Inhibition of TGF-β signaling enables long-term proliferation of mouse primary epithelial stem/progenitor cells of the tympanic membrane and the middle ear mucosa

The surface of the middle ear is composed of the tympanic membrane (TM) and the middle ear mucosa (MEM). A number of diseases and conditions such as otitis media, middle ear cholesteatoma, and perforation of the TM have been reported to cause dysfunction of the middle ear, ultimately leading to high-frequency hearing loss. Despite its importance in repairing the damaged tissues, the stem/progenitor cells of the TM and the MEM epithelia remains largely uncharacterized due, in part, to the lack of an optimal methodology to expand and maintain stem/progenitor cells long-term. Here, we show that suppression of TGF-beta signaling in a low Ca2+ condition enables long-term proliferation of p63-positive epithelial stem/progenitor cells of the TM and the MEM while avoiding their malignant transformation. Indeed, our data show that the expanded TM and MEM stem/progenitor cells respond to Ca2+ stimulation and differentiate into the mature epithelial cell lineages marked by cytokeratin (CK) 1/8/18 or Bpifa1, respectively. These results will allow us to expand epithelial stem/progenitor cells of the TM and MEM in quantity for large-scale analyses and will enhance the use of mouse models in developing stem cell-mediated therapeutic strategies for the treatment of middle ear diseases and conditions.