Myofibroblasts differentiate from fibroblasts when plated at low density.

TLDR

Myofibroblasts, defined by smooth muscle α‑actin expression, appear at corneal and dermal incisions and promote wound contraction. The study reports that fibroblasts differentiate into myofibroblasts in a cell density‑dependent manner and proposes a model involving loss of cell contact and TGF‑β. The authors model differentiation as driven by loss of cell contact at low density and the presence of TGF‑β. Low‑density plating produced 70–80 % myofibroblasts after 5–7 days, whereas high‑density plating yielded only 5–10 %; high‑density passage caused loss of the phenotype within 3 days, low‑density cells showed higher proliferation and higher TGF‑β levels, and exogenous TGF‑β did not significantly increase myofibroblast numbers, supporting that low‑density culture induces differentiation.

Abstract

Myofibroblasts, defined by their expression of smooth muscle alpha-actin, appear at corneal and dermal incisions and promote wound contraction. We report here that cultured fibroblasts differentiate into myofibroblasts by a cell density-dependent mechanism. Fibroblasts seeded at low density (5 cells per mm2) produced a cell culture population consisting of 70-80% myofibroblasts, 5-7 days after seeding. In contrast, fibroblasts seeded at high density (500 cells per mm2) produced cultures 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-enriched 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 addition of exogenous TGF-beta, the results did not reach statistical significance. We conclude that myofibroblast differentiation can be induced in fibroblasts by plating at low density. We propose a cell density-dependent model of myofibroblast differentiation during wounding and healing in which at least two factors interact: loss of cell contact and the presence of TGF-beta.