PRIMARY-CELL CULTURE OF HUMAN TYPE-II PNEUMONOCYTES - MAINTENANCE OF A DIFFERENTIATED PHENOTYPE AND TRANSFECTION WITH RECOMBINANT ADENOVIRUSES

Studies of the regulation of surfactant lipoprotein metabolism and sec retion and surfactant protein gene expression have been hampered by th e lack of a cell culture system in which the phenotypic properties of type II cells are maintained, We have developed a primary culture syst em that facilitates the maintenance of a number of morphologic and bio chemical properties of type II pneumonocytes for up to 2 wk. Cells wer e isolated by collagenase digestion of midgestation human fetal lung t issue that had been maintained in organ culture in the presence of dib utyryl cyclic AMP (Bt(2)cAMP) for 5 days. The isolated cells were enri ched for epithelial components by treatment with DEAE-dextran, plated on an extracellular matrix (ECM) derived from Madin-Darby canine kidne y (MDCK) cells, and incubated at an air/liquid interface in a minimal amount of culture medium containing Bt(2)cAMP. The cell cultures were comprised of islands of round epithelial-like cells containing numerou s dense osmiophilic granules had the appearance of lamellar bodies, th e distinguishing feature of type II pneumonocytes. Additionally, the c ultures maintained elevated levels of SP-A gene expression for up to 2 wk. The expression of mRNAs encoding SP-A, SP-B, and SP-C were regula ted in the cultured cells by glucocorticoids and cyclic AMP in a manne r similar to that observed in fetal lung tissue in organ culture. The differentiated phenotype was most apparent when the cells were culture d at an air/liquid interface. In order to utilize the cultured type Il cells for study of the effects of overexpression of various proteins and for promoter analysis, it is of essence to transfect DNA construct s into these cells with high efficiency. Unfortunately, we found the c ells to be refractory to efficient transfer of DNA using conventional methods (i.e., lipofection, electroporation, or calcium phosphate-medi ated transfection). However, replication-defective recombinant human a denoviruses were found to provide a highly efficient means of introduc ing DNA into the type II pneumonocytes. Furthermore, we observed in ty pe II cell-enriched cultures infected with recombinant adenoviruses co ntaining the lacZ gene under control of a cytomegalovirus promoter, th at beta-galactosidase was expressed uniformly in the islands of type I l cells and surrounding fibroblasts. By contrast, in cultures infected with recombinant adenoviruses containing the human growth hormone (hG H) gene under control of the SP-A gene promoter and 5'-flanking region , hGH was expressed only in the type Il cells, Thus, this culture syst em provides an excellent means for identifying genomic elements that m ediate type II cell-specific gene expression.