miR-122-5p is involved in posttranscriptional regulation of the mitochondrial thiamin pyrophosphate transporter (SLC25A19) in pancreatic acinar cells.

Thiamin (vitamin B1) plays a vital role in cellular energy metabolism/ATP production. Pancreatic acinar cells (PACs) obtain thiamin from circulation and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. TPP is then taken up by the mitochondria via a carrier-mediated process that involves the mitochondrial TPP transporter (MTPPT; encoded by the gene SLC25A19). We have previously characterized different aspects of the mitochondrial carrier-mediated TPP uptake process, but nothing is known about its possible regulation at the posttranscriptional level. We address this issue in the current investigations focusing on the role of miRNAs in this regulation. First, we subjected the human (and rat) 3'-untranslated region (3'-UTR) of the SLC25A19 to three in-silico programs, and all have identified putative binding sites for miR-122-5p. Transfecting pmirGLO-hSLC25A19 3'-UTR into rat PAC AR42J resulted in a significant reduction in luciferase activity compared with cells transfected with pmirGLO-empty vector. Mutating as well as truncating the putative miR-122-5p binding sites in the hSLC25A19 3'-UTR led to abrogation of inhibition in luciferase activity in PAC AR42J. Furthermore, transfecting/transducing PAC AR42J and human primary PACs with mimic of miR-122-5p led to a significant inhibition in the level of expression of the MTPPT mRNA and protein as well as in mitochondrial carrier-mediated TPP uptake. Conversely, transfecting PAC AR42J with an inhibitor of miR-122-5p increased MTPPT expression and function. These findings show, for the first time, that expression and function of the MTPPT in PACs are subject to posttranscriptional regulation by miR-122-5p.NEW & NOTEWORTHY This study shows that the expression and function of mitochondrial TPP transporter (MTPPT) are subject to posttranscriptional regulation by miRNA-122-5p in pancreatic acinar cells.

Progress in individualizing autologous islet isolation techniques for pediatric islet autotransplantation after total pancreatectomy in children for chronic pancreatitis.

Total pancreatectomy with islet autotransplantation is performed to treat chronic pancreatitis in children. Successful islet isolation must address the challenges of severe pancreatic fibrosis and young donor age. We have progressively introduced modifications to optimize enzymatic and mechanical dissociation of the pancreas during islet isolation. We evaluated 2 islet isolation metrics in 138 children-digest islet equivalents per gram pancreas tissue (IEQ/g) and digest IEQ per kilogram body weight (IEQ/kg), using multiple regression to adjust for key disease and patient features. Islet yield at digest had an average 4569 (standard deviation 2949) islet equivalent (IEQ)/g and 4946 (4009) IEQ/kg, with 59.1% embedded in exocrine tissue. Cases with very low yield (<2000 IEQ/g or IEQ/kg) have decreased substantially over time, 6.8% and 9.1%, respectively, in the most recent tertile of time compared to 19.2% and 23.4% in the middle and 34.1% and 36.4% in the oldest tertile. IEQ/g and IEQ/kg adjusted for patient and disease factors improved in consistency and yield in the modern era. Minimal mechanical disruption during digestion, warm enzymatic digestion using enzyme collagenase:NP activity ratio < 10:1, coupled with extended distension and trimming time during islet isolation of younger and fibrotic pediatric pancreases, gave increased islet yield with improved patient outcomes.

The demise of islet allotransplantation in the United States: A call for an urgent regulatory update.

