Single cell transcriptome profiling of Lrig1 quiescent stem cell populations

Identifying progenitor cells in laryngeal and vocal fold (VF) mucosa is essential for advancing stem cell-based tissue engineering and therapies for treatment of VF diseases. In this study, we used the Lrig1 gene as a marker for tissue-resident stem cells within murine laryngeal and VF mucosa. We show that Lrig1? cells exhibited hallmark features of quiescent stem cells: slow-cycling, persistence and differentiation potential. Transcriptionally, they suppress transcription of cell type–specific genes and proliferation, while activating a global transcriptional program related to RNA metabolism, epigenetic regulation and protein ubiquitination – supporting quiescence and readiness for activation. Lrig1+ cells were enriched in basal, parabasal, and immature secretory populations and were spatially shielded from direct stress exposure to prevent their premature or chronic mobilization. Notably, we detected analogous Lrig1-expressing cells in human laryngeal and VF tissues, emphasizing the translational relevance of our findings. To investigate how Lrig1? cell quiescence is regulated, we conditionally deleted Notch1, leading to epithelial hyperplasia, expansion of secretory cell populations, and mucus hyperproduction. In summary, Lrig1 marks a conserved, quiescent stem cell population in the larynx and VFs, with quiescence maintained through Notch1 signaling. Overall design: We first genetically labeled Lrig1? cells in Lrig1CreERT2/+;ROSALSL-tdTomato/+reporter mice, induced the Cre-recombination using tamoxifen injections and collected tissue at various timepoints - 1day, 3days, 1week, 2weeks and 2 months post recombination (for lineage tracing experiments). For transcriptional profiling, we used the same labeling and recombination approach and collected tissue 24 hours post-recombination. WT and ROSALSL-tdTomato/+ were included as negative and background controls for flow cytometry cell sorting. Cells were dissociated to generate single-cell suspensions, sorted based on tdTomato signal, and subjected to single-cell RNA sequencing using the 10x Genomics pipeline. To experimentally disrupt Lrig1? cell quiescence in vivo, we conditionally deleted the Notch1 gene in Lrig1? cells and investigated whether Notch1 deletion can lead to pathological mucosal remodeling, including epithelial hyperplasia and/or increased mucus secretion.