Publications by authors named "Tatsuya Kin"

105 Publications

Glucose metabolism and pyruvate carboxylase enhance glutathione synthesis and restrict oxidative stress in pancreatic islets.

Cell Rep 2021 Nov;37(8):110037

Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, MA 02115, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston 02115, MA, USA; Department of Medicine, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115, USA. Electronic address:

Glucose metabolism modulates the islet β cell responses to diabetogenic stress, including inflammation. Here, we probed the metabolic mechanisms that underlie the protective effect of glucose in inflammation by interrogating the metabolite profiles of primary islets from human donors and identified de novo glutathione synthesis as a prominent glucose-driven pro-survival pathway. We find that pyruvate carboxylase is required for glutathione synthesis in islets and promotes their antioxidant capacity to counter inflammation and nitrosative stress. Loss- and gain-of-function studies indicate that pyruvate carboxylase is necessary and sufficient to mediate the metabolic input from glucose into glutathione synthesis and the oxidative stress response. Altered redox metabolism and cellular capacity to replenish glutathione pools are relevant in multiple pathologies beyond obesity and diabetes. Our findings reveal a direct interplay between glucose metabolism and glutathione biosynthesis via pyruvate carboxylase. This metabolic axis may also have implications in other settings where sustaining glutathione is essential.
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http://dx.doi.org/10.1016/j.celrep.2021.110037DOI Listing
November 2021

Imeglimin ameliorates β-cell apoptosis by modulating the endoplasmic reticulum homeostasis pathway.

Diabetes 2021 Sep 29. Epub 2021 Sep 29.

Laboratory of Diabetes and Metabolic Disorders, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8510, Japan;

The effects of imeglimin, a novel anti-diabetes agent, on β-cell function remain unclear. Here, we unveiled the impact of imeglimin on β-cell survival. Treatment with imeglimin augmented mitochondrial function, enhanced insulin secretion, promoted β-cell proliferation, and improved β-cell survival in mouse islets. Imeglimin upregulated the expression of endoplasmic reticulum (ER)-related molecules including , and decreased eIF2α phosphorylation, after treatment with thapsigargin, and restored global protein synthesis in β-cells under ER stress. Imeglimin failed to protect ER stress-induced β-cell apoptosis in CHOP-deficient islets or in the presence of GADD34 inhibitor. Treatment with imeglimin showed a significant decrease in the number of apoptotic β-cells and increased β-cell mass in Akita mice. Imeglimin also protected against β-cell apoptosis in both human islets and human pluripotent stem cell (hPSC)-derived β-like cells. Taken together, imeglimin modulates ER homeostasis pathway, which results in the prevention of β-cell apoptosis both and .
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http://dx.doi.org/10.2337/db21-0123DOI Listing
September 2021

Silencing the G-protein coupled receptor 3-salt inducible kinase 2 pathway promotes human β cell proliferation.

Commun Biol 2021 07 23;4(1):907. Epub 2021 Jul 23.

Sunnybrook Research Institute, Toronto, Canada.

Loss of pancreatic β cells is the hallmark of type 1 diabetes, for which provision of insulin is the standard of care. While regenerative and stem cell therapies hold the promise of generating single-source or host-matched tissue to obviate immune-mediated complications, these will still require surgical intervention and immunosuppression. Here we report the development of a high-throughput RNAi screening approach to identify upstream pathways that regulate adult human β cell quiescence and demonstrate in a screen of the GPCRome that silencing G-protein coupled receptor 3 (GPR3) leads to human pancreatic β cell proliferation. Loss of GPR3 leads to activation of Salt Inducible Kinase 2 (SIK2), which is necessary and sufficient to drive cell cycle entry, increase β cell mass, and enhance insulin secretion in mice. Taken together, our data show that targeting the GPR3-SIK2 pathway is a potential strategy to stimulate the regeneration of β cells.
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http://dx.doi.org/10.1038/s42003-021-02433-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8302759PMC
July 2021

Total pancreatectomy with islet cell autotransplantation in a 2-year-old child with hereditary pancreatitis due to a PRSS1 mutation.

Am J Transplant 2021 11 4;21(11):3790-3793. Epub 2021 Aug 4.

Department of Surgery, Clinical Islet Transplant Program and Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada.

Acute recurrent and chronic pancreatitis in children carries high morbidity and burden. Compared to adults, ~75% of the cases of chronic pancreatitis in children are associated with underlying genetic mutations. The decision to intervene and the optimal timing poses unique challenges. Total pancreatectomy and islet cell autotransplantation (TPIAT) provides definitive therapy to relieve pain and improve quality of life while minimizing the risk of pancreatogenic diabetes. Substantial clinical data are available for adults; however, information on clinical outcomes in children remains scarce, particularly for very young children. Herein, we present an unusual, complex case of a 2-year-old child that underwent a successful TPIAT due to hereditary pancreatitis with an underlying mutation in PRSS1 gene, complicated by unremitting pancreatic ascites, hemorrhage, and sepsis. This is the youngest case to be reported in the literature. We provide a comprehensive report of the course and procedures implemented in this patient to guide other teams when considering these extraordinary measures in similar cases.
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http://dx.doi.org/10.1111/ajt.16723DOI Listing
November 2021

CD82 is a marker to isolate β cell precursors from human iPS cells and plays a role for the maturation of β cells.

Sci Rep 2021 05 5;11(1):9530. Epub 2021 May 5.

