Abstract
Isolated human islets do not always meet the quality standards required for transplant survival and reliable functional in vitro studies. The formation of pseudoislets, i.e. the reaggregation of a defined number of islet cells after dissociation, improves insulin secretion. We present a simple method of pseudoislet formation from human islet cells and assess the transcriptome and function of isolated human islets and pseudoislets from the same organ donors. Following pseudoislet formation, insulin content/DNA and mRNA/RPS13 resembled that of islets. In pseudoislets, glucose-stimulated insulin secretion (GSIS) was significantly higher (8–13-fold) than in islets (2–4-fold). GSIS of pseudoislets was partly inhibited by the glucagon-like peptide-1 receptor (GLP-1R) antagonist exendin-9. The stimulatory effects of palmitate and forskolin at 12 mM glucose were also significantly higher in pseudoislets than in islets. Further analysis of pseudoislets revealed that regulation of secretion and insulin and glucagon content was maintained over a longer culture period (6–14 d). While adrenaline inhibited GSIS, adrenaline together with palmitate stimulated glucagon secretion 2-fold at low glucose, an effect suppressed by high glucose. Transcriptome analysis revealed that, unlike islets, pseudoislets were deprived of exocrine and endothelial cells. In conclusion, pseudoislet formation restores functional integrity of human islet cells and allows long-term in vitro testing.
Highlights
Up to now, isolated human islets have been used for transplantation with only limited success[1]
While an elevated body mass index (BMI) of the donor correlated with higher insulin content and secretion, the major adverse effect was the contamination of the islet preparation with exocrine tissue
The results reveal a specific restitution of glucose-stimulated insulin secretion (GSIS), together with a higher expression of β-cell markers, glucose transporter type 2 (GLUT-2), paired box gene 4 (PAX4) and islet-amyloid-polypeptide (IAPP) and a simultaneous reduction of acinar, ductal and endocrine cell markers
Summary
Up to now, isolated human islets have been used for transplantation with only limited success[1]. Jean-Claude Henquin’s careful evaluation of glucose-responsiveness of isolated human islets using a perifusion setting revealed no dependence of glucose-stimulated insulin secretion (GSIS) on donor age and islet size[8]. The role of matrix and spreading for endocrine cell survival and function of human islet transplants has already been summarized elsewhere[14,15]. Another approach to improve glucose-responsiveness is the formation of pseudoislets[11]. This study was initiated to evaluate the improvement of GSIS upon the formation of pseudoislets This entailed comparing the glucose-responsiveness of the isolated human islets prior to dissociation with that of pseudoislets originating from the same organ donors. We ascertained that glucagon secretion is highest at low glucose in the presence of both palmitate and adrenaline, a stimulation inhibited by high glucose
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