Generation and characterization of human insulin-releasing cell lines

Aims/hypothesis Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process. Methods Human pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays. ResultsThe proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3. Conclusions/interpretation Our study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.

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“…trypsin solution (Invitrogen, Paisley, UK) for 5 min at 37°C, pelleted, washed in ice-cold PBS and centrifuged at 1,000 g for 3 min [22].…”

Section: Methodsmentioning

confidence: 99%

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

et al. 2011

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“…5A). Previous reports suggest that insulinoma lines are polyhormonal, expressing glucagon in addition to insulin (1,14,19). In both culture systems, the mRNA levels of glucagon and somatostatin (SST) were expressed at levels 100 times lower than insulin (Fig.…”

Section: Pi Formation Induces the Expression Of ␤-Cell-associated Genementioning

confidence: 87%

Human EndoC-βH1 β-cells form pseudoislets with improved glucose sensitivity and enhanced GLP-1 signaling in the presence of islet-derived endothelial cells

Spelios

Afinowicz

Tipon

et al. 2018

American Journal of Physiology-Endocrinology and Metabolism

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Three-dimensional (3D) pseudoislets (PIs) can be used for the study of insulin-producing β-cells in free-floating islet-like structures similar to that of primary islets. Previously, we demonstrated the ability of islet-derived endothelial cells (iECs) to induce PIs using murine insulinomas, where PI formation enhanced insulin production and glucose responsiveness. In this report, we examined the ability of iECs to spontaneously induce the formation of free-floating 3D PIs using the EndoC-βH1 human β-cell line murine MS1 iEC. Within 14 days, the coculturing of both cell types produced fully humanized EndoC-βH1 PIs with little to no contaminating murine iECs. The size and shape of these PIs were similar to primary human islets. iEC-induced PIs demonstrated reduced dysregulated insulin release under low glucose levels and higher insulin secretion in response to high glucose and exendin-4 [a glucagon-like peptide-1 (GLP-1) analog] compared with monolayer cells cultured alone. Interestingly, iEC-PIs were also better at glucose sensing in the presence of extendin-4 compared with PIs generated on a low-adhesion surface plate in the absence of iECs and showed an overall improvement in cell viability. iEC-induced PIs exhibited increased expression of key genes involved in glucose transport, glucose sensing, β-cell differentiation, and insulin processing, with a concomitant decrease in glucagon mRNA expression. The enhanced responsiveness to exendin-4 was associated with increased protein expression of GLP-1 receptor and phosphokinase A. This rapid coculture system provides an unlimited number of human PIs with improved insulin secretion and GLP-1 responsiveness for the study of β-cell biology.

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“…This strategy offers a potentially unlimited source of readily available functionally competent insulin-producing cells for b cell and transplantation research (Efrat 1998, Limbert et al 2007. To date, this approach has mainly focused on use of rodent insulinoma cell lines (Santere et al 1981, McClenaghan et al 1996, Efrat 1998, Limbert et al 2007 due to limited access to functional human islets or derived b-cell lines (Baroni et al 1999, Narushima et al 2005, Gartner et al 2006, Labriola et al 2009, Ravassard et al 2011. However, in addition to species complications, use of rodent cell lines suffers the disadvantage that cells are routinely grown in culture as monolayers that differ fundamentally from the natural aggregation of b cells as islet micro-organs.…”

Section: Introductionmentioning

confidence: 99%

Configuration of electrofusion-derived human insulin-secreting cell line as pseudoislets enhances functionality and therapeutic utility

Guo-Parke¹,

McCluskey²,

Kelly³

et al. 2012

Journal of Endocrinology

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Formation of pseudoislets from rodent cell lines has provided a particularly useful model to study homotypic islet cell interactions and insulin secretion. This study aimed to extend this research to generate and characterize, for the first time, functional human pseudoislets comprising the recently described electrofusion-derived insulin-secreting 1.1B4 human b-cell line. Structural pseudoislets formed readily over 3-7 days in culture using ultra-low-attachment plastic, attaining a static size of 100-200 mm in diameter, corresponding to w6000 b cells. This was achieved by decreases in cell proliferation and integrity as assessed by BrdU ELISA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, and lactate dehydrogenase assays. Insulin content was comparable between monolayers and pseudoislets. However, pseudoislet formation enhanced insulin secretion by 1 . 7-to 12 . 5-fold in response to acute stimulation with glucose, amino acids, incretin hormones, or drugs compared with equivalent cell monolayers. Western blot and RT-PCR showed expression of key genes involved in cell communication and the stimulus-secretion pathway. Expression of E-Cadherin and connexin 36 and 43 was greatly enhanced in pseudoislets with no appreciable connexin 43 protein expression in monolayers. Comparable levels of insulin, glucokinase, and GLUT1 were found in both cell populations. The improved secretory function of human 1.1B4 cell pseudoislets over monolayers results from improved cellular interactions mediated through gap junction communication. Pseudoislets comprising engineered electrofusion-derived human b cells provide an attractive model for islet research and drug testing as well as offering novel therapeutic application through transplantation.

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Generation and characterization of human insulin-releasing cell lines

Cited by 11 publications

References 44 publications

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Human EndoC-βH1 β-cells form pseudoislets with improved glucose sensitivity and enhanced GLP-1 signaling in the presence of islet-derived endothelial cells

Configuration of electrofusion-derived human insulin-secreting cell line as pseudoislets enhances functionality and therapeutic utility

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