MYOFIBROBLASTS DIFFERENTIATE FROM FIBROBLASTS WHEN PLATED AT LOW-DENSITY

Myofibroblasts, defined by their expression of smooth muscle alpha-act in, appear at corneal and dermal incisions and promote wound contracti on. We report here that cultured fibroblasts differentiate into myofib roblasts by a cell density-dependent mechanism. Fibroblasts seeded at low density (5 cells per mm(2)) produced a cell culture population con sisting of 70-80% myofibroblasts, 5-7 days after seeding. In contrast, fibroblasts seeded at high density (500 cells per mm(2)) produced cul tures with only 5-10% myofibroblasts. When the myofibroblast-enriched cultures were subsequently passaged at high density, the smooth muscle alpha-actin phenotype was lost within 3 days. Furthermore, initially 60% of the low-density-cultured cells incorporated BrdUrd compared to 30% of cells passaged at high density. Media from myofibroblast-enrich ed cultures had more latent and active transforming growth factor beta (TGF-beta) than did media from fibroblast-enriched cultures. Although there was a trend towards increased numbers of myofibroblasts after a ddition of exogenous TGF-beta, the results did not reach statistical s ignificance. We conclude that myofibroblast differentiation can be ind uced in fibroblasts by plating at low density. We propose a cell densi ty-dependent model of myofibroblast differentiation during wounding an d healing in which at least two factors interact: loss of cell contact and the presence of TGF-beta.