One-step purification of functional human and rat pancreatic alpha cells

Abstract

Pancreatic alpha cells contribute to glucose homeostasis by the regulated secretion of glucagon, which increases glycogenolysis and hepatic gluconeogenesis in response to hypoglycemia. Alterations of glucagon secretion are observed in diabetic patients and exacerbate the disease. The restricted availability of purified primary alpha cells has limited our understanding of their function in health and disease. This study was designed to establish convenient protocols for the purification of viable alpha cells from rat and human pancreatic islets by FACS, using intrinsic cellular properties. Islets were isolated from the pancreata of Wistar rats or deceased human organ donors. Dispersed islet cells were separated by FACS based on light scatter and autofluorescence. Purity of sorted cells was evaluated by immunocytochemistry using hormone specific antibodies. Relative hormone expression was further determined by quantitative RT-PCR. Viability was determined by Annexin V and propidium iodide staining and function was assessed by monitoring cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) using Fura-2/AM. We developed species-specific FACS gating strategies that resulted in populations consisting mainly of alpha cells (96.6 ± 1.4%, n = 3 for rat; 95.4 ± 1.7%, n = 4 for human, mean ± SEM). These cell fractions showed ~5-fold and ~4-fold enrichment (rat and human, respectively) of glucagon mRNA expression compared to total ungated islet cells. Most of the sorted cells were viable and functional, as they responded with an increase in [Ca(2+)](i) upon stimulation with L-arginine (10 mM). The majority of the sorted human alpha cells responded also to stimulation with kainate (100 μM), whereas this response was infrequent in rat alpha cells. Using the same sample preparation, but a different gating strategy, we were also able to sort rat and human populations enriched in beta cells. In conclusion, we have simplified and optimized a method for the purification of rat alpha cells, as well as established a novel approach to separate human alpha cells using neither antibodies nor dyes possibly interfering with cellular functions.

Effects of activin A on survival, function and gene expression of pancreatic islets from non-diabetic and diabetic human donors

“…(N D 4). After sorting using the same parameters reported in Kohler et al, 21 the recovered a-and b-cell fractions (A and B) were similar to Kohler et al, 21 with a high purity and viability and low overall yield but sufficient for PCR (C) and ICC analysis (Fig. 3).…”

“…To confirm the protein expression and the cellular location, we used sorted primary human a-and b-cells from a subset of the islet shipments. The sorting procedure was adapted from Kohler, et al 21 and produced highly enriched populations of a-and b-cells based on qPCR of recovered cells ( Fig. 2A-C) and verified by immunostaining (Fig.…”

“…The dispersed cells were then sorted into primary b-cells and a-cells by FACS (BD FACSAria, BD Biosciences, San Jose, CA) using auto-fluorescence induced by low glucose solution and gating strategies as previously described. 21 Cells were then processed for RNA isolation and RT-PCR as described above, as well as, immunostaining for cellular location of the activin A and activin B proteins.…”

Transcriptome landmarks of the functional maturity of rat beta-cells, from lactation to adulthood

“…Fluorescence-activated cell sorting is a useful tool to study a wide variety of biological processes in a particular type of cell. In previous work, different research groups used this approach to study beta-cells obtained from adult rats (Pipeleers et al 1985, Kohler et al 2012 and, recently, from neonatal rats (Martens et al 2014). Table 4 List of genes that contribute to the leading-edge subset within the GO calcium channel activity gene set.…”

Activins A and B Regulate Fate-Determining Gene Expression in Islet Cell Lines and Islet Cells From Male Mice

“…Enriched populations of ␣-and ␤-cells were obtained using the method of Kohler et al (28) adapted for mouse islets. Briefly, isolated mouse islets were dispersed into single cells using trypsin-EDTA solution (450 mL, 37°C, 7-9 min), washed, and then placed in a low-glucose sorting buffer (Hanks' balanced salt solution plus 2.5 mM glucose; 1% BSA; 100 U/mL penicillin G; 100 g/mL streptomycin-sulfate; and 25 mM HEPES, pH 7.4) and filtered through a 0.2-m mesh screen.…”