Single-cell transcriptomics to study the effects of small molecules on pancreatic β-cell proliferation
Jérémie Charbord, Nadja Schulz, Dominika Tworus and Olov Andersson
Department of Cell and Molecular Biology, Karolinska Institutet, von Eulers väg 3, 17177 Stockholm, Sweden
Diabetes can be controlled with insulin injections, but a cure is still lacking. One potentially curative approach is to increase the number of insulin-producing β–cells by proliferation. Although the basal rate of β-cell proliferation is very low, recent chemical screening approaches have allowed the identification of β-cell mitogens that potently increase β-cell proliferation. The mechanisms of action of these small molecules and the processes controlling β-cell proliferation are still quite elusive. By a screen for small molecules inducing β-cell proliferation and single-cell transcriptomics we aim to identify β–cell mitogens and have better insight into what molecular mechanisms regulates β-cell proliferation. Screening of more than 3000 small molecules in transgenic zebrafish enabled us to identify hits increasing β-cells proliferation. Treatment of dissociated mouse islets further validated the effect of three small molecules. To analyze their mechanisms of action, we treated mouse islets for 6 and 48 hours followed by cell-sorting and single-cell RNA sequencing (SMARTseq2). After the 6h treatment, weighted gene correlation network and gene ontology analyses enabled us to identify activated mitochondrial genes and downregulated genes implicated in β-cell maturation for one potent β–cell mitogen. After the 48h treatment, a similar analysis of cells selected for their responsiveness to the treatment (as determined by a principal component analyses) displayed one major cluster of activated cell-cycle genes. This highlights the importance of analyzing the inductive mechanism of β-cell proliferation at time points prior to cell cycle entry. In conclusion, these studies can lead to the identification of exogenous small molecules that stimulate β-cell proliferation, as well as a better understanding of the endogenous molecular networks regulating both basal and induced β-cell proliferation.