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

Abstract

Restoration of β-cell mass through the induction of proliferation represents an attractive therapeutic approach for the treatment of diabetes. However, intact and dispersed primary islets suffer from rapidly deteriorating viability and function ex vivo, posing a significant challenge for their experimental use in proliferation studies. Here, we describe a novel method for the assessment of compound effects on β-cell proliferation and count using reaggregated primary human islets, or islet microtissues (MTs), which display homogeneous size and tissue architecture as well as robust and stable functionality and viability for 4 weeks in culture. We utilized this platform to evaluate the dose-dependent short- and long-term effects of harmine on β-cell proliferation and function. Following compound treatment and EdU incorporation, islet MTs were stained and confocal-imaged for DAPI (nuclear marker), NKX6.1 (β-cell marker), and EdU (proliferation marker), allowing automated 3D-analysis of number of total cells, β-cells, and proliferating β- and non-β-cells per islet MT. In parallel, insulin secretion, intracellular insulin and ATP contents, and Caspase 3/7 activity were analyzed to obtain a comprehensive overview of islet MT function and viability. We observed that 4-day harmine treatment increased β- and non-β-cell proliferation, NKX6.1 expression, and basal and stimulated insulin secretion in a dose-dependent manner, while fold-stimulation of secretion peaked at intermediate harmine doses. Interestingly, 15-day harmine treatment led to a general reduction in harmine’s proliferative effects as well as altered dose-dependent trends. The described methodology provides a unique tool for in vitro high-throughput evaluation of short- and long-term changes in human β-cell proliferation, count and fraction along with a variety of functional parameters, in a representative 3D human islet model.

Biomaterials‐based nanoparticles conjugated to regulatory T cells provide a modular system for localized delivery of pharmacotherapeutic agents

“…To address this, we utilized BDC2.5 transgenic NOD T cells, which express a pancreatic islet‐specific TCR that promotes cell trafficking to the pancreatic islets 69 . The NP‐Treg formulation was further optimized by dual loading of IL‐2 with harmine, which stimulates insulin‐producing β‐cell replication 70,71 . IL‐2/harmine NPs were coupled to BDC2.5 Tregs and administered to 12‐week‐old, pre‐diabetic female NOD mice, representing late stage intervention when significant insulin‐producing cell loss has already occurred 72 .…”

“… 69 The NP‐Treg formulation was further optimized by dual loading of IL‐2 with harmine, which stimulates insulin‐producing β‐cell replication. 70 , 71 IL‐2/harmine NPs were coupled to BDC2.5 Tregs and administered to 12‐week‐old, pre‐diabetic female NOD mice, representing late stage intervention when significant insulin‐producing cell loss has already occurred. 72 While unconjugated IL‐2/harmine NPs did not prevent diabetes, animals that received IL‐2/harmine NP‐coupled BDC2.5 Tregs had significantly reduced T1D incidence versus the saline control group (Figure 8B ).…”

Single Cell RNA-Seq Analysis of Regenerative Drug-Treated Human Pancreatic Islets Identifies A Cycling Alpha Cell Population As Key Beta Cell Progenitors

“…Clearly, drug treatment studies at multiple time points (e.g., 3, 6, 9, 12 days) may be of interest, but human islets survive and retain phenotype poorly over more than a few days. In this regard, Title et al reported that reaggregated pseudoislets can be cultured and subjected to DYRK1A inhibitor treatment for up to 15 days 71 . These reaggregated pseudoislet models hold promise for longer term human islet studies.…”

Stem cell-derived intestinal organoids: a novel modality for IBD

“…It can add different cells or microorganisms to mediate cell-cell and cell-microbe interactions [ 100 – 102 ]. Microarray technology can also be combined with imaging instruments to monitor cell biological changes in real time, record behavioral changes of cells in disease states, and record the whole process of cellular response to drugs [ 103 – 105 ]. Microarray technology enables more systematic and mechanized organoid culture, increases throughput, expands the scope of research, and takes a firm step toward clinical translation [ 69 , 100 , 106 – 108 ].…”