Institute for Quantitative Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Generation of pancreatic β cells from pluripotent stem cells is a key technology to develop cell therapy for insulin-dependent diabetes and considerable efforts have been made to produce β cells. However, due to multiple and lengthy differentiation steps, production of β cells is often unstable. It is also desirable to eliminate undifferentiated cells to avoid potential risks of tumorigenesis. To isolate β cell precursors from late stage pancreatic endocrine progenitor (EP) cells derived from iPS cells, we have identified CD82, a member of the tetraspanin family. CD82 cells at the EP stage differentiated into endocrine cells more efficiently than CD82 EP stage cells. We also show that CD82 cells in human islets secreted insulin more efficiently than CD82 cells. Furthermore, knockdown of CD82 expression by siRNA or inhibition of CD82 by monoclonal antibodies in NGN3 cells suppressed the function of β cells with glucose-stimulated insulin secretion, suggesting that CD82 plays a role in maturation of EP cells to β cells.
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http://dx.doi.org/10.1038/s41598-021-88978-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100138PMC
May 2021

Clinical islet transplantation: Current progress and new frontiers.

J Hepatobiliary Pancreat Sci 2021 Mar 11;28(3):243-254. Epub 2021 Feb 11.

Clinical Islet Transplant Program, University of Alberta, Edmonton, AB, Canada.

Islet transplantation (IT) is now a robust treatment for selected patients with type 1 diabetes suffering from recurrent hypoglycemia and impaired awareness of hypoglycemia. A global soar of clinical islet transplant programs attests to the commitment of many institutions and researchers to advance IT as a potential cure for this devastating disease. However, many challenges limiting the widespread applicability of clinical IT remain. In this review, we will touch on the milestones in the history of IT and its path to clinical success, discuss the current challenges around IT, propose some possible solutions, and elaborate on the frontiers envisioned in the future of clinical IT.
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http://dx.doi.org/10.1002/jhbp.891DOI Listing
March 2021

Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation.

Nat Metab 2020 05 11;2(5):432-446. Epub 2020 May 11.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

Chronic inflammation is linked to diverse disease processes, but the intrinsic mechanisms that determine cellular sensitivity to inflammation are incompletely understood. Here, we show the contribution of glucose metabolism to inflammation-induced changes in the survival of pancreatic islet β-cells. Using metabolomic, biochemical and functional analyses, we investigate the protective versus non-protective effects of glucose in the presence of pro-inflammatory cytokines. When protective, glucose metabolism augments anaplerotic input into the TCA cycle via pyruvate carboxylase (PC) activity, leading to increased aspartate levels. This metabolic mechanism supports the argininosuccinate shunt, which fuels ureagenesis from arginine and conversely diminishes arginine utilization for production of nitric oxide (NO), a chief mediator of inflammatory cytotoxicity. Activation of the PC-urea cycle axis is sufficient to suppress NO synthesis and shield cells from death in the context of inflammation and other stress paradigms. Overall, these studies uncover a previously unappreciated link between glucose metabolism and arginine-utilizing pathways via PC-directed ureagenesis as a protective mechanism.
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http://dx.doi.org/10.1038/s42255-020-0199-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568475PMC
May 2020

Frequency of Obliteration of the Dorsal and Ventral Ducts of the Pancreas in Islet Transplantation.

Dig Dis Sci 2021 01 21;66(1):218-223. Epub 2020 Feb 21.

Clinical Islet Transplant Program, University of Alberta, 210 College Plaza 8215-112 St, Edmonton, AB, T6G2C8, Canada.

Background: Islet isolation is an essential process in every human islet transplantation protocol. Intraductal enzyme delivery followed by adequate distention of the pancreas is the most critical step in islet isolation. Anomalies of the pancreatic duct system can significantly affect this process. Thus, identification and characterization of ductal patency is of paramount importance to achieve optimal islet isolation.

Aims: To investigate the frequency of duct obliteration in the human pancreas and explore donor/patient characteristics associated with specific ductal variations.

Methods: We examined ductal patency of pancreata allocated for islet allotransplantation (n = 597) and autotransplantation (n = 21) after removal of the duodenum during islet isolation procedure. Donor/patient factors were reviewed from the batch files.

Results: Among 559 deceased donor pancreata without pancreas divisum (n = 38, 6.4%), both ducts were patent in 50.1%, only ventral duct was patent in 46.7%, and only dorsal duct was patent in 3.2%. Donor age was not associated with the frequency of obliterated dorsal duct. Black race tended to have the higher frequency of patent dorsal duct. As expected, pancreas divisum was more frequent in chronic pancreatitis cases (n = 6, 28.6%). Within 7 cases of chronic pancreatitis with unknown etiology, we encountered one case of ventral duct obliteration.

Conclusions: The minor duodenal papilla and aging do not likely play an important role in the occurrence of dorsal duct obliteration. Although frequency of obliterated ventral duct was low in our population, physicians, including gastroenterologists and endoscopists, as well as islet transplantation researchers should be aware of this possibility.
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http://dx.doi.org/10.1007/s10620-020-06145-1DOI Listing
January 2021

Luseogliflozin increases beta cell proliferation through humoral factors that activate an insulin receptor- and IGF-1 receptor-independent pathway.

Diabetologia 2020 03 3;63(3):577-587. Epub 2020 Jan 3.

Department of Endocrinology and Metabolism, Graduate School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan.

Aims/hypothesis: Sodium-glucose cotransporter 2 (SGLT2) inhibitors, which prevent the renal reabsorption of glucose, decrease blood glucose levels in an insulin-independent manner. We previously reported creating a mouse model of systemic inhibition of target receptors for both insulin and IGF-1 by treating animals with OSI-906, a dual insulin/IGF-1 receptor inhibitor, for 7 days. The OSI-906-treated mice exhibited an increased beta cell mass, hepatic steatosis and adipose tissue atrophy, accompanied by hyperglycaemia and hyperinsulinaemia. In the present study, we investigated the effects of an SGLT2 inhibitor, luseogliflozin, on these changes in OSI-906-treated mice.

Methods: We treated C57BL/6J male mice either with vehicle, luseogliflozin, OSI-906 or OSI-906 plus luseogliflozin for 7 days, and phenotyping was performed to determine beta cell mass and proliferation. Subsequently, we tested whether serum-derived factors have an effect on beta cell proliferation in genetically engineered beta cells, mouse islets or human islets.

