a-combinatorial-single-cell-approach-to-characterize-molecular

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Lund University

Insulin is secreted from the pancreatic β-cells in response to elevated glucose. In intact islets the capacity for insulin release is determined by a complex interplay between different cell types. This has made it difficult to specifically assess the role of β-cell defects to the insulin secretory impairment in type 2 diabetes. Here we describe a new approach, based on optogenetics, that enables specific investigation of β-cells in intact islets. We used transgenic mice expressing the light-sensitive cation channel Channelrhodopsin-2 (ChR2) under control of the insulin promoter. Glucose tolerance in vivo was assessed using intraperitoneal glucose tolerance tests, and glucose-induced insulin release was measured from static batch incubations. ChR2 localization was determined by fluorescence confocal microscopy. The effect of ChR2 stimulation with blue LED light was assessed using Ca(2+) imaging and static islet incubations. Light stimulation of islets from transgenic ChR2 mice triggered prompt increases in intracellular Ca(2+). Moreover, light stimulation enhanced insulin secretion in batch-incubated islets at low and intermediate but not at high glucose concentrations. Glucagon release was not affected. Beta-cells from mice rendered diabetic on a high-fat diet exhibited a 3.5-fold increase in light-induced Ca(2+) influx compared with mice on a control diet. Furthermore, light enhanced insulin release also at high glucose in these mice, suggesting that high-fat feeding leads to a compensatory potentiation of the Ca(2+) response in β-cells. The results demonstrate the usefulness and versatility of optogenetics for studying mechanisms of perturbed hormone secretion in diabetes with high time-resolution and cell-specificity. 

Fatemeh Safi

Division of Molecular Hematology,
Lund Stem Cell Center,
Division of Molecular Hematology, Lund Stem Cell Center

Lund University

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Fatemeh Safi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Molecular Hematology,Lund Stem Cell Center,Division of Molecular Hematology, Lund Stem Cell Center

Lund University

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Division of Molecular Hematology,
Lund Stem Cell Center,
Division of Molecular Hematology, Lund Stem Cell Center