A Novel Method for the Assessment of Cellular Composition and Beta-Cell Viability in Human Islet Preparations

Ichii, Hirohito; Inverardi, Luca; Pileggi, Antonello; Molano, R. Damaris; Cabrera, Over; Caicedo, Alejandro; Messinger, Shari; Kuroda, Yoshikazu; Berggren, Per Olof; Ricordi, Camillo

doi:10.1111/j.1600-6143.2005.00913.x

Cited by 190 publications

(240 citation statements)

References 31 publications

(46 reference statements)

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“…By using confocal microscopy and multiple immunofluorescence to enumerate the proportional contribution of the different cell types in one and the same section, we have avoided the potential inaccuracies of counting cells in consecutive sections. Our immunohistochemical data are in line with results of recent reports using laser scanning confocal microscopy on whole isolated islets (10) and laser scanning cytometry on dispersed islet cells (16). Finally, the cell composition of mouse islets described in our study is consistent with that reported in the literature (e.g., refs.…”

Section: Discussionsupporting

confidence: 83%

The unique cytoarchitecture of human pancreatic islets has implications for islet cell function

Cabrera

Berman

Kenyon

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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1,109

967

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The cytoarchitecture of human islets has been examined, focusing on cellular associations that provide the anatomical framework for paracrine interactions. By using confocal microscopy and multiple immunofluorescence, we found that, contrary to descriptions of prototypical islets in textbooks and in the literature, human islets did not show anatomical subdivisions. Insulin-immunoreactive ␤ cells, glucagon-immunoreactive ␣ cells, and somatostatin-containing ␦ cells were found scattered throughout the human islet. Human ␤ cells were not clustered, and most (71%) showed associations with other endocrine cells, suggesting unique paracrine interactions in human islets. Human islets contained proportionally fewer ␤ cells and more ␣ cells than did mouse islets. In human islets, most ␤, ␣, and ␦ cells were aligned along blood vessels with no particular order or arrangement, indicating that islet microcirculation likely does not determine the order of paracrine interactions. We further investigated whether the unique human islet cytoarchitecture had functional implications. Applying imaging of cytoplasmic free Ca 2؉ concentration, [Ca 2؉ ]i, we found that ␤ cell oscillatory activity was not coordinated throughout the human islet as it was in mouse islets. Furthermore, human islets responded with an increase in [Ca 2؉ ]i when lowering the glucose concentration to 1 mM, which can be attributed to the large contribution of ␣ cells to the islet composition. We conclude that the unique cellular arrangement of human islets has functional implications for islet cell function.␣ cell ͉ ␤ cell ͉ cytoplasmic free Ca 2ϩ concentration ͉ insulin ͉ glucagon I n the last three decades, hundreds of individuals with type 1 diabetes mellitus have received allogeneic transplants of endocrine pancreas, the islets of Langerhans, to cure their chronic condition. In these patients, diabetes is reversed by transplanting cells capable of physiologically regulating insulin secretion. Determining the quality of islets obtained from cadaveric pancreata should be indispensable in this context. However, it is not known which physiological parameters correlate best with a fully functional islet capable of reversing diabetes after transplantation. There is a wealth of information about the physiology of rodent islets, but the biology of human islets remains poorly understood. As assays for determining islet quality are being developed by many laboratories in the field of islet transplantation, a reassessment of the structure and function of human islets is warranted.The islets of Langerhans are small organs located in the pancreas that are crucial for glucose homeostasis. Islets typically consist of four types of secretory endocrine cells, namely, the insulin-containing ␤ cells, the glucagon-containing ␣ cells, the somatostatin-containing ␦ cells, and the pancreatic polypeptideproducing (PP) cells. In rodent islets, the vastly predominating ␤ cells are clustered in the core of a generally round islet, surrounded by a mantle of ␣, ␦, and PP cells. Thus, ...

