CELL-SIZE, CELL-CYCLE, AND ALPHA-SMOOTH MUSCLE ACTIN EXPRESSION BY PRIMARY HUMAN LUNG FIBROBLASTS

Primary human lung fibroblasts were separated into small (group I), in termediate (group II), and large (group III) subpopulations by unit gr avity sedimentation (1 G). The three subsets retained differences in c ell size for up to 15 days of primary culture. Flow cytometric (fluore scence-activated cell sorter) measurements of forward-angle light scat ter agreed well with fibroblast volume measured by image analysis and confirmed the utility of forward-angle light scatter for discriminatin g size subpopulations. Group II fibroblasts accumulated most rapidly b y 8 days of culture and also contained the greatest proportion of S an d G(2)/M phase cells as determined by fluorescence-activated cell sort er. Fibroblasts that were immunoreactive with antibodies to alpha r-sm ooth muscle actin (alpha-SMA) were found only in group III. In situ en d labeling of fragmented DNA detected apoptotic cells in both groups I I and III, but double labeling for in situ end labeling and alpha-SMA revealed apoptotic cells in both the alpha-SMA-positive and -negative populations. These results demonstrate that primary human lung fibrobl asts behave as predicted by classic models of cell cycle progression a nd differentiation However, they do not support the hypothesis that th e expression of alpha-actin is related to apoptosis. We also describe a simple and reproducible method for the high-yield isolation of human lung fibroblast subsets of differing proliferative potential and phen otype.