islet graft function.

Improvement of massive human islet isolation techniques and the evaluation of isolated human islets

To obtain massive human pancreatic islets with modified techniques and evaluation of the islets for the clinical allo-transplantation to treat type I and II diabetes. 28 consecutive adult human pancreata were isolated with modified automated techniques. Islets were purified using continuous density gradient. The islet yield was counted with international standard known as islet equivalent (IEQ). The function of the isolated islets was evaluated by measuring DNA/insulin ratio, static glucose stimulating test in vitro and transplanting the islets into diabetic nude mice in vivo followed by abdominal glucose tolerance test and C peptide measurement. The yield of 28 consecutive human pancreata isolations ranged from 5 000 to 1 030 000 IEQs/pancreas with the average of 291 635 IEQs/pancreas. The first 13 isolations yielded 49 123 IEQs/pancreas, 846 IEQs/g and, purity 87% in average. The remained 15 isolations after the modifications yielded 501 813 IEQs/pancreas, 7 003 IEQs/g and purity 89% in average. The results of in vitro SGS showed good response to the different glucose concentration. 34 diabetic nude mice were transplanted under the renal capsule with the freshly isolated islets. 29 out of 34 diabetic mice obtained normoglycemia within 12 hours and the glucose tolerance tests were near normal. Serum C peptide level of transplanted mice is close to that of the control group. Massive human islets can be isolated with the modified techniques. Quality assessment of these islets both in vitro and in vivo has indicated that these high quality human islets could be used for the clinical allogeneic islet transplantation.

LONG-TERM PRESERVATION OF THE PIG PANCREAS BY THE ONE-LAYER METHOD FOR SUCCESSFUL ISLET ISOLATION

P33 Aims: The oxygenation of the human pancreas during cold storage was recently established for clinical islet transplantation by introduction of the two-layer method (TLM). Nevertheless, simplification of TLM would facilitate its application as a broadly used pancreas preservation method for subsequent islet transplantation. Preliminary data about vascularized pancreas transplantation in rats and dogs indicate that successful preservation by a one-layer method (OLM) omitting UW solution (UWS) as the second layer is also possible. The purpose of the present study was therefore to examine the efficiency of OLM for pig islet isolation after 7 hours of cold ischemia. Methods: After resection adult pig pancreata were intraductally flushed with 2 mL/g of cold UWS. Subsequently, pancreata were either continuously oxygenated during cold ischemia by the TLM (n=10) or OLM (n=8), or processed promptly (n=6). Immediately before digestion-filtration pancreata were intraductally distended with cold UWS supplemented with Collagenase NB 8 and Neutral Protease NB (SERVA). Isolated islets were isolated and purified as previously described. Quality control included determination of islet yield expressed as islet equivalents (IEQ), isolation index (IEQ/islet number), trypan-blue exclusion, glucose stimulation index during static incubation (20 vs 2.8 mM glucose), insulin and ATP content, and mitochondrial activity measured as formazan production at 490 nm and expressed as optical density (OD). Islet in vivo function was evaluated after transplantation into diabetic nude mice. Results: No significant differences were found regarding islet purity (>90%) and viability (>95%). Compared to freshly procured pancreata (429200±86700 IEQ) postpurification islet yield was significantly decreased (P<0.05) after prolonged cold storage by TLM (238000±26600 IEQ). In contrast, islet yield was significantly higher (P<0.05) in cold-stored organs using OLM (338600±42100 IEQ, NS vs fresh). Metabolic islet integrity was not different between unstored pancreata and organs preserved by OLM or TLM as indicated by the intracellular insulin content (710±130 vs 830±120 vs 750±70 μU/IEQ), formazan production (0.7±0.1 vs 0.6±0.1 vs 0.7±0.1 OD/1000 IEQ) and ATP content (390±70 vs 370±80 vs 410±170 pg/IEQ). In contrast, glucose stimulation index was significantly decreased in islets isolated after TLM-storage (1.8±0.2, P<0.05) but not after OLM-storage (2.3±0.6, NS) compared to freshly isolated islets (2.5±0.4). Transplantation into diabetic nude mice demonstrated islet graft function in all experimental groups. Conclusions: The present study demonstrates clearly that viable pig islets can be successfully isolated from long-term stored pig pancreata utilizing perfluorochemicals as a single layer for oxygenation of pancreatic tissue during cold storage. This methodological simplification of the TLM might be of relevance for broad application in clinical islet transplantation.

