REGULATION OF CILIATED CELL-DIFFERENTIATION IN CULTURES OF RAT TRACHEAL EPITHELIAL-CELLS

The cellular pathway of ciliated cell differentiation and its regulati on is poorly defined. To begin to understand the process of ciliated c ell. differentiation, we sought to identify factors regulating ciliate d cell development in vitro. Rat tracheal epithelial (RTE) cells were cultured on collagen gel-coated membranes at an air-liquid interface i n hormone- and growth factor-supplemented medium (complete medium [CM] ). Under these conditions, RTE cells first proliferate and then differ entiate into a pseudostratified mucociliary epithelium. Ciliated cell differentiation was measured using a monoclonal antibody, RTE(3), whic h was shown to specifically react with the plasma membrane of ciliated cells. Cultures were immunostained in situ, and the percentage of the culture surface covered with ciliated cells was estimated using video microscopy and an image analysis program. If an air-liquid interface w as not created and the cells were maintained in the submerged state, c iliated cell differentiation was suppressed 25-fold. Culture in the ab sence of mitogenic components present in CM, including epidermal growt h factor (EGF), cholera toxin (CT), or bovine pituitary extract, resul ted in 2- to 4-fold increases in the percentage of ciliated cells. Whe n both EGF and CT were removed from the media, DNA synthesis and total cell number was reduced, while ciliated cell differentiation increase d as much as 5-fold. These results demonstrate that submersion inhibit s, while withdrawal of mitogenic compounds promotes, ciliated cell dif ferentiation in vitro.