Results: SGLT2 inhibition with luseogliflozin significantly ameliorated hyperglycaemia, but not hyperinsulinaemia, in the OSI-906-treated mice. Liver steatosis and adipose tissue atrophy induced by OSI-906 were not altered by treatment with luseogliflozin. Beta cell mass and proliferation were further increased by SGLT2 inhibition with luseogliflozin in the OSI-906-treated mice. Luseogliflozin upregulated gene expression related to the forkhead box M1 (FoxM1)/polo-like kinase 1 (PLK1)/centromere protein A (CENP-A) pathway in the islets of OSI-906-treated mice. The increase in beta cell proliferation was recapitulated in a co-culture of Irs2 knockout and Insr/IR knockout (βIRKO) beta cells with serum from both luseogliflozin- and OSI-906-treated mice, but not after SGLT2 inhibition in beta cells. Circulating factors in both luseogliflozin- and OSI-906-treated mice promoted beta cell proliferation in both mouse islets and cadaveric human islets.

Conclusions/interpretation: These results suggest that luseogliflozin can increase beta cell proliferation through the activation of the FoxM1/PLK1/CENP-A pathway via humoral factors that act in an insulin/IGF-1 receptor-independent manner.
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http://dx.doi.org/10.1007/s00125-019-05071-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574158PMC
March 2020

Abnormal regulation of glucagon secretion by human islet alpha cells in the absence of beta cells.

EBioMedicine 2019 Dec 26;50:306-316. Epub 2019 Nov 26.

Columbia Center for Translational Immunology, Department of Surgery, Columbia University Medical Center, 650 West 168th Street, BB1701, New York, NY 10032, USA. Electronic address:

Background: The understanding of the regulation of glucagon secretion by pancreatic islet α-cells remains elusive. We aimed to develop an in vitro model for investigating the function of human α-cells under direct influence of glucose and other potential regulators.

Methods: Highly purified human α-cells from islets of deceased donors were re-aggregated in the presence or absence of β-cells in culture, evaluated for glucagon secretion under various treatment conditions, and compared to that of intact human islets and non-sorted islet cell aggregates.

Findings: The pure human α-cell aggregates maintained proper glucagon secretion capability at low concentrations of glucose, but failed to respond to changes in ambient glucose concentration. Addition of purified β-cells, but not the secreted factors from β-cells at low or high concentrations of glucose, partly restored the responsiveness of α-cells to glucose with regulated glucagon secretion. The EphA stimulator ephrinA5-fc failed to mimic the inhibitory effect of β-cells on glucagon secretion. Glibenclamide inhibited glucagon secretion from islets and the α- and β-mixed cell-aggregates, but not from the α-cell-only aggregates, at 2.0 mM glucose.

Interpretation: This study validated the use of isolated and then re-aggregated human islet cells for investigating α-cell function and paracrine regulation, and demonstrated the importance of cell-to-cell contact between α- and β-cells on glucagon secretion. Loss of proper β- and α-cell physical interaction in islets likely contributes to the dysregulated glucagon secretion in diabetic patients. Re-aggregated select combinations of human islet cells provide unique platforms for studying islet cell function and regulation.
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http://dx.doi.org/10.1016/j.ebiom.2019.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6921359PMC
December 2019

Induction of Expandable Tissue-Specific Progenitor Cells from Human Pancreatic Tissue through Transient Expression of Defined Factors.

Mol Ther Methods Clin Dev 2019 Jun 29;13:243-252. Epub 2019 Jan 29.

Clinical Islet Transplant Program and Department of Surgery, University of Alberta, Edmonton, AB, Canada.

We recently demonstrated the generation of mouse induced tissue-specific stem (iTS) cells through transient overexpression of reprogramming factors combined with tissue-specific selection. Here we induced expandable tissue-specific progenitor (iTP) cells from human pancreatic tissue through transient expression of genes encoding the reprogramming factors OCT4 (octamer-binding transcription factor 4), p53 small hairpin RNA (shRNA), SOX2 (sex-determining region Y-box 2), KLF4 (Kruppel-like factor 4), L-MYC, and LIN28. Transfection of episomal plasmid vectors into human pancreatic tissue efficiently generated iTP cells expressing genetic markers of endoderm and pancreatic progenitors. The iTP cells differentiated into insulin-producing cells more efficiently than human induced pluripotent stem cells (iPSCs). iTP cells continued to proliferate faster than pancreatic tissue cells until days 100-120 (passages 15-20). iTP cells subcutaneously inoculated into immunodeficient mice did not form teratomas. Genomic bisulfite nucleotide sequence analysis demonstrated that the and promoters remained partially methylated in iTP cells. We compared the global gene expression profiles of iPSCs, iTP cells, and pancreatic cells (islets >80%). Microarray analyses revealed that the gene expression profiles of iTP cells were similar, but not identical, to those of iPSCs but different from those of pancreatic cells. The generation of human iTP cells may have important implications for the clinical application of stem/progenitor cells.
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http://dx.doi.org/10.1016/j.omtm.2019.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383192PMC
June 2019

Posttransplant Characterization of Long-term Functional hESC-Derived Pancreatic Endoderm Grafts.

Diabetes 2019 May 19;68(5):953-962. Epub 2018 Nov 19.