ADAPTION OF NEUTRAL PROTEASE ACTIVITY FOR ISLET ISOLATION FROM THE LONG-TERM TLM-STORED PIG PANCREAS

P32 Aims: The enzymatic dissociation of pancreatic tissue by collagenase is a key step in the islet isolation procedure. Previous investigations clearly showed that purified collagenase needs additional protease activities to successfully release islets from the acinar tissue. We speculate that adjustment of neutral protease (NP) activity as a critical effector of islet integrity might be useful to reduce islet damage during digestion after long-term storage of the pancreas. To prove this hypothesis adult pig pancreata were digested by means of a new two-component collagenase blend consisting of Collagenase NB 8 and Neutral Protease NB (SERVA) after 7 hours of cold ischemia. Methods: After resection pancreata were intraductally flushed with 2 mL/g cold UW solution (UWS). Subsequently, pancreata were intraductally distended with Collagenase NB-8 (4.3 PZ-U/g) dissolved in cold UWS either before or after cold storage by the two-layer method (TLM). NP activity was adjusted according to collagenase administration to respectively 1.1, 0.2 or 0.8 DMC-U/g for pancreata freshly procured (n=6) or distended with collagenase before (TLM-preloaded, n=7) or after TLM storage (TLM-postloaded, n=10). Islets were isolated as previously described. Quality control included determination of islet yield expressed as islet equivalents (IEQ), isolation index (IEQ/islet number), trypan-blue exclusion, glucose stimulation index during static incubation (20 vs 2.8 mM glucose), insulin content, and mitochondrial activity measured as formazan production at 490 nm and expressed as optical density (OD). Islet in vivo function was evaluated after transplantation into diabetic nude mice. Results: Postpurification islet yield decreased significantly (P<0.05) from 429200±86700 IEQ in freshly procured pancreata to respectively 210400±22900 and 238000±26600 IEQ in TLM-preloaded or postloaded organs. As indicated by the isolation index (IEQ/islet number) islet integrity was preserved in TLM-preloaded (0.7±0.1) as well as in postloaded organs (0.7±0.1) compared to freshly procured pancreata (0.9±0.1). No significant differences were found regarding islet purity (>90%) and viability (>95%). Cold storage significantly reduced glucose stimulation index of islets isolated from TLM-preloaded (1.5±0.2) and postloaded organs (1.8±0.2) compared to freshly isolated islets (2.5±0.4, P<0.05). No differences were observed between unstored organs, TLM-preloaded or postloaded pancreata regarding insulin content (710±130 vs 680±30 vs 750±70 μU/IEQ) and mitochondrial activity (0.7±0.1 vs 0.8±0.1 vs 0.7±0.1 OD/1000 IEQ). Transplantation into diabetic nude mice revealed islet graft function in all experimental groups. Conclusions: To our knowledge pig pancreata have never been subjected to 7 hours of cold storage prior to islet isolation before. The present study demonstrates that pig islets can be successfully isolated after prolonged periods of cold storage by the TLM if proteolytic activity is adapted according to the timing of collagenase administration.

Human Pancreatic Islet-Derived Progenitor Cell Engraftment in Immunocompetent Mice

The potential for the use of stem/progenitor cells for the restoration of injured or diseased tissues has garnered much interest recently, establishing a new field of research called regenerative medicine. Attention has been focused on embryonic stem cells derived from human fetal tissues. However, the use of human fetal tissue for research and transplantation is controversial. An alternative is the isolation and utilization of multipotent stem/progenitor cells derived from adult donor tissues. We have previously reported on the isolation, propagation, and partial characterization of a population of stem/progenitor cells isolated from the pancreatic islets of Langerhans of adult human donor pancreata. Here we show that these human adult tissue-derived cells, nestin-positive islet-derived stem/progenitor cells, prepared from human adult pancreata survive engraftment and produce tissue chimerism when transplanted into immunocompetent mice either under the kidney capsule or by systemic injection. These xenografts seem to induce immune tolerance by establishing a mixed chimerism in the mice. We propose that a population of stem/progenitor cells isolated from the islets of the pancreas can cross xenogeneic transplantation immune barriers, induce tissue tolerance, and grow.

Changing distribution of islets in the developing human pancreas: a computer-assisted three-dimensional reconstruction study.

Tissue specimens of nine normal human pancreata from fetuses, neonates, and adults were subjected to serial sectioning and computer-assisted three-dimensional (3-D) reconstruction in an effort to study the growth of Langerhans' islets based on their distribution in the lobule and their relationship with the ducts and arterioles. Also, islet volumes were obtained by 3-D morphometry. In 24-week-old fetuses, the islets were shown to be in direct contact with the ducts, whereas in the neonates, they gradually became separated from the ducts, finally dispersing throughout the lobule in the adults. This transition seemed to allow islet hormones to have physiological effects on the exocrine function of the acinar tissue, making the pancreas achieve the endocrine-exocrine correlation. However, half of the islets remained next to the ducts even in the adult pancreata. With regard to the relationship between the islets and arterioles, "arteriolar" islets having an afferent arteriole accounted for approximately 20% in number, but their volume cumulated to approximately 70% of the total, comprising a greater part of the pancreatic endocrine gland.