Islet allotransplantation in the United States (US) is facing an imminent demise. Despite nearly three decades of progress in the field, an archaic regulatory framework has stymied US clinical practice. Current regulations do not reflect the state-of-the-art in clinical or technical practices. In the US, islets are considered biologic drugs and "more than minimally manipulated" human cell and tissue products (HCT/Ps). In contrast, across the world, human islets are appropriately defined as "minimally manipulated tissue" and not regulated as a drug, which has led to islet allotransplantation (allo-ITx) becoming a standard-of-care procedure for selected patients with type 1 diabetes mellitus. This regulatory distinction impedes patient access to islets for transplantation in the US. As a result only 11 patients underwent allo-ITx in the US between 2016 and 2019, and all as investigational procedures in the settings of a clinical trials. Herein, we describe the current regulations pertaining to islet transplantation in the United States. We explore the progress which has been made in the field and demonstrate why the regulatory framework must be updated to both better reflect our current clinical practice and to deal with upcoming challenges. We propose specific updates to current regulations which are required for the renaissance of ethical, safe, effective, and affordable allo-ITx in the United States.

Preoperative ERCP has no impact on islet yield following total pancreatectomy and islet autotransplantation (TPIAT): Results from the Prospective Observational Study of TPIAT (POST) cohort.

BACKGROUND AND AIMS: Many patients undergoing total pancreatectomy with islet autotransplant (TPIAT) for severe, refractory chronic pancreatitis or recurrent acute pancreatitis have a history of endoscopic retrograde cholangiopancreatography (ERCP). Using data from the multicenter POST (Prospective Observational Study of TPIAT) cohort, we aimed to determine clinical characteristics associated with ERCP and the effect of ERCP on islet yield. METHODS: Using data from 230 participants (11 centers), demographics, pancreatitis history, and imaging features were tested for association with ERCP procedures. Logistic and linear regression were used to assess association of islet yield measures with having any pre-operative ERCPs and with the number of ERCPs, adjusting for confounders. RESULTS: 175 (76%) underwent ERCPs [median number of ERCPs (IQR) 2 (1-4). ERCP was more common in those with obstructed pancreatic duct (p = 0.0009), pancreas divisum (p = 0.0009), prior pancreatic surgery (p = 0.005), and longer disease duration (p = 0.004). A greater number of ERCPs was associated with disease duration (p < 0.0001), obstructed pancreatic duct (p = 0.006), and prior pancreatic surgery (p = 0.006) and increased risk for positive islet culture (p < 0.0001). Mean total IEQ/kg with vs. without prior ERCP were 4145 (95% CI 3621-4669) vs. 3476 (95% CI 2521-4431) respectively (p = 0.23). Adjusting for confounders, islet yield was not significantly associated with prior ERCP, number of ERCPs, biliary or pancreatic sphincterotomy or stent placement. CONCLUSIONS: ERCP did not appear to adversely impact islet yield. When indicated, ERCP need not be withheld to optimize islet yield but the risk-benefit ratio of ERCP should be considered given its potential harms, including risk for excessive delay in TPIAT.

Differential cytotoxicity, ER/oxidative stress, dysregulated AMPKα signaling, and mitochondrial stress by ethanol and its metabolites in human pancreatic acinar cells.

BACKGROUND: Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disorder of the exocrine pancreatic gland. A previous study from this laboratory showed that ethanol (EtOH) causes cytotoxicity, dysregulates AMPKα and ER/oxidative stress signaling, and induces inflammatory responses in primary human pancreatic acinar cells (hPACs). Here we examined the differential cytotoxicity of EtOH and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters; FAEEs) metabolites in hPACs was examined to understand the metabolic basis and mechanism of ACP. METHODS: We evaluated concentration-dependent cytotoxicity, AMPKα inactivation, ER/oxidative stress, and inflammatory responses in hPACs by incubating them for 6 h with EtOH, acetaldehyde, or FAEEs at clinically relevant concentrations reported in alcoholic subjects using conventional methods. Cellular bioenergetics (mitochondrial stress and a real-time ATP production rate) were determined using Seahorse XFp Extracellular Flux Analyzer in AR42J cells treated with acetaldehyde or FAEEs. RESULTS: We observed concentration-dependent increases in LDH release, inactivation of AMPKα along with upregulation of ACC1 and FAS (key lipogenic proteins), downregulation of p-LKB1 (an oxidative stress-sensitive upstream kinase regulating AMPKα) and CPT1A (involved in ß-oxidation of fatty acids) in hPACs treated with EtOH, acetaldehyde, or FAEEs. Concentration-dependent increases in oxidative stress and ER stress as measured by GRP78, unspliced XBP1, p-eIF2α, and CHOP along with activation of p-JNK1/2, p-ERK1/2, and p-P38MAPK were present in cells treated with EtOH, acetaldehyde, or FAEEs, respectively. Furthermore, a significant decrease was observed in the total ATP production rate with subsequent mitochondrial stress in AR42J cells treated with acetaldehyde and FAEEs. CONCLUSIONS: EtOH and its metabolites, acetaldehyde and FAEEs, caused cytotoxicity, ER/oxidative and mitochondrial stress, and dysregulated AMPKα signaling, suggesting a key role of EtOH metabolism in the etiopathogenesis of ACP. Because oxidative EtOH metabolism is negligible in the exocrine pancreas, the pathogenesis of ACP could be attributable to the formation of FAEEs and related pancreatic acinar cell injury.