Clinical Islet Transplant Program, University of Alberta, Edmonton, Alberta, Canada

The paucity of human donors limits broadened application of β-cell replacement therapy. Insulin-producing cells derived from human embryonic stem cells (hESCs) have recently been investigated clinically as a feasible surrogate to primary tissue. Herein, we examine the long-term efficacy of hESC-derived pancreatic endoderm cells (PECs) to maintain normoglycemia posttransplant and characterize the phenotype of the PEC grafts. Mice with chemically induced diabetes were transplanted with PECs into the subcutaneous device-less site. Transplant function was assessed through nonfasting blood glucose measurements, intraperitoneal glucose tolerance testing (IPGTT), and human C-peptide secretion for 517 days. Explanted grafts were assessed for ex vivo function and immunohistochemically. All PEC recipients ( = 8) maintained normoglycemia until graft retrieval. IPGTTs at 365 and 517 days posttransplant did not differ ( > 0.05), however, both demonstrated superior glucose clearance compared with nondiabetic and transplant controls ( < 0.001). Serum C-peptide levels demonstrated significant glucose responsiveness (fasted vs. stimulated) ( < 0.01). Small intragraft cysts were palpable in all mice, which resolved but recurred after aspiration. Cysts showed monomorphic neuroendocrine proliferation and lined by ductal epithelium. Explanted grafts demonstrated similar insulin secretory capacity as human islets and stained positively for endocrine cells. Our results demonstrate the ability of PECs to differentiate in vivo and restore glycemic control while confirming minimal proliferation and absence of neoplastic change within the grafts during the time evaluated.
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http://dx.doi.org/10.2337/db18-0788DOI Listing
May 2019

Oxygen Perfusion (Persufflation) of Human Pancreata Enhances Insulin Secretion and Attenuates Islet Proinflammatory Signaling.

Transplantation 2019 01;103(1):160-167

Physiological Sciences, University of Arizona, Tucson, AZ.

Background: All human islets used in research and for the clinical treatment of diabetes are subject to ischemic damage during pancreas procurement, preservation, and islet isolation. A major factor influencing islet function is exposure of pancreata to cold ischemia during unavoidable windows of preservation by static cold storage (SCS). Improved preservation methods may prevent this functional deterioration. In the present study, we investigated whether pancreas preservation by gaseous oxygen perfusion (persufflation) better preserved islet function versus SCS.

Methods: Human pancreata were preserved by SCS or by persufflation in combination with SCS. Islets were subsequently isolated, and preparations in each group matched for SCS or total preservation time were compared using dynamic glucose-stimulated insulin secretion as a measure of β-cell function and RNA sequencing to elucidate transcriptomic changes.

Results: Persufflated pancreata had reduced SCS time, which resulted in islets with higher glucose-stimulated insulin secretion compared to islets from SCS only pancreata. RNA sequencing of islets from persufflated pancreata identified reduced inflammatory and greater metabolic gene expression, consistent with expectations of reducing cold ischemic exposure. Portions of these transcriptional responses were not associated with time spent in SCS and were attributable to pancreatic reoxygenation. Furthermore, persufflation extended the total preservation time by 50% without any detectable decline in islet function or viability.

Conclusions: These data demonstrate that pancreas preservation by persufflation rather than SCS before islet isolation reduces inflammatory responses and promotes metabolic pathways in human islets, which results in improved β cell function.
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http://dx.doi.org/10.1097/TP.0000000000002400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371803PMC
January 2019

Circumportal pancreas accompanied with pancreas divisum in a deceased donor for islet transplantation.

Surg Radiol Anat 2018 Nov 24;40(11):1323-1325. Epub 2018 Jul 24.

Clinical Islet Transplant Program, University of Alberta, Alberta, Canada.

Circumportal pancreas is a rare and previously not well-recognized anatomical variant. In contrast, pancreas divisum is the most frequent anatomical variant in the pancreas. We report a case in which circumportal pancreas was accompanied with pancreas divisum in a deceased donor for islet transplantation. A unique aspect of our case is that the connecting portion behind the portal vein between the pancreas head and body originated from the ventral pancreas. This is the first case report describing a ventral embryogenic origin of the connecting portion of a circumportal pancreas accompanied with pancreas divisum.
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http://dx.doi.org/10.1007/s00276-018-2072-7DOI Listing
November 2018

Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice.

Diabetologia 2018 09 3;61(9):2016-2029. Epub 2018 Jul 3.

Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB, Canada.

Aims/hypothesis: Islet transplantation is a treatment option that can help individuals with type 1 diabetes become insulin independent, but inefficient oxygen and nutrient delivery can hamper islet survival and engraftment due to the size of the islets and loss of the native microvasculature. We hypothesised that size-controlled pseudoislets engineered via centrifugal-forced-aggregation (CFA-PI) in a platform we previously developed would compare favourably with native islets, even after taking into account cell loss during the process.

Methods: Human islets were dissociated and reaggregated into uniform, size-controlled CFA-PI in our microwell system. Their performance was assessed in vitro and in vivo over a range of sizes, and compared with that of unmodified native islets, as well as islet cell clusters formed by a conventional spontaneous aggregation approach (in which dissociated islet cells are cultured on ultra-low-attachment plates). In vitro studies included assays for membrane integrity, apoptosis, glucose-stimulated insulin secretion assay and total DNA content. In vivo efficacy was determined by transplantation under the kidney capsule of streptozotocin-treated Rag1 mice, with non-fasting blood glucose monitoring three times per week and IPGTT at day 60 for glucose response. A recovery nephrectomy, removing the graft, was conducted to confirm efficacy after completing the IPGTT. Architecture and composition were analysed by histological assessment via insulin, glucagon, pancreatic polypeptide, somatostatin, CD31 and von Willebrand factor staining.

