Scientists from the Van Oudenaarden and Robin group discovered that a widely-used experimental protocol induces a stress response in a subpopulation of the studied cells. This discovery was made when they for the first time applied a new technique, named single-cell sequencing, to muscle stem cells. The stress response changes gene expression (how much genes are ‘on’ in a cell) in some of the studied cells. One important implication of this finding is that several previously published studies, were similar experimental protocols have been used, might need to be partly corrected and reinterpreted. These results were published in Nature Methods on September 28th.

 Muscle stem cells

Muscles in adults contain stem cells. In uninjured and healthy muscles, these stem cells are normally inactive. It is only upon injury of the muscle that the stem cells are activated to repair the injury. Muscle stem cells are thus essential for muscle repair after injury or sports, and defects in these stem cells result in muscle diseases.

Gene expression in individual cells

The human body consists of trillions of cells. Gene expression is different in all these cells, and depends on the cell type. The Van Oudenaarden laboratory is specialized in measuring gene expression in single cells, which allows them to investigate cells in more detail than was previously possible. In this study, the authors investigated gene expression in single muscle stem cells.

Cells get ‘stressed’ during experiment

In order to measure gene expression in cells, these cells first have to be extracted (‘taken out’) from their organ. The cell-extraction technique (a combination of dissociation and FACS) used to isolate cells from organs has been used for many years. The authors used this extraction technique to isolate single muscle stem cells form muscles to study their gene expression. Unexpectedly, they found that some of the studied cells express many genes that are linked to stress (immediate early response genes, including Fos and Jun, and heat shock protein genes). A more detailed analysis via microscopy then showed that these stress genes are turned ‘on’ in response to the cell-extraction technique, and that these genes are normally not expressed in the cells when they are still in the muscle.

 Relevance for research

These results thus show that this widely-used cell-extraction technique causes a stress response in some of the cells, which then turn on their stress genes. These findings are important for scientists that study gene expression, as this cell-extraction method is used a lot in their field. Alexander van Oudenaarden: “We found similar subpopulations where stress genes are ‘on’ in other datasets, for example in the pancreas and in the zebrafish fin. This suggests that we found a general effect of the cell-extraction method, and scientists that use this technique have to keep this in mind.” Susanne van den Brink: “We now developed two new methods that can be used to remove the stressed cells prior to measuring their gene expression. These methods will need to be developed further in the coming years.” This discovery implies that results from previous gene expression studies might need to be partly revised. These revisions are relatively small, as only a small subset of the cells gets stressed during the experiment.

Biological explanation

The scientists also found a biological explanation for their findings. Susanne van den Brink: “Interestingly, muscle injury induces the expression of the exact same genes as the cell-extraction method does. This similarity suggests that the stem cells ‘think’ that the muscle is injured and therefore react the same as upon injury when we extract them from muscles”

Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations

Susanne C van den Brink, Fanny Sage, Ábel Vértesy, Bastiaan Spanjaard, Josi Peterson-Maduro, Chloé S Baron, Catherine Robin & Alexander van Oudenaarde. Nature Methods 2017

Prof.dr.ir. Alexander van Oudenaarden is director and group leader at the Hubrecht Institute (KNAW) and professor of quantitative biology of gene regulation at the Faculty of Science and the Faculty of Medicine at Utrecht University.

Dr. Catherine Robin is group leader at the Hubrecht Institute and is also associated with the UMC Utrecht.