Single-cell RNA-seq addresses key issues in islet cell biology and diabetes research

The pancreas is an abdominal organ that produces digestive enzymes and hormones that regulate blood sugar. This hormone-producing function is confined to the islets of Langerhans, which form clusters of various endocrine cell types.

Among them are beta cells, which produce the hormone insulin, which is required to lower blood glucose (a type of sugar) levels, and alpha cells, which produce the hormone glucagon, which is responsible for raising blood glucose levels. ..

Type 1 Diabetes is a chronic disease The body’s immune system mistakenly attacks and destroys the insulin-producing beta cells of the pancreas. Regenerative medicine aims to replace the amount of beta cells and thus supports and ultimately replaces current insulin replacement therapy.

Altered islet composition, including poor beta cell function and beta cell dedifferentiation, also contributes to type II diabetes.

Therefore, a deeper understanding of the identity and crosstalk of different islet cell types may lead to better characterization of both types of diabetes and contribute to the development of new therapeutic concepts.

Single-cell transcriptomics is a powerful technique for characterizing cell identity. Previously, CeMM researchers from Christoph Bock and Stefan Kubicek’s group at CeMM published the first single-cell transcriptome from primary human islet cells.

Advances in technology have potential applications in the generation of global human and mouse single-cell transcriptome atlases. Despite these advances, the single cell approach remains technically challenging given the very small amounts of RNA present that have been completely exhausted in the experiment. Therefore, it is essential to ensure the quality and purity of the resulting single cell transcriptome.

CeMM researchers from two contributing laboratories confirmed unexpectedly high hormone expression in non-endocrine cell types, both in their own dataset and in other published single-cell studies.

They sought to elucidate whether this was the result of contamination with RNA molecules, for example from dying cells, and how to remove it to obtain a more reliable dataset.

Such contamination appeared to be present in single-cell RNA-seq data in most tissues, but was most pronounced in islets. Islet endocrine cells are dedicated to producing a single hormone, with beta-cell insulin and alpha-cell glucagon being expressed at higher levels than typical “housekeeping” genes.

Therefore, the redistribution of these transcripts into other cell types was very marked. Based on this observation, their goal was to develop, validate, and apply methods to ly determine and computationally remove such contamination.

In the study, CeMM researchers used spike-in cells of different cell types in both mouse and human samples, which were added to the islet samples. Importantly, the transcriptome of these spike-in cells has been fully characterized.

This allows internal and precise control of the level of RNA contamination of single-cell RNA-seq, allowing human transcripts detected in mouse spike-in cells to make up the contaminating RNA.

In this way, they found that the sample had a contamination level of up to 20% and were able to define the contamination of each sample. Next, we developed a new bioinformatics approach to computationally remove contaminated reads from the single-cell transcriptome.

Here, “decontaminated” transcriptomes are obtained, from which spurious signals are removed, and how they respond to treatment with three different drugs, the identity of cells in different cell types Began to characterize.

They found that a small molecule inhibitor of the transcription factor FOXO1 induces dedifferentiation of both alpha and beta cells.

In addition, they were shown to impair alpha cell function and studied artemether, which may induce insulin production, in both in vivo and in vitro studies. The effects of the drug Artemether were species-specific and cell-type specific.

In alpha cells, some of the cells increase insulin expression, acquiring the aspect of beta cell identity in both mouse and human samples. Importantly, the researchers found that there was no significant change in insulin expression in human beta cells, but in mouse pancreatic islets, beta cells shared insulin expression with global beta cell identity. Lower.

This work is the result of an interdisciplinary collaboration between SteMM Kubicek and Christoph Bock’s laboratories at CeMM and Patrick Collombat at the Valrose Biology Institute in France.

This was the first study to apply single cell sequencing to analyze the kinetic drug response in intact dissociated tissue and benefited from the high quantitative accuracy of the decontamination method.

Therefore, it not only provides a new method for single-cell decontamination and highly quantitative single-cell analysis of drug response in intact tissues, but also addresses important current questions in islet cell biology and diabetes research. Will also deal with. These findings may open up potential treatments for future treatment of type 1 diabetes.