Complement 1q-like-3 protein inhibits insulin secretion from pancreatic ß-cells via the cell adhesion G protein-coupled receptor BAI3.

Secreted proteins are important metabolic regulators in both healthy and disease states. Here, we sought to investigate the mechanism by which the secreted protein complement 1q-like-3 (C1ql3) regulates insulin secretion from pancreatic ß-cells, a key process affecting whole-body glucose metabolism. We found that C1ql3 predominantly inhibits exendin-4- and cAMP-stimulated insulin secretion from mouse and human islets. However, to a lesser extent, C1ql3 also reduced insulin secretion in response to KCl, the potassium channel blocker tolbutamide, and high glucose. Strikingly, C1ql3 did not affect insulin secretion stimulated by fatty acids, amino acids, or mitochondrial metabolites, either at low or submaximal glucose concentrations. Additionally, C1ql3 inhibited glucose-stimulated cAMP levels, and insulin secretion stimulated by exchange protein directly activated by cAMP-2 and protein kinase A. These results suggest that C1ql3 inhibits insulin secretion primarily by regulating cAMP signaling. The cell adhesion G protein-coupled receptor, brain angiogenesis inhibitor-3 (BAI3), is a C1ql3 receptor and is expressed in ß-cells and in mouse and human islets, but its function in ß-cells remained unknown. We found that siRNA-mediated Bai3 knockdown in INS1(832/13) cells increased glucose-stimulated insulin secretion. Furthermore, incubating the soluble C1ql3-binding fragment of the BAI3 protein completely blocked the inhibitory effects of C1ql3 on insulin secretion in response to cAMP. This suggests that BAI3 mediates the inhibitory effects of C1ql3 on insulin secretion from pancreatic ß-cells. These findings demonstrate a novel regulatory mechanism by which C1ql3/BAI3 signaling causes an impairment of insulin secretion from ß-cells, possibly contributing to the progression of type 2 diabetes in obesity.

Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix.

Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability. These results showed a reduced percentage of mantled islets (17% ± 7.5%) and higher postpurification islet recovery (83.8% ± 5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase-protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

Polychlorinated biphenyl exposures differentially regulate hepatic metabolism and pancreatic function: Implications for nonalcoholic steatohepatitis and diabetes.

