The tracheobronchial epithelium is composed of three major cell types, ciliated cells, secretory cells and basal cells. The pathway of differentiation in this epithelium is not well understood. Particularly unclear is the function of the basal cells and the role they play in the normal and pathological forms of regeneration. The goals of our studies are 1) to obtain immunological and biochemical markers for the different cell-subpopulations, to define the different cell types with the aid of these markers and to elucidate the pathways of differentiation during development and during regeneration of the airway epithelium. 2) to develop an in vitro cell culture system suited for the study of cell differentiation under defined conditions. (Presently, we are using an in vivo cell culture system which permits us to examine the differentiation capacity of different cell types but without the ability to control regulating factors e.g. growth factors). We raised several new cell type specific monoclonal antibodies useful for identifying secretory and ciliated cells and demonstrated that cyto-keratin 14 antibody and the lectin GS1B4 (from griffonia simplicifolia) specifically react with basal cells in adult, steady state tracheal epithelium. The epithelial regeneration studies using the in vivo cell culture model indicated that during the regeneration process, both secretory and basal cells differentiated into a "poorly differentiated" cell type (designated PD-cell) which does not express secretory cell or ciliated cell markers. (Whether ciliated cells also participate in this process is currently not known). These PD-cells do however express two basal cell markers namely, keratin 14 and GS1B4 binding sites. The PD-cells subsequently generate new basal, secretory and ciliated cells. The latter two gradually lose the basal cell markers as they mature and express their own cell type specific markers. The role of the PD-cell in tracheal cell differentiation will be further defined in future studies including ongoing developmental studies. The development of the in vitro differentiation model has progressed to the point that the role of factors such as retinoids and TGF-beta in mucous and ciliated cell differentiation can now be studied in defined conditions. This system will be used in the future in the regeneration/differentiation studies.