biostudies-literatureFrumm SMNIDCR NIH HHSNational Institute of Diabetes and Digestive and Kidney DiseasesNIDDK NIH HHSAmerican Otological SocietyNIDCD NIH HHSNational Institute on Deafness and Other Communication DisordersNational Institute of Dental and Craniofacial ResearchNIH HHS315-330.e5https://www.ebi.ac.uk/biostudies/studies/S-EPMC8962966biostudies-literatureUnknown28(2)The tympanic membrane (TM) is critical for hearing and requires continuous clearing of cellular debris, but little is known about homeostatic mechanisms in the TM epidermis. Using single-cell RNA sequencing, lineage tracing, whole-organ explant, and live-cell imaging, we show that homeostatic TM epidermis is distinct from other epidermal sites and has discrete proliferative zones with a three-dimensional hierarchy of multiple keratinocyte populations. TM stem cells reside in a discrete location of the superior TM and generate long-lived clones and committed progenitors (CPs). CP clones exhibit lateral migration, and their proliferative capacity is supported by Pdgfra+ fibroblasts, generating migratory but non-proliferative progeny. Single-cell sequencing of the human TM revealed similar ceCell stem cellA Hierarchy of Proliferative and Migratory Keratinocytes Maintains the Tympanic Membrane.PMC8962966R01 DE029890R01 DK118421S10 OD021664R01 DC018076Frumm SMTward ADYu SKLee KPByrnes LEChang JArtichoker JAScaria SMSneddon JBfalseA Hierarchy of Proliferative and Migratory Keratinocytes Maintains the Tympanic Membrane.The tympanic membrane (TM) is critical for hearing and requires continuous clearing of cellular debris, but little is known about homeostatic mechanisms in the TM epidermis. Using single-cell RNA sequencing, lineage tracing, whole-organ explant, and live-cell imaging, we show that homeostatic TM epidermis is distinct from other epidermal sites and has discrete proliferative zones with a three-dimensional hierarchy of multiple keratinocyte populations. TM stem cells reside in a discrete location of the superior TM and generate long-lived clones and committed progenitors (CPs). CP clones exhibit lateral migration, and their proliferative capacity is supported by Pdgfra+ fibroblasts, generating migratory but non-proliferative progeny. Single-cell sequencing of the human TM revealed similar ce2021-01-01T00:00:00Z2021 Feb2026-05-30T15:02:41.041Z2025-04-04T23:04:24.025ZS-EPMC89629663318107810.1016/j.stem.2020.10.006