Behavior of embryonic chick heart cells in culture. 1. Cellular responses to insulin-transferrin-selenium

Muscle cell-enriched primary cell cultures were prepared from 8-day embryonic chick heart ventricles (74% of these cells showed positive staining with anti-cardiac myosin antibody). To determine if ITS (a commercial mixture of insulin, transferrin, and selenium) affects these cardiac muscle cells, immunostaining and autoradiography were performed to determine the Muscle Cell Labeling Index (MLI). MLI represents the proportion of cardiac myosin-positive cells that specifically incorporated [3H]thymidine. The MLI for ITS-treated cells was 52%. Controls in Serum-free Nutrient Medium (SFNM) had a MLI of 27%. Combinations of growth signals also were tested. Whereas 5% Fetal Bovine Serum (FBS) was optimal for stimulation of [3H]thymidine incorporation, 10 and 20% FBS elicited an inhibitory effect. Addition of ITS enhanced the stimulatory effect of FBS and relieved some of the inhibitory effect. TGF-beta also was shown to have inhibitory effect on [3H]thymidine incorporation in these heart cells, but the inhibitory effect was not seen when it was added with ITS. Staining with anti-cardiac myosin antibody revealed that when the cells were cultured with ITS for 6 or 10 days, the percentages of muscle cells were 65 and 59%, whereas the percentages of muscle cells of controls in SFNM dropped to 44 and 31% respectively. Additional experiments showed that cell number increased in the presence of 5% FBS. In contrast, although ITS stimulated DNA synthesis, it did not immediately stimulate complete cell division. The percentage of muscle cells remained around 74% in the presence of 5% FBS, whereas it fell slightly (to 65%) in SFNM. This study showed that cardiac muscle cells from 8-day embryos in culture were responsive to ITS, FBS and TGF-beta and that ITS may be permissive for continued expression of differentiation of embryonic cardiac muscle cells.