Identification of dormant cells using a novel fluorescent reporter of RNA-polymerase II activity

Dormancy is a reversible cellular state in which the cell cycle and metabolic activity of a cell is dramatically decreased, leading to protection from genetic damage and prolonged survival. One example of dormant cells is the somatic stem cell, which is crucial for tissue homeostasis and response to injury or transplantation. However, while dormant cells have been well characterized in some tissues, the ability to identify them irrespective of tissue of origin remains elusive. Moreover, in some tissues (e.g. the intestinal epithelium) dormant stem cells showed heterogeneity in the marker’s profile that makes their identification and isolation challenging. Here, we developed a live cell reporter of dormancy based on our observation that phosphorylation of RNA Polymerase II (RNApII), a hallmark of active mRNA transcription elongation, is largely absent in dormant stem cells from multiple lineages. We show that insertion of a short RNApII kinase target peptide directly into the yellow fluorescent protein Venus generates a live cell Optical Stem Cell Activity Reporter (OSCAR) for detection of dormant cells. We found, using small intestinal crypt cultures, that OSCAR is able to show the dynamics of dormancy induction and cellular differentiation in real time. Generation and analysis of mice ubiquitously expressing OSCAR showed several populations of OSCARhigh and OSCARlow cells. Microscopy, RNA-Seq and single-cell culture confirmed that OSCAR enables direct identification and isolation of cell populations bearing different transcriptional states and provides a useful tool for understanding dormant cell biology.