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Section: Discussionsupporting

confidence: 83%

The unique cytoarchitecture of human pancreatic islets has implications for islet cell function

Cabrera

Berman

Kenyon

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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1,109

967

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“…2c). In order to test the hypothesis that L-JNKI specifically protects beta cells, we evaluated the mitochondrial membrane potential of both beta cell and non beta cell subsets of dissociated human islets, since dissipation of the mitochondrial membrane potential is a feature of apoptotic and necrotic beta cell damage [23]. We found that cytokines predominantly affect beta cells and that pretreatment with L-JNKI prevents this phenomenon (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…A cocktail of cytokines (IL-1β 50 U/ml, TNF-α 1,000 U/ml and INF-γ 1,000 U/ml) was added 18 h prior to islet dissociation and analysis. Measurement of viable beta cells was performed as described [23]. Briefly, the zinc-binding dye Newport green (NG) allows identification and quantification of islet beta cells on dissociated islets, based on the abundant zinc content in beta cell secretory granules [24].…”

Section: Methodsmentioning

confidence: 99%

“…Data were expressed as TMRE + cells on gated NG + 7AAD − cells (viable beta cells). Analysis of cellular composition was performed by immunofluorescence in the first five preparations and quantified by laser scanning cytometry (LSC) as previously described [23]. Briefly, dispersed cells were incubated for 1 h with the following antibodies: mouse monoclonal antibody to insulin and rabbit polyclonal antibody to somatostatin (1:100; both: Neo Markers, Fremont, CA, USA); mouse monoclonal antibody to glucagon (1:500; Sigma, St Louis, MO, USA); and undiluted rabbit polyclonal antibody to pancreatic polypeptide (PP; Bio-Genex, San Ramon, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation

et al. 2007

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Aims/hypothesis Activation of c-jun N-terminal kinase (JNK) has been described in islet isolation and engraftment, making JNK a key target in islet transplantation. The objective of this study was to investigate if JNK inhibition with a cellpermeable TAT peptide inhibitor (L-JNKI) protects functional beta cell mass in human islets and affects AKT and its substrates in islet cells. Methods The effect of L-JNKI (10 μmol/l) on islet count, mitochondrial membrane potential, glucose-stimulated insulin release and phosphorylation of both AKT and its substrates, as well as on reversal of diabetes in immunodeficient diabetic Nu/Nu mice was studied. Results In vitro, L-JNKI reduced the islet loss in culture and protected from cell death caused by acute cytokine exposure. In vivo, treatment of freshly isolated human islets and diabetic Nu/Nu mice recipients of such islets resulted in improved functional beta cell mass. We showed that L-JNKI activates AKT and downregulates glycogen synthase kinase-3 beta (GSK-3B) in human islets exposed to cytokines, while other AKT substrates were unaffected, suggesting that a specific AKT/GSK-3B regulation by L-JNKI may represent one of its mechanisms of cytoprotection. Conclusions/interpretation In conclusion, we have demonstrated that targeting JNK in human pancreatic islets results in improved functional beta cell mass and in the regulation of AKT/GSK3B activity.

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“…4 The FDA considers somatic cell therapy in the United States to be "experimental," rather than a standard medical practice. Therefore, allogeneic islet cells cannot be used clinically without an Investigational New Drug application or an approved Biologics License Application (BLA) issued to licensed products.…”

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confidence: 99%

Regulatory Challenges in Manufacturing of Pancreatic Islets

Linetsky

Ricordi

2008

Transplantation Proceedings

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At the present time, transplantation of pancreatic islet cells is considered an experimental therapy for a selected cohort of patients with type 1 diabetes, and is conducted under an Investigational New Drug (IND) application. Encouraging results of the Edmonton Protocol published in the year 2000 sparked a renewed interest in clinical transplantation of allogeneic islets, triggering a large number of IND applications for phase I clinical trials. Promising results reported by a number of centers since then prompted the Food and Drug Administration (FDA) to consider the possibility of licensing allogeneic islets as a therapeutic treatment for patients with type 1 diabetes. However, prior to licensure, issues such as safety, purity, efficacy, and potency of the islet product must be addressed. This is complicated by the intricate nature of pancreatic islets and limited characterization prior to transplantation. In this context, control of the manufacturing process plays a critical role in the definition of the final product. Despite significant progress made in standardization of the donor organ preservation methods, reagents used, and characterization assays performed to qualify an islet cell product, control of the isolation process remains a challenge. Within the scope of the FDA regulations,

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A Novel Method for the Assessment of Cellular Composition and Beta-Cell Viability in Human Islet Preparations

Cited by 190 publications

References 31 publications

The unique cytoarchitecture of human pancreatic islets has implications for islet cell function

The unique cytoarchitecture of human pancreatic islets has implications for islet cell function

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation

Regulatory Challenges in Manufacturing of Pancreatic Islets

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