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 cell types and transcriptional programming. Thus, during homeostasis, TM keratinocytes transit through a proliferative CP state and exhibit directional lateral migration. This work forms a foundation for understanding TM disorders and modeling keratinocyte biology.

鼓膜 (TM) 对听力至关重要，需要不断清除细胞碎片，但对 TM 表皮中的稳态机制知之甚少。使用单细胞 RNA 测序、谱系追踪、全器官外植体和活细胞成像，我们表明稳态 TM 表皮不同于其他表皮部位，并且具有离散的增殖区，具有多个角质形成细胞群的三维层次结构。TM 干细胞位于上级 TM 的离散位置，并产生长寿命克隆和定型祖细胞 (CP)。CP 克隆表现出横向迁移，其增殖能力由 Pdgfra+ 成纤维细胞支持，产生迁移但不增殖的后代。人类 TM 的单细胞测序揭示了相似的细胞类型和转录程序。因此，在稳态过程中，TM 角质形成细胞通过增殖性 CP 状态并表现出定向横向迁移。这项工作为理解 TM 疾病和模拟角质形成细胞生物学奠定了基础。