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), intermediate (group II), and large (group III) subpopulations by unit gravity sedimentation (1 G). The three subsets retained differences in cell size for up to 15 days of primary culture. Flow cytometric (fluorescence-activated cell sorter) measurements of forward-angle light scatter agreed well with fibroblast volume measured by image analysis and confirmed the utility of forward-angle light scatter for discriminating size subpopulations. Group II fibroblasts accumulated most rapidly by 8 days of culture and also contained the greatest proportion of S and G2/M phase cells as determined by fluorescence-activated cell sorter. Fibroblasts that were immunoreactive with antibodies to alpha-smooth muscle actin (alpha-SMA) were found only in group III. In situ end labeling of fragmented DNA detected apoptotic cells in both groups II 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 fibroblasts behave as predicted by classic models of cell cycle progression and differentiation. However, they do not support the hypothesis that the 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 phenotype.