IN-VITRO INDUCTION OF TYPE-II PNEUMOCYTE-RELATED DIFFERENTIATION IN ACLONAL FETAL BRONCHIOLOALVEOLAR EPITHELIAL-CELL LINE (M3E3 C3)/

The aim of the present study is to investigate the differentiation of a cloned fetal Syrian hamster lung epithelial cell line, M3E3/C3, to a ssume morphological and biochemical features of Type II pneumocytes (p hospholipid synthesis). The use of a soft agar overlay and a different iation medium, based on RPMI 1640 combined with hormone supplements, i ncreased the cellular content of phosphatidylcholine (PC) from 48.6 % in the conventional culture without any of these factors (referred to as 'control') to 64.7 % (p < 0.02). The other cell membrane-associated components, phosphatidylethanolamine (p < 0.05), sphingomyelin (p < 0 .001), phosphatidylserine (n. s.), phosphatidic acid (p < 0.02) and ph osphatidylinositol (p < 0.02) decreased. The content of phosphatidylgl ycerol showed no essential change (from 11.2 % to 8.4 %) and the conte nt of disaturated phospholipids decreased from 32.0 to 23.4 mug/10(6) cells (p < 0.002). The phospholipid pattern of these differentiated ce lls is in rough accordance with that of primary isolated Type II pneum ocytes. They incorporated H-3-choline over a period of four hours at a higher rate in the Type II pneumocyte-specific phospholipids, PC and dipalmitoyl-phosphatidylcholine (DPPC), than the undifferentiated cont rol. The radiolabeling of PC and DPPC in the differentiated cells, aft er 3 hours of incubation with H-3-choline, was about 3.2-fold and 2.2- fold, respectively, higher than that in the control cells (p < 0.001). Intracytoplasmatic phospholipid granules were evident in the differen tiated cells by light and fluorescence microscopy (modified PAPANICOLA OU stain, Phosphin 3 R fluorescence). Furthermore, the differentiated cells had a high activity of alkaline phosphatase, whereas the control cells showed only little activity of this enzyme. Ultrastructurally, many concentric multilayered osmiophilic bodies, well developed Golgi apparatuses and many cytoplasmic protrusions comparable to microvilli, were detectable in the cuboidal shaped differentiated cells. The cont rol cells remained wide and flattened on the plastic surface and produ ced a fibrillar extracellular matrix. In the simultaneously studied fe tal lung fibroblasts none of these specific features were noted. These results indicate a specific differentiation capacity of the clonal fe tal cell line, M3E3/C3, by closely resembling Type II pneumocytes.