Effects of Fibroblast Growth Factor and Epidermal Growth Factor on the Rate of Growth of Amniotic Fluid-Derived Cells DENIS GOSPODAROWICZ,* JOHN S. MORAN, AND NORMAN D. OWASHI The Salk Institute for Biological Studies, La Jolla, California ABSTRACT. Primary cultures of cells derived from human and bovine amniotic fluid were cultivated in Dulbecco's modified Eagles medium with 20% fetal calf serum. Whereas increased concentrations of fetal calf or other types of serum bring about no further mitogenic response, the rate of growth of these cells was nevertheless sharply increased when 100 ng/ml of FGF was added to the culture media. The FGF induced mitogenic effect was statistically
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significant. EGF at 100 ng/ml had a less pronounced effect which, when analyzed statistically, was not significant. Amniotic fluid-derived cells are used for the prenatal detection of genetic disorders. The use of FGF to increase the rate of growth of these cells should reduce the time required between amniocentesis and diagnosis. (J Clin Endocrinol Metab 44: 651, 1977)
HE RATE of proliferation of animal cells in culture is limited by the availability of nutrients (1) and other factors including hormones (2,3) and mitogenic polypeptides (3,4). For many cell types, a rich medium supplemented with 10% to 20% serum induces rapid growth. Certain cell types, however, divide rapidly in serum-supplemented medium only when special mitogenic factors are added. For example, granulocytes require colony stimulating factor (5) and proerythroblasts require erythropoietin (6). Recently, it has been shown that human foreskin fibroblasts, a cell type that proliferates rapidly in 10% serum-supplemented medium, can be induced to grow to higher cell densities by the addition of low concentrations (10~ 9 M) of either epidermal growth factor (EGF) (7) or fibroblast growth factor (FGF) (8). Also, it was shown that the population doubling time of these fibroblasts in 10% calf serum could be reduced 50% by the addition of FGF to the culture medium (8). The findings sig-
naled a breakthrough in the study of cell growth since earlier studies of fibroblast mitogens showed mitogenic effects only on serum-deprived cells. The studies with EGF and FGF showed, for the first time, an enhancement of growth in medium supplemented with an optimal concentration of serum. However, these studies were performed with only one cell type, human foreskin fibroblasts, and those cells were in early passage rather than in primary culture. Also, cell growth at clonal density (
T
significant. EGF at 100 ng/ml had a less pronounced effect which, when analyzed statistically, was not significant. Amniotic fluid-derived cells are used for the prenatal detection of genetic disorders. The use of FGF to increase the rate of growth of these cells should reduce the time required between amniocentesis and diagnosis. (J Clin Endocrinol Metab 44: 651, 1977)
HE RATE of proliferation of animal cells in culture is limited by the availability of nutrients (1) and other factors including hormones (2,3) and mitogenic polypeptides (3,4). For many cell types, a rich medium supplemented with 10% to 20% serum induces rapid growth. Certain cell types, however, divide rapidly in serum-supplemented medium only when special mitogenic factors are added. For example, granulocytes require colony stimulating factor (5) and proerythroblasts require erythropoietin (6). Recently, it has been shown that human foreskin fibroblasts, a cell type that proliferates rapidly in 10% serum-supplemented medium, can be induced to grow to higher cell densities by the addition of low concentrations (10~ 9 M) of either epidermal growth factor (EGF) (7) or fibroblast growth factor (FGF) (8). Also, it was shown that the population doubling time of these fibroblasts in 10% calf serum could be reduced 50% by the addition of FGF to the culture medium (8). The findings sig-
naled a breakthrough in the study of cell growth since earlier studies of fibroblast mitogens showed mitogenic effects only on serum-deprived cells. The studies with EGF and FGF showed, for the first time, an enhancement of growth in medium supplemented with an optimal concentration of serum. However, these studies were performed with only one cell type, human foreskin fibroblasts, and those cells were in early passage rather than in primary culture. Also, cell growth at clonal density (
