Retinoic acid promotes tissue regeneration by resolving stem cell lineage plasticity

Stem cells upended from their niche upon injury display lineage plasticity, a transient multi-lineage state essential for tissue repair. Employing high-throughput approaches and three-dimensional cultures of hair follicle stem cells (HFSCs), we investigate the signals that govern the transition between homeostatic regeneration and lineage plasticity. We identify retinoic acid (RA) as a master orchestrator of HFSC behavior during these two processes. In the hair follicle, RA signals within defined niches and interacts with WNT and BMP cues to drive hair regeneration. In wounded skin, reduced RA signaling prompts HFSCs to prioritize epidermal re-epithelialization and must be restored to promote hair regrowth. Substantiated in vivo, our findings have profound therapeutic implications for hair growth and for chronic wounds and cancers, where lineage plasticity is unresolved. Overall design: scRNA-seq: Mouse skin and hair follicle cells were FACS-purified from AnaVI back skin and submitted for single cell RNA-sequencing using the 10x Genomics platform. In parallel, autologous hair follicle stem cells (HFSCs) were FACS-purified, propagated in culture, and apportioned into 3D cultures as described. Cultured HFSC colonies from each condition were dissociated, multiplexed using TotalSeq-B hashtag antibodies, and submitted for single cell RNA-sequencing using the 10x Genomics platform. ATAC-seq: Mouse hair follicle stem cells (HFSCs) from wild-type and Sox9-CreER;Rxra-fl/fl;R26-YFP mice were FACS-purified from telogen back skin, propagated in culture, and apportioned into 3D cultures. ATAC-seq was performed as described in Buenrostro et al., 2013. bulkRNA-seq: Mouse hair follicle stem cells (HFSCs) from wild-type and Sox9-CreER;Rxra-fl/fl;R26-YFP mice were FACS-purified from telogen back skin, propagated in culture, and apportioned into 3D cultures. Cells were collected in Trizol and bulk RNA-seq was performed. Cut&Run: Mouse hair follicle stem cells (HFSCs) from wild-type and Sox9-CreER;Rxra-fl/fl;R26-YFP mice were FACS-purified from telogen back skin, propagated in culture, and apportioned into 3D cultures. Cells were collected to perform Cut-N-Run experiments for different antibodies.