Results: CFA-PI exhibit markedly increased uniformity over native islets, as well as substantially improved glucose-stimulated insulin secretion (8.8-fold to 11.1-fold, even after taking cell loss into account) and hypoxia tolerance. In vivo, CFA-PI function similarly to (and potentially better than) native islets in reversing hyperglycaemia (55.6% for CFA-PI vs 20.0% for native islets at 500 islet equivalents [IEQ], and 77.8% for CFA-PI vs 55.6% for native islets at 1000 IEQ), and significantly better than spontaneously aggregated control cells (55.6% for CFA-PI vs 0% for spontaneous aggregation at 500 IEQ, and 77.8% CFA-PI vs 33.4% for spontaneous aggregation at 1000 IEQ; p < 0.05). Glucose clearance in the CFA-PI groups was improved over that in the native islet groups (CFA-PI 18.1 mmol/l vs native islets 29.7 mmol/l at 60 min; p < 0.05) to the point where they were comparable with the non-transplanted naive normoglycaemic control mice at a low IEQ of 500 IEQ (17.2 mmol/l at 60 min).

Conclusions/interpretation: The ability to efficiently reformat dissociated islet cells into engineered pseudoislets with improved properties has high potential for both research and therapeutic applications.
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http://dx.doi.org/10.1007/s00125-018-4672-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096633PMC
September 2018

Role of Egr1 on Pancreatic Endoderm Differentiation.

Cell Med 2018 29;10:2155179017733177. Epub 2018 May 29.

Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan.

The low efficiency of in vitro differentiation of human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (iPSCs) into insulin-producing cells is a crucial hurdle for the clinical implementation of human pluripotent stem cells (PSCs). Our previous investigation into the key factors for the differentiation of PSCs into insulin-producing cells suggested that the expression of GATA binding protein 6 (GATA6) and Gremlin 1 (GREM1) and inhibition of early growth response protein 1 (Egr1) may be important factors. In this study, we investigated the role of Egr1 in pancreas development. The transfection of small interfering RNA (siRNA) of Egr1 in the early phase induced the differentiation of iPSCs derived from fibroblasts (FiPSCs) into pancreatic endoderm and insulin-producing cells. In contrast, the downregulation of Egr1 in the late phase suppressed the differentiation of FiPSCs into pancreatic endoderm and insulin-producing cells. In addition, the overexpression of Egr1 suppressed the differentiation of iPSCs derived from pancreatic cells into pancreatic endoderm and insulin-producing cells. These data suggest that the downregulation of Egr1 in the early phase can efficiently induce the differentiation of iPSCs into insulin-producing cells.
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http://dx.doi.org/10.1177/2155179017733177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172987PMC
May 2018

Ferroptosis-inducing agents compromise in vitro human islet viability and function.

Cell Death Dis 2018 05 22;9(6):595. Epub 2018 May 22.

Clinical Islet Transplant Program, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada.

Human islet transplantation has been hampered by donor cell death associated with the islet preparation procedure before transplantation. Regulated necrosis pathways are biochemically and morphologically distinct from apoptosis. Recently, ferroptosis was identified as a non-apoptotic form of iron-dependent regulated necrosis implicated in various pathological conditions. Mediators of islet oxidative stress, including glutathione peroxidase-4 (GPX4), have been identified as inhibitors of ferroptosis, and mechanisms that affect GPX4 function can impact islet function and viability. Ferroptosis has not been investigated directly in human islets, and its relevance in islet transplantation remains unknown. Herein, we sought to determine whether in vitro human islet viability and function is compromised in the presence of two distinct ferroptosis-inducing agents (FIA), erastin or RSL3, and whether these effects could be rescued with ferroptosis inhibitors, ferrostatin-1 (Fer-1), or desferrioxamine (DFO). Viability, as assessed by lactate dehydrogenase (LDH) release, revealed significant death in erastin- and RSL3-treated islets, 20.3% ± 3.8 and 24.4% ± 2.5, 24 h post culture, respectively. These effects were ameliorated in islets pre-treated with Fer-1 or the iron chelator, desferrioxamine (DFO). Stimulation index, a marker of islet function revealed a significant reduction in function in erastin-treated islets (control 1.97 ± 0.13 vs. 50 μM erastin 1.32 ± 0.1) (p < 0.05). Fer-1 and DFO pre-treatment alone did not augment islet viability or function. Pre-treatment of islets with erastin or Fer-1 did not impact in vivo engraftment in an immunodeficient mouse transplant model. Our data reveal that islets are indeed susceptible to ferroptosis in vitro, and induction of this novel cell death modality leads to compromised islet function, which can be recoverable in the presence of the ferroptosis inhibitors. The in vivo impact of this pathway in islet transplantation remains elusive given the constraints of our study, but warrants continued investigation.
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http://dx.doi.org/10.1038/s41419-018-0506-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964226PMC
May 2018

Self-Condensation Culture Enables Vascularization of Tissue Fragments for Efficient Therapeutic Transplantation.

Cell Rep 2018 05;23(6):1620-1629

Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Kanazawa-ku 3-9, Yokohama, Kanagawa 236-0004, Japan; Advanced Medical Research Center, Yokohama City University, Kanazawa-ku 3-9, Yokohama, Kanagawa 236-0004, Japan. Electronic address:

Clinical transplantation of tissue fragments, including islets, faces a critical challenge because of a lack of effective strategies that ensure efficient engraftment through the timely integration of vascular networks. We recently developed a complex organoid engineering method by "self-condensation" culture based on mesenchymal cell-dependent contraction, thereby enabling dissociated heterotypic lineages including endothelial cells to self-organize in a spatiotemporal manner. Here, we report the successful adaptation of this method for generating complex tissues from diverse tissue fragments derived from various organs, including pancreatic islets. The self-condensation of human and mouse islets with endothelial cells not only promoted functionalization in culture but also massively improved post-transplant engraftment. Therapeutically, fulminant diabetic mice were more efficiently treated by a vascularized islet transplant compared with the conventional approach. Given the general limitations of post-transplant vascularization associated with 3D tissue-based therapy, our approach offers a promising means of enhancing efficacy in the context of therapeutic tissue transplantation.
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http://dx.doi.org/10.1016/j.celrep.2018.03.123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8289710PMC
May 2018

An engineered cell sheet composed of human islets and human fibroblast, bone marrow-derived mesenchymal stem cells, or adipose-derived mesenchymal stem cells: An in vitro comparison study.

