Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes.
Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14-15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin and alpha-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycnic centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells may provide a useful experimental system for the study of human cardiac muscle cell biology.
Goldman
B I
BI
Department of Pathology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Wurzel
J
J
D000199:Actins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002469:Cell Separation; D002500:Centrifugation, Isopycnic; D003470:Culture Media; D016895:Culture Media, Serum-Free; D006133:Growth Substances / Q000494:pharmacology; D006321:Heart / Q000196:embryology; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D009200:Myocardial Contraction; D009206:Myocardium / Q000166:cytology*; D009218:Myosins / Q000378:metabolism
D000199:Actins; D003470:Culture Media; D016895:Culture Media, Serum-Free; D006133:Growth Substances; D009218:Myosins