Dose‐dependent initiation of myogenesis by neurotrophic factor

Abstract

The kinetics of primary chick muscle cells in response to partially purified neurotrophic factor (NTF) were investigated. Chick muscle cells are routinely plated in the absence of NTF but in the presence of horse serum to allow cell attachment to the substratum; a maximum number of cells attaches 5–8 hr after plating, but cells do not proliferate in the absence of NTF, or do so extremely slowly. Subsequent to a routine attachment period of 6 hr, the medium is aspirated and replaced with medium containing 2–20 μg/ml of NTF as well as horse serum. Dose responsiveness at elevated NTF concentrations is observed only if F12 medium, supplemented with serum, insulin, transferrin, and selenium is used. In the presence of commercial F10, although supplemented with serum, dose responsiveness is not apparent at more than 2 μg/ml of NTF. Upon exposure to NTF, in the presence of supplemented F12 and serum, an 8‐hr lag ensues; then, thymidine is incorporated at NTF dose‐dependent rates by growing cell populations. The rates of incorporation into cell populations depend on the initial number of cells plated and the amount of NTF supplied. Cell counting indicates that dose‐dependent proliferation of muscle cells has occurred during the first 48 hr of exposure to NTF. The specific activity of NTF is repeatedly shown to be at least 10‐fold greater than unfractionated nerve extract. Shortly after 48 hr of exposure to NTF, myoblasts characteristically begin to fuse with one another, and myogenesis is visible. These studies show that the initiation of in vitro myogenesis in primary chick muscle cells depends on the amount of NTF supplied. The work also indicates a requirement for optimum medium conditions in order to detect dose–responses to NTF.