Islets 2018 05 2;10(3):e1445948. Epub 2018 Apr 2.

a Department of Surgery , Nagasaki University Graduate School of Biomedical Sciences , Nagasaki , Japan.

Background: We previously reported the utility of engineered cell sheets composed of human islets and supporting cells in vitro and in vivo. It is unclear which type of supporting cell is most suitable for constructing cell sheets with human islets. The present study aimed to compare human fibroblasts, bone marrow-derived mesenchymal stem cells (BM-MSCs), and adipose-derived mesenchymal stem cells (ADSCs) as a supporting source for cell sheets.

Methods: Engineered cell sheets were fabricated with human islets using human fibroblasts, BM-MSCs, or ADSCs as supporting cells. The islet viability, recovery rate, glucose-stimulated insulin release (determined by the stimulation index), and cytokine secretion (TGF-β1, IL-6, and VEGF) of groups-including an islet-alone group as a control-were compared.

Results: All three sheet groups consistently exhibited higher viability, recovery rate, and stimulation index values than the islet-alone group. The ADSC group showed the highest viability and recovery rate among the three sheet groups. There were no discernible differences in the stimulation index values of the groups. The fibroblast group exhibited significantly higher TGF-β1 values in comparison to the other groups. The IL-6 level of the ADSC group was more than five times higher than that of the other groups. The ADSC group showed the VEGF level; however, it did not differ from that of the BM-MSC group to a statistically significant extent.

Conclusion: Engineered cell sheets composed of islets and supporting cells had a cytoprotective effect on islets. These results suggest that individual cell types could be a more attractive source for crafting engineered cell sheets in comparison to islets alone.
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http://dx.doi.org/10.1080/19382014.2018.1445948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989879PMC
May 2018

Single-cell analyses of human islet cells reveal de-differentiation signatures.

Cell Death Discov 2018 Dec 9;4:14. Epub 2018 Feb 9.

1Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, Proteos, Singapore, Singapore.

Human pancreatic islets containing insulin-secreting β-cells are notoriously heterogeneous in cell composition. Since β-cell failure is the root cause of diabetes, understanding this heterogeneity is of paramount importance. Recent reports have cataloged human islet transcriptome but not compared single β-cells in detail. Here, we scrutinized ex vivo human islet cells from healthy donors and show that they exhibit de-differentiation signatures. Using single-cell gene expression and immunostaining analyses, we found healthy islet cells to contain polyhormonal transcripts, and INS cells to express decreased levels of β-cell genes but high levels of progenitor markers. Rare cells that are doubly positive for progenitor markers/exocrine signatures, and endocrine/exocrine hormones were also present. We conclude that ex vivo human islet cells are plastic and can possibly de-/trans-differentiate across pancreatic cell fates, partly accounting for β-cell functional decline once isolated. Therefore, stabilizing β-cell identity upon isolation may improve its functionality.
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http://dx.doi.org/10.1038/s41420-017-0014-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841351PMC
December 2018

BMX-001, a novel redox-active metalloporphyrin, improves islet function and engraftment in a murine transplant model.

Am J Transplant 2018 08 31;18(8):1879-1889. Epub 2018 Mar 31.

Clinical Islet Transplant Program, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada.

Islet transplantation has become a well-established therapy for select patients with type 1 diabetes. Viability and engraftment can be compromised by the generation of oxidative stress encountered during isolation and culture. We evaluated whether the administration of BMX-001 (MnTnBuOE-2-PyP [Mn(III) meso-tetrakis-(N-b-butoxyethylpyridinium-2-yl)porphyrin]) and its earlier derivative, BMX-010 (MnTE-2-PyP [Mn(III) meso-tetrakis-(N-methylpyridinium-2-yl)porphyrin]) could improve islet function and engraftment outcomes. Long-term culture of human islets with BMX-001, but not BMX-010, exhibited preserved in vitro viability. Murine islets isolated and cultured for 24 hours with 34 μmol/L BMX-001 exhibited improved insulin secretion (n = 3 isolations, P < .05) in response to glucose relative to control islets. In addition, 34 μmol/L BMX-001-supplemented murine islets exhibited significantly reduced apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling, compared with nontreated control islets (P < .05). Murine syngeneic islets transplanted under the kidney capsule at a marginal dose of 150 islets revealed 58% of 34 μmol/L BMX-001-treated islet recipients became euglycemic (n = 11 of 19) compared with 19% of nontreated control islet recipients (n = 3 of 19, P < .05). Of murine recipients receiving a marginal dose of human islets cultured with 34 μmol/L BMX-001, 92% (n = 12 of 13) achieved euglycemia compared with 57% of control recipients (n = 8 of 14, P = .11). These results demonstrate that the administration of BMX-001 enhances in vitro viability and augments murine marginal islet mass engraftment.
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http://dx.doi.org/10.1111/ajt.14705DOI Listing
August 2018

Beta Cell Death by Cell-free DNA and Outcome After Clinical Islet Transplantation.

Transplantation 2018 06;102(6):978-985

Department of Surgery and Clinical Islet Transplant Program, University of Alberta, Edmonton, AB, Canada.

Background: Optimizing engraftment and early survival after clinical islet transplantation is critical to long-term function, but there are no reliable, quantifiable measures to assess beta cell death. Circulating cell-free DNA (cfDNA) derived from beta cells has been identified as a novel biomarker to detect cell loss and was recently validated in new-onset type 1 diabetes and in islet transplant patients.

Methods: Herein we report beta cell cfDNA measurements after allotransplantation in 37 subjects and the correlation with clinical outcomes.

