Pancreatic Islets - Flow Cytometry for Large (10-1500 micron) Particles

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Islet dimension and its impact on transplant outcome: A systematic review

Background: Not all islet transplants desirably achieve insulin independence. This can be attributed to the microarchitecture and function of the islets influenced by their dimensions. Large islets enhance insulin secretion through paracrine effects but are more susceptible to hypoxic injury post-transplant, while small islets offer better viability and insulin independence. In vivo studies suggest large islets are essential for maintaining euglycemia, though smaller islets are typically preferred in transplantation for better outcomes.

Aim: To document the impact of islet dimension on clinical and preclinical transplant outcomes to optimize procedures.

Methods: PubMed, Scopus and EMBASE platforms were searched for relevant literature up to 9 April 2024. Articles reported on either glucose-stimulated insulin-secreting (GSIS) capacity, islet viability and engraftment, or insulin independence based on the islet dimension were included. The risk of bias was measured using the Appraisal Tool for Cross-Sectional Studies. Extracted data was analyzed via a narrative synthesis.

Results: Nineteen studies were included in the review. A total of sixteen studies reported the GSIS, of which nine documented the increased insulin secretion in the small islet, where the majority reported insulin secretion per islet equivalent (IEQ). Seven studies documented increased GSIS in large-sized islets that measure insulin secretion per cell or islet. All the articles that compared small and large islets reported poor viability and engraftment of large islets.

Conclusion: Small islets with a diameter < 125 µm have desired transplantation outcomes due to their better survival following isolation. Large-sized islets receive blood supply directly from arterioles in vivo to meet their higher metabolic demands. The large islet undergoes central necrosis soon after the isolation (devascularization); failing to maintain the viability and glucose stimuli leads to a decline in GSIS and the overall function of the islet. Improved preservation of large islets after islet isolation, enhances the islet yield (IEQ), thereby reducing the likelihood of failed islet isolation and potentially improves transplant outcome.

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Loss of RREB1 in pancreatic beta cells reduces cellular insulin content and affects endocrine cell gene expression

Aims/hypothesis: Genome-wide studies have uncovered multiple independent signals at the RREB1 locus associated with altered type 2 diabetes risk and related glycaemic traits. However, little is known about the function of the zinc finger transcription factor Ras-responsive element binding protein 1 (RREB1) in glucose homeostasis or how changes in its expression and/or function influence diabetes risk.

Methods: A zebrafish model lacking rreb1a and rreb1b was used to study the effect of RREB1 loss in vivo. Using transcriptomic and cellular phenotyping of a human beta cell model (EndoC-βH1) and human induced pluripotent stem cell (hiPSC)-derived beta-like cells, we investigated how loss of RREB1 expression and activity affects pancreatic endocrine cell development and function. Ex vivo measurements of human islet function were performed in donor islets from carriers of RREB1 type 2 diabetes risk alleles.

Results: CRISPR/Cas9-mediated loss of rreb1a and rreb1b function in zebrafish supports an in vivo role for the transcription factor in beta cell mass, beta cell insulin expression and glucose levels. Loss of RREB1 also reduced insulin gene expression and cellular insulin content in EndoC-βH1 cells and impaired insulin secretion under prolonged stimulation. Transcriptomic analysis of RREB1 knockdown and knockout EndoC-βH1 cells supports RREB1 as a novel regulator of genes involved in insulin secretion. In vitro differentiation of RREB1KO/KO hiPSCs revealed dysregulation of pro-endocrine cell genes, including RFX family members, suggesting that RREB1 also regulates genes involved in endocrine cell development. Human donor islets from carriers of type 2 diabetes risk alleles in RREB1 have altered glucose-stimulated insulin secretion ex vivo, consistent with a role for RREB1 in regulating islet cell function.

Conclusions/interpretation: Together, our results indicate that RREB1 regulates beta cell function by transcriptionally regulating the expression of genes involved in beta cell development and function.

Applications & Publications

Technical Notes

An Overview of COPAS ™ Large Particle Flow Cytometry for the Analysis and Sorting of Mouse, Rat, Pig and Human Pancreatic Islets (QTN-014)

Publications

Islet dimension and its impact on transplant outcome: A systematic review

Loss of RREB1 in pancreatic beta cells reduces cellular insulin content and affects endocrine cell gene expression

Functional assessment of automatically sorted pancreatic islets using large particle flow cytometry.

Acute antibody-mediated complement activation mediates lysis of pancreatic islets cells and may cause tissue loss in clinical islet transplantation.

Validation of Large Particle Flow Cytometry for the Analysis and Sorting of Intact Pancreatic Islets.