The endocrine disrupting chemicals, polychlorinated biphenyls (PCBs), have been associated with nonalcoholic steatohepatitis (NASH) and diabetes. However, an integrative analysis of the effects of PCBs on the liver and pancreas has never been performed for the two major PCB subtypes, dioxin-like (DL) and nondioxin-like (NDL), and a mixture of NDL/DL PCBs. Therefore, male C57BL/6 J mice fed a control synthetic diet were treated with either a NDL PCB mixture, Aroclor 1260 (20 mg/kg); a single DL PCB congener, PCB 126 (20 µg/kg); a NDL/DL mixture, Aroclor 1260 plus PCB 126; or vehicle control for 2 weeks. PCB126 had the greatest impact on hepatic lipid metabolism. It caused steatosis due to increased hepatic lipid import with associated hypolipidemia. However, all PCB exposures impacted expression of hepatic lipid metabolism genes in different manners. The 'NASH gene', Pnpla3, was elevated by Aroclor 1260, but decreased by all other exposures. The expression of hepatokines implicated in metabolic syndrome (Fgf21, Igf1, and betatrophin) were differentially regulated. The NDL/DL PCB mixture had the greatest effects on pancreatic histology, including acinar cell atrophy, mild steatosis, and fibrosis without ductal changes or immune cell infiltration. It decreased expression of insulin and altered the expression of genes regulating islet identity. None of these exposures was associated with altered HOMA-IR or HOMA-B. In summary, PCB exposures differentially regulated liver and pancreas structure and function. Novel mechanisms for PCB-induced endocrine/metabolic disruption included altered hepatokines and Pnpla3 as well as 'PCB pancreatopathy' that was associated with altered expression of pancreatic islet identity factors. More research is required to understand fully these findings in the context of human NASH and diabetes.

Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield.

A high number of human islets can be isolated by using modern purified tissue dissociation enzymes; however, this requires the use of >20 Wunsch units (WU)/g of pancreas for digestion. Attempts to reduce this dose have resulted in pancreas underdigestion and poor islet recovery but improved islet function. In this study, we achieved a high number of functional islets using a low dose of recombinant collagenase enzyme mixture (RCEM-1200 WU rC2 and 10 million collagen-degrading activity [CDA] U of rC1 containing about 209 mg of collagenase to digest a 100-g pancreas). The collagenase dose used in these isolations is about 42% of the natural collagenase enzyme mixture (NCEM) dose commonly used to digest a 100-g pancreas. Low-dose RCEM was efficient in digesting entire pancreases to obtain higher yield (5535 ± 830 and 2582 ± 925 islet equivalent/g, P < .05) and less undigested tissue (16.7 ± 5% and 37.8 ± 3%, P < .05) compared with low-dose NCEM (12WU/g). Additionally, low-dose RCEM islets retained better morphology (confirmed with scanning electron microscopy) and higher in vitro basal insulin release (2391 ± 1342 and 1778 ± 978 µU/mL; P < .05) compared with standard-dose NCEM. Nude mouse bioassay demonstrated better islet function for low-dose RCEM (area under the curve [AUC] 24 968) compared with low-dose (AUC-38 225) or standard-dose NCEM (AUC-38 685), P < .05. This is the first report indicating that islet function can be improved by using low-dose rC1rC2 (RCEM).

Pig islet xenotransplantation.

PURPOSE OF REVIEW: The current article reviews the rationale, sources and preparation of pig islets for xenotransplantation, and presents current progress in solving the problems associated with establishing pig islet transplant as a clinical treatment for type 1 diabetes. SUMMARY: Islet transplantation is an effective treatment option for type 1 diabetes, but the available supply of human pancreases is insufficient to meet the need and demand for obtaining islets. Pig islets provide a readily available source for islet transplantation, with trials in non-human primates demonstrating their potential to reverse diabetes. The risk of zoonosis can be reduced by designated pathogen-free breeding of the donor pigs, but porcine endogenous retroviruses (PERVs) that are integrated into the genome of all pigs are especially difficult to eliminate. However, clinical trials have demonstrated an absence of PERV transmission with a significant reduction in the number of severe hypoglycemic episodes and up to 30% reduction in exogenous insulin doses. A number of methods such as production of various transgenic pigs to better xenotransplantation efficiency and the encapsulation of islets to isolate them from the host immune system are currently being tested to overcome the xenograft immune rejection. Furthermore, ongoing research is also shedding light on factors such as the age and breed of the donor pig to determine the optimal islet quantity and function.