Results: A distinctive peak of cfDNA was observed 1 hour after transplantation in 31 (83.8%) of 37 subjects. The presence and magnitude of this signal did not correlate with transplant outcome. The 1-hour signal represents dead beta cells carried over into the recipient after islet isolation and culture, combined with acute cell death post infusion. Beta cell cfDNA was also detected 24 hours posttransplant (8/37 subjects, 21.6%). This signal was associated with higher 1-month insulin requirements (P = 0.04), lower 1-month stimulated C-peptide levels (P = 0.01), and overall worse 3-month engraftment, by insulin independence (receiver operating characteristic-area under the curve = 0.70, P = 0.03) and beta 2 score (receiver operating characteristic-area under the curve = 0.77, P = 0.006).

Conclusions: cfDNA-based estimation of beta cell death 24 hours after islet allotransplantation correlates with clinical outcome and could predict early engraftment.
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http://dx.doi.org/10.1097/TP.0000000000002083DOI Listing
June 2018

Neuronal PAS Domain Protein 4 Suppression of Oxygen Sensing Optimizes Metabolism during Excitation of Neuroendocrine Cells.

Cell Rep 2018 01;22(1):163-174

Diabetes Research Group, BC Children's Hospital Research Institute, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada. Electronic address:

Depolarization of neuroendocrine cells results in calcium influx, which induces vesicle exocytosis and alters gene expression. These processes, along with the restoration of resting membrane potential, are energy intensive. We hypothesized that cellular mechanisms exist to maximize energy production during excitation. Here, we demonstrate that NPAS4, an immediate early basic helix-loop-helix (bHLH)-PAS transcription factor, acts to maximize energy production by suppressing hypoxia-inducible factor 1α (HIF1α). As such, knockout of Npas4 from insulin-producing β cells results in reduced OXPHOS, loss of insulin secretion, β cell dedifferentiation, and type 2 diabetes. NPAS4 plays a similar role in the nutrient-sensing cells of the hypothalamus. Its knockout here results in increased food intake, reduced locomotor activity, and elevated peripheral glucose production. In conclusion, NPAS4 is critical for the coordination of metabolism during the stimulation of electrically excitable cells; its loss leads to the defects in cellular metabolism that underlie the cellular dysfunction that occurs in metabolic disease.
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http://dx.doi.org/10.1016/j.celrep.2017.12.033DOI Listing
January 2018

Encapsulation of Human Islets Using a Biomimetic Self-Assembled Nanomatrix Gel for Protection against Cellular Inflammatory Responses.

ACS Biomater Sci Eng 2017 Sep 11;3(9):2110-2119. Epub 2017 Jul 11.

Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, Alabama 35294, United States.

The major concern of pancreatic islet transplantation is that the implanted islets are exposed to the immune system of the recipient. To overcome this challenge, the peptide amphiphile (PA) nanomatrix gel was used for immunoisolation of islets through microencapsulation. The PA can self-assemble to form a nanomatrix gel with an extracellular matrix-mimicking, islet nurturing microenvironment and a semipermeable immune barrier. In this study, the islet protective effect of the PA nanomatrix gel was evaluated by coculture of PA-encapsulated human islets with differentiated U937 cells (human monocyte cell-line) for 3 and 7 days. The coculture of the bare islets with the differentiated U937 cells stimulated proinflammatory cytokine (IL-1 and TNF-) secretion and caused islet death after 7 days, which simulated an early inflammatory response environment after islet transplantation. The PA-encapsulated islets, however, did not stimulate proinflammatory cytokine secretion and maintained islet viability up to 7 days. More insulin-producing cells were observed when islets were PA-encapsulated than control islets with the differentiated U937 cells for 7 days compared to the bare islets. This result was also confirmed by dithizone staining analysis. Further evaluation of islet functionality was assessed by a glucose-stimulated insulin secretion test. The PA-encapsulated islets showed greater insulin secretion response to glucose stimulation than the bare islets with the differentiated U937 cells after 3 and 7 days. These results demonstrated that islet encapsulation with the PA nanomatrix gel was able to improve islet survival and function in the presence of inflammatory responses, which will increase the success rate of islet engraftment and the efficacy of islet transplantation.
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http://dx.doi.org/10.1021/acsbiomaterials.7b00261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615894PMC
September 2017

Transplantation of Human Pancreatic Endoderm Cells Reverses Diabetes Post Transplantation in a Prevascularized Subcutaneous Site.

Stem Cell Reports 2017 06;8(6):1689-1700

Clinical Islet Transplant Program, University of Alberta, 112 Street & 87 Avenue, Edmonton, AB T6G 2E1, Canada; Department of Surgery, University of Alberta, 8440 Walter C Mackenzie Health Centre 112 Street, Edmonton, AB T5G 2B7, Canada. Electronic address:

Beta-cell replacement therapy is an effective means to restore glucose homeostasis in select humans with autoimmune diabetes. The scarcity of "healthy" human donor pancreata restricts the broader application of this effective curative therapy. "β-Like" cells derived from human embryonic stem cells (hESC), with the capacity to secrete insulin in a glucose-regulated manner, have been developed in vitro, with limitless capacity for expansion. Here we report long-term diabetes correction in mice transplanted with hESC-derived pancreatic endoderm cells (PECs) in a prevascularized subcutaneous site. This advancement mitigates chronic foreign-body response, utilizes a device- and growth factor-free approach, facilitates in vivo differentiation of PECs into glucose-responsive insulin-producing cells, and reliably restores glycemic control. Basal and stimulated human C-peptide secretion was detected throughout the study, which was abolished upon graft removal. Recipient mice demonstrated physiological clearance of glucose in response to metabolic challenge and safely retrieved grafts contained viable glucose regulatory cells.
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http://dx.doi.org/10.1016/j.stemcr.2017.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470173PMC
June 2017

A case of double common bile duct in a deceased donor for transplantation.

Surg Radiol Anat 2017 Dec 18;39(12):1409-1411. Epub 2017 May 18.

