Pronounced proliferation of non-beta cells in response to beta-cell mitogens in isolated human islets of Langerhans

Abstract

The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labeling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.

Evaluation of the Effects of Harmine on β-cell Function and Proliferation in Standardized Human Islets Using 3D High-Content Confocal Imaging and Automated Analysis

“…Harmine also efficiently induced b-cell proliferation in our platform, with quite low donor-to-donor variation. The observed proliferative rates identified based on the quantification of EdU and NKX6.1 were within a similar range to those previously reported (32,33,36,37,53,54). Our data also confirmed the non-specificity of harmine: we and others have found that harmine induces high levels of proliferation of non-b-cells (32,36,37,53), although this has been suggested to be rectified through combinatorial treatment with GLP-1 receptor agonists (32).…”

“…The observed proliferative rates identified based on the quantification of EdU and NKX6.1 were within a similar range to those previously reported ( 32 , 33 , 36 , 37 , 53 , 54 ). Our data also confirmed the non-specificity of harmine: we and others have found that harmine induces high levels of proliferation of non-β-cells ( 32 , 36 , 37 , 53 ), although this has been suggested to be rectified through combinatorial treatment with GLP-1 receptor agonists ( 32 ). Perplexingly, one of the most interesting observations we made was the significantly increased β-cell count and fraction with high harmine dose treatment, despite low β-cell and high non-β-cell proliferation rates.…”

“…Several previous studies have investigated the proliferative potential of harmine in rodent and human islets. 10 μM harmine treatment was found to induce β-cell proliferation rates of ~0.25-2.5% ( 32 , 33 , 36 , 37 , 53 , 54 ) in dispersed human islets in vitro , and rates of 0.8-2% were observed in human islets transplanted into immunodeficient mice treated with 10 mg/kg harmine ( 32 , 36 , 37 ), with some of the variation accounted for by differences in labeling method of proliferating cells (KI67, EdU) and β-cells (PDX1, NKX6.1, Insulin, C-peptide). Harmine also efficiently induced β-cell proliferation in our platform, with quite low donor-to-donor variation.…”

β Cell mass expansion during puberty involves serotonin signaling and determines glucose homeostasis in adulthood

“…Following treatment, islets were dissociated into single cells with accutase (1 μL/islet; Innovative Cell Technologies Inc.) for 10 minutes at 37°C, and β cell proliferation was assessed by flow cytometry as described previously ( 76 ). Dead cells were labeled using the LIVE/DEAD Aqua (405 nm) (BD Bioscience).…”

Beta-cell mass expansion during puberty involves serotonin signaling and determines glucose homeostasis in adulthood

“…Following treatment islets were dissociated into single cells with accutase (1 μl/islet; Innovative Cell Technologies Inc., San Diego, CA) for 10 min at 37° and β-cell proliferation assessed by flow cytometry as described previously (66). Dead cells were labeled using the LIVE/DEAD™ Aqua (405 nm) (BD Bioscience).…”