Magnetic resonance imaging: a tool to monitor and optimize enzyme distribution during porcine pancreas distention for islet isolation.

Porcine islet xenotransplantation is emerging as a potential alternative for allogeneic clinical islet transplantation. Optimization of porcine islet isolation in terms of yield and quality is critical for the success and cost-effectiveness of this approach. Incomplete pancreas distention and inhomogeneous enzyme distribution have been identified as key factors for limiting viable islet yield per porcine pancreas. The aim of this study was to explore the utility of magnetic resonance imaging (MRI) as a tool to investigate the homogeneity of enzyme delivery in porcine pancreata. Traditional and novel methods for enzyme delivery aimed at optimizing enzyme distribution were examined. Pancreata were procured from Landrace pigs via en bloc viscerectomy. The main pancreatic duct was then cannulated with an 18-g winged catheter and MRI performed at 1.5-T. Images were collected before and after ductal infusion of chilled MRI contrast agent (gadolinium) in physiological saline. Regions of the distal aspect of the splenic lobe and portions of the connecting lobe and bridge exhibited reduced delivery of solution when traditional methods of distention were utilized. Use of alternative methods of delivery (such as selective re-cannulation and distention of identified problem regions) resolved these issues, and MRI was successfully utilized as a guide and assessment tool for improved delivery. Current methods of porcine pancreas distention do not consistently deliver enzyme uniformly or adequately to all regions of the pancreas. Novel methods of enzyme delivery should be investigated and implemented for improved enzyme distribution. MRI serves as a valuable tool to visualize and evaluate the efficacy of current and prospective methods of pancreas distention and enzyme delivery.

The future treatment for type 1 diabetes: Pig islet- or stem cell-derived ß cells?

Replacement of ß cells is only a curative approach for type 1 diabetes (T1D) patients to avoid the threat of iatrogenic hypoglycemia. In this pursuit, islet allotransplantation under Edmonton's protocol emerged as a medical miracle to attain hypoglycemia-free insulin independence in T1D. Shortage of allo-islet donors and post-transplantation (post-tx) islet loss are still unmet hurdles for the widespread application of this therapeutic regimen. The long-term survival and effective insulin independence in preclinical studies have strongly suggested pig islets to cure overt hyperglycemia. Importantly, CRISPR-Cas9 technology is pursuing to develop "humanized" pig islets that could overcome the lifelong immunosuppression drug regimen. Lately, induced pluripotent stem cell (iPSC)-derived ß cell approaches are also gaining momentum and may hold promise to yield a significant supply of insulin-producing cells. Theoretically, personalized ß cells derived from a patient's iPSCs is one exciting approach, but ß cell-specific immunity in T1D recipients would still be a challenge. In this context, encapsulation studies on both pig islet as well as iPSC-ß cells were found promising and rendered long-term survival in mice. Oxygen tension and blood vessel growth within the capsules are a few of the hurdles that need to be addressed. In conclusion, challenges associated with both procedures, xenotransplantation (of pig-derived islets) and stem cell transplantation, are required to be cautiously resolved before their clinical application.

Pancreatic Stellate Cells Prolong Ex Vivo Islet Viability and Function and Improve Engraftment.

Preserving islet health and function is critical during pretransplant culture to improve islet transplantation outcome and for ex vivo modeling of diabetes for pharmaceutical drug discovery. The limited islet engraftment potential is primarily attributable to loss of extracellular matrix (ECM) support and interaction. Multipotent cells with ECM depositing competency improve islet survival during short coculture period. However, role of pancreatic stellate cells (PSCs) and their ECM support in preserving ex vivo islet physiology remains largely unknown. Here, we report novel cytoprotective effects of culture-adapted porcine PSCs and role of their ECM-mediated intercellular communication on pig, mouse and human islets ex vivo. Using direct-contact coculture system, we demonstrate that porcine PSCs preserve and significantly prolong islet viability and function from 7 ± 3 days to more than 28 ± 5 (P < .001) days in vitro. These beneficial effects of PSCs on islet health are not species-specific. Using NSC47924 to specifically inhibit 37/67 kDa laminin receptor (LR), we identified that LR-mediated intercellular communication is essential for PSCs to protect functional viability of islets in vitro. Finally, our results demonstrate that PSC co-transplantation improved function and enhanced capacity of syngeneic islets to reverse hyperglycemia in mice with preexisiting diabetes. Cumulatively, our findings unveil novel effects of culture-adapted PSCs on islet health likely mirroring in vivo niche interaction. Furthermore, islet and PSC coculture may aid in development of ex vivo diabetes modeling and also suggests that a combined islet-PSC tissue engineered implant may significantly improve islet transplantation outcome.