Clinical Islet Transplantation Program, University of Alberta, Edmonton, AB, Canada.

A double common bile duct is extremely rare among the anatomical variations in the biliary tract system. We report an incidentally encountered case of the double common bile duct and discuss the novel anatomical findings of the accessory common bile duct from the viewpoint of embryology. A unique point of our case is that the accessory common bile duct bifurcated at the level of the intrapancreatic bile duct. There is no similar case in the previous literature among type II double common bile duct in the viewpoint of anatomical findings of the accessory common bile duct. We assume that this asymptomatic anatomical variation may be present more commonly, but not diagnosed.
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http://dx.doi.org/10.1007/s00276-017-1874-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681599PMC
December 2017

Engraftment Site and Effectiveness of the Pan-Caspase Inhibitor F573 to Improve Engraftment in Mouse and Human Islet Transplantation in Mice.

Transplantation 2017 10;101(10):2321-2329

1 Clinical Islet Transplant Program, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada. 2 Department of Surgery, University of Alberta, Edmonton, AB, Canada.

Background: Islet transplantation is an effective therapy in type 1 diabetes and recalcitrant hypoglycemia. However, there is an ongoing need to circumvent islet loss posttransplant. We explore herein the potential of the pan-caspase inhibitor F573 to mitigate early apoptosis-mediated islet death within portal and extrahepatic portal sites in mice.

Methods: Mouse or human islets were cultured in standard media ±100 μM F573 and subsequently assessed for viability and apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. Diabetic mice were transplanted with syngeneic islets placed under the kidney capsule (KC) or into the subcutaneous deviceless (DL) site at a marginal islet dose (150 islets), or into the portal vein (PV) at a full dose (500 islets). Human islets were transplanted under the KC of diabetic immunodeficient mice at a marginal dose (500 islet equivalents). Islets were cultured in the presence of F573, and F573 was administered subcutaneously on days 0 to 5 posttransplant. Control mice were transplanted with nontreated islets and were injected with saline. Graft function was measured by nonfasting blood glucose and glucose tolerance testing.

Results: F573 markedly reduced human and mouse islet apoptosis after in vitro culture (P < 0.05 and P < 0.05, respectively). Furthermore, F573 improved human islet function when transplanted under the KC (P < 0.05); whereas F573 did not enhance murine islet marginal KC transplants. Conversely, F573 significantly improved mouse islet engraftment in the PV and DL site (P < 0.05 and P < 0.05, respectively).

Conclusions: The pan-caspase inhibitor F573 markedly reduces human and mouse islet apoptosis and improves engraftment most effectively in the portal and DL subcutaneous sites.
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http://dx.doi.org/10.1097/TP.0000000000001638DOI Listing
October 2017

Long-term function and optimization of mouse and human islet transplantation in the subcutaneous device-less site.

Islets 2016 11 7;8(6):186-194. Epub 2016 Nov 7.

a Clinical Islet Transplant Program, Alberta Diabetes Institute, University of Alberta , Edmonton , AB , Canada.

Clinical islet transplantation has routinely been demonstrated to be an efficacious means of restoring glycemic control in select patients with autoimmune diabetes. Notwithstanding marked progress and improvements, the broad-spectrum application of this treatment option is restricted by the complications associated with intrahepatic portal cellular infusion and the scarcity of human donor pancreata. Recent progress in stem cell biology has demonstrated that the potential to expand new β cells for clinical transplantation is now a reality. As such, research focus is being directed toward optimizing safe extrahepatic transplant sites to house future alternative β cell sources for clinical use. The present study expands on our previous development of a prevascularized subcutaneous device-less (DL) technique for cellular transplantation, by demonstrating long-term (>365 d) durable syngeneic murine islet graft function. Furthermore, histological analysis of tissue specimens collected immediately post-DL site creation and acutely post-human islet transplantation demonstrates that this technique results in close apposition of the neovascularized collagen to the transplanted cells without dead space, thereby avoiding hypoxic luminal dead-space. Murine islets transplanted into the DL site created by a larger luminal diameter (6-Fr.) (n = 11), reversed diabetes to the similar capacity as our standard DL method (5-Fr.)(n = 9). Furthermore, glucose tolerance testing did not differ between these 2 transplant groups (p > 0 .05). Taken together, this further refinement of the DL transplant approach facilitates a simplistic means of islet infusion, increases the transplant volume capacity and may provide an effective microenvironment to house future alternative β cell sources.
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http://dx.doi.org/10.1080/19382014.2016.1253652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161146PMC
November 2016

National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities.

Diabetes 2016 11 27;65(11):3418-3428. Epub 2016 Jul 27.

Schulze Diabetes Institute and Department of Surgery, University of Minnesota, Minneapolis, MN.

Eight manufacturing facilities participating in the National Institutes of Health-sponsored Clinical Islet Transplantation (CIT) Consortium jointly developed and implemented a harmonized process for the manufacture of allogeneic purified human pancreatic islet (PHPI) product evaluated in a phase 3 trial in subjects with type 1 diabetes. Manufacturing was controlled by a common master production batch record, standard operating procedures that included acceptance criteria for deceased donor organ pancreata and critical raw materials, PHPI product specifications, certificate of analysis, and test methods. The process was compliant with Current Good Manufacturing Practices and Current Good Tissue Practices. This report describes the manufacturing process for 75 PHPI clinical lots and summarizes the results, including lot release. The results demonstrate the feasibility of implementing a harmonized process at multiple facilities for the manufacture of a complex cellular product. The quality systems and regulatory and operational strategies developed by the CIT Consortium yielded product lots that met the prespecified characteristics of safety, purity, potency, and identity and were successfully transplanted into 48 subjects. No adverse events attributable to the product and no cases of primary nonfunction were observed.
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http://dx.doi.org/10.2337/db16-0234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079635PMC
November 2016
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