Predictors of Glycemic Outcomes at 1 Year Following Pediatric Total Pancreatectomy With Islet Autotransplantation.

OBJECTIVE: Total pancreatectomy with islet autotransplantation (TPIAT) is indicated to alleviate debilitating pancreas-related pain and mitigate diabetes in patients with acute recurrent and chronic pancreatitis when medical/endoscopic therapies fail. Our aim was to evaluate predictors of insulin requirement at 1 year following TPIAT in a cohort of children. RESEARCH DESIGN AND METHODS: This was a review of 43 pediatric patients followed after TPIAT for 1 year or longer. Primary outcome was insulin use at 1 year, categorized as follows: insulin independent, low insulin requirement (<0.5 units/kg/day), or high insulin requirement (≥0.5 units/kg/day). RESULTS: At 1 year after TPIAT, 12 of 41 (29%) patients were insulin independent and 21 of 41 (51%) had low and 8 of 41 (20%) had high insulin requirement. Insulin-independent patients were younger than those with low and high insulin requirement (median age 8.2 vs. 14.6 vs. 13.1 years, respectively; P = 0.03). Patients with insulin independence had a higher number of transplanted islet equivalents (IEQ) per kilogram body weight (P = 0.03) and smaller body surface area (P = 0.02), compared with those with insulin dependence. Preoperative exocrine insufficiency was associated with high insulin requirement (P = 0.03). Higher peak C-peptide measured by stimulated mixed-meal tolerance testing (MMTT) at 3 and 6 months post-TPIAT was predictive of lower insulin requirement at 1 year (P = 0.006 and 0.03, respectively). CONCLUSIONS: We conclude that insulin independence following pediatric TPIAT is multifactorial and associated with younger age, higher IEQ per kilogram body weight transplanted, and smaller body surface area at time of operation. Higher peak C-peptide measured by MMTT following TPIAT confers a higher likelihood of low insulin requirement.

Correction to: trisected pancreas model for testing tissue dissociation enzyme combinations: a novel methodology for improving human islet yield for clinical islet transplantation.

[This corrects the article DOI: 10.1007/s40200-020-00519-y.].

Circulating miRNA in Patients Undergoing Total Pancreatectomy and Islet Autotransplantation.

Circulating microRNAs (miRNAs) can be biomarkers for diagnosis and progression of several pathophysiological conditions. In a cohort undergoing total pancreatectomy with islet autotransplantation (TPIAT) from the multicenter Prospective Observational Study of TPIAT (POST), we investigated associations between a panel of circulating miRNAs (hsa-miR-375, hsa-miR-29b-3p, hsa-miR-148a-3p, hsa-miR-216a-5p, hsa-miR-320d, hsa-miR-200c, hsa-miR-125b, hsa-miR-7-5p, hsa-miR-221-3p, hsa-miR-122-5p) and patient, disease and islet-isolation characteristics. Plasma samples (n = 139) were collected before TPIAT and miRNA levels were measured by RTPCR. Disease duration, prior surgery, and pre-surgical diabetes were not associated with circulating miRNAs. Levels of hsa-miR-29b-3p (P = 0.03), hsa-miR-148a-3p (P = 0.04) and hsa-miR-221-3p (P = 0.01) were lower in those with genetic risk factors. Levels of hsa-miR-148a-3p (P = 0.04) and hsa-miR-7-5p (P = 0.04) were elevated in toxic/metabolic disease. Participants with exocrine insufficiency had lower hsa-miR-29b-3p, hsa-miR-148a-3p, hsa-miR-320d, hsa-miR-221-3p (P < 0.01) and hsa-miR-375, hsa-miR-200c-3p, and hsa-miR-125b-5p (P < 0.05). Four miRNAs were associated with fasting C-peptide before TPIAT (hsa-miR-29b-3p, r = 0.18; hsa-miR-148a-3p, r = 0.21; hsa-miR-320d, r = 0.19; and hsa-miR-221-3p, r = 0.21; all P < 0.05), while hsa-miR-29b-3p was inversely associated with post-isolation islet equivalents/kg and islet number/kg (r = -0.20, P = 0.02). Also, hsa-miR-200c (r = 0.18, P = 0.03) and hsa-miR-221-3p (r = 0.19, P = 0.03) were associated with islet graft tissue volume. Further investigation is needed to determine the predictive potential of these miRNAs for assessing islet autotransplant outcomes.

Trisected pancreas model for testing tissue dissociation enzyme combinations: a novel methodology for improving human islet yield for clinical islet transplantation.

PURPOSE: Human islet isolation requires a defined collagenase-protease enzyme combination for obtaining a successful islet yield. While different islet laboratories use different enzyme combinations, a systematic methodology to identify optimal enzyme combinations and their concentrations within a single donor pancreas has not been tested. In this study, we designed a trisected pancreas model to test efficacy of three clinical grade enzyme blends (VitaCyte, Roche, SERVA) within a single pancreas. METHODS: Islet isolations were performed using brain-dead donor pancreases (n = 15) applying the enzyme-related design of experiments (DOEs) and the trisected model approach. After trimming, split each pancreas into three individual lobes (head, body, tail). As per the DOEs, the lobes were altered between different experiments, to minimize anatomical bias. Islets isolated from each lobe (27 lobes totally) were subjected to functional assessments. Insulin staining and islet area fraction were determined for tissue sections obtained from each lobe. RESULTS: Utilizing the trisected model, we identified that the collagenase dose from three different vendors did not affect the pancreas digestion and islet yield, but islet morphology after isolation with the neutral protease and BP-protease was better than thermolysin. In addition, the head lobe yielded a lower islet mass and higher tissue volume compared to other two lobes, irrespective of enzyme combination used. CONCLUSIONS: This study demonstrates that the trisected model is a promising methodology in assessing donor and isolation associated parameters. Based on this study, we conclude that the donor characteristics and an optimal enzyme dose play a critical role in achieving higher islet yields.

Human pancreatic tissue dissociation enzymes for islet isolation: Advances and clinical perspectives.

BACKGROUND AND AIMS: Successful clinical human allo or auto-islet transplantation requires the recovery of a sufficient number of functional islets from either brain-dead or chronic pancreatitis pancreases respectively. METHODS: In the last two decades (2000-2019), significant progress has been made in improving the human islet isolation procedures and in standardizing the use of different tissue dissociation enzyme (TDE; a mixture of collagenase and protease enzymes) blends to recover higher islet yields. RESULTS AND CONCLUSIONS: This review presents information focusing on properties and role of TDE blends during the islet isolation process, particularly emphasizing on the current developments, associated challenges and future perspectives within the field.

Islet cell transplantation in children.

This paper aims to provide an overview of islet cell transplantation in children, with specific attention to pediatric total pancreatectomy with islet autotransplantation (TPIAT). We will summarize the definition and causes of chronic pancreatitis in children, the TPIAT procedure and potential complications, the process of islet cell isolation and autotransplantation, and long-term results after TPIAT. Lastly, we will briefly discuss islet cell allotransplantation in the adult population and its potential role in treating children.