In culture medium containing heparinized, heatinactivated, chicken plasma, normal chicken heart mesenchymal cells do not proliferate but their Rous sarcoma virus-infected counterparts proliferate maximally. In medium containing serum derived from chicken whole blood or plasma, on the other hand, normal chicken heart mesenchymal cells proliferate actively, at similar overall rates and to similar extents. The rate and extent of normal cell proliferation are decreased by a factor of approximately 'A with whole blood-derived serum that is heparinized and inactivated; proliferation ceases in plasma-derived serum that is heparinized and inactivated. Heparinization and inactivation of serum does not affect the proliferation of Rous sarcoma virus-infected cells, indicating that this combined treatment eliminates a mitogenic (regulatory) rather than a supportive (nutrient) factor(s) for cell replication. We hypothesize that mitogen(s) is released from plasma protein precursors when plasma clots in the presence of formed elements of the blood or when plasma-derived serum is exposed to cultured cells; heparinization and inactivation, within the framework of this hypothesis, would render nonfunctional the plasma protein precursor(s) from which the mitogen(s) is generated. Alternatively, our data are consistent with the release of two mitogens during blood clotting, one from plasma protein precursors and the other from formed elements of the blood. We also have studied the proliferative behavior of Swiss and BALB/c 3T3 cells in whole blood-derived and plasma-derived human serum. Our studies suggest that the platelet-derived growth factor has an artifactual supportive (nutrient) role, rather than an authentic mitogenic role, in cell replication.Between 1971 and 1973, Balk and coworkers (1-3) demonstrated that normal chicken pectoral muscle fibroblasts did not proliferate in low-calcium culture medium containing chicken plasma but proliferated actively when this medium contained chicken serum. On the basis of that observation, it was postulated that a mitogen might be released from plasma protein precursors or from formed elements of the blood during the clotting process and that this mitogen, found in serum but not in plasma, might represent a "wound hormone" capable of initiating cell proliferation at sites of tissue injury. Because of uncertainties inherent in the interpretation of data derived from studies using a culture medium with a low calcium concentration (4), however, further studies of the serum-plasma difference were deferred until a culture system was available in which normal cells were quiescent in a plasma-containing medium with physiological ion concentrations.Other workers subsequently have claimed that the mitogenic property present in serum, but not plasma, resided in a specific polypeptide "platelet-derived growth factor" (for general re-views, see refs. 5 and 6; for critical reviews, see refs. 7 and 8). Ross and Vogel (5) Because the activity ofthe platelet-derived growth factor has been defin...

Normal chicken heart mesenchymal cells at low culture density are proliferatively quiescent in a physiological culture medium containing heparinized, heat-inactivated, chicken plasma at 10%. Rous sarcoma virus-infected chicken heart mesenchymal cells, on the other hand, proliferate maximally in this same medium, undergoing a 60-fold increase in cell number during 4 days of exponential growth. When normal heart mesenchymal cells are cultured for 4 days in the presence of epidermal growth factor at 1 ,ug/ml they undergo a 16-fold increase in number, with graded responses to lower concentrations of the factor.In the presence ofinsulin at 10 ,g/ml, normal heart mesenchymal cells multiply by a factor of 3 over a 4-day period. The addition of epidermal growth factor (1 ,ug/ml) and insulin (10 ,ug/ml) to cultures of normal chicken heart mesenchymal cells causes these cells to proliferate at a rate comparable to that of their RSV-infected counterparts.Epidermal growth factor (EGF) is a 6045-dalton polypeptide first isolated by Cohen from the submaxillary glands of adult male mice (1). A human form of EGF has been isolated and is probably identical to f-urogastrone (2-4). EGF is mitogenic for skin and corneal epithelium in vivo and in organ cultures (5). In addition, EGF stimulates the proliferation ofcultured human fibroblasts and mouse cells under conditions of limiting serum concentration (0.1-1.0%) or of limiting cell density (5-7). At hyperphysiological concentrations, insulin stimulates the proliferation of various cultured cells under similar conditions (8,9). Insulin is thought to cause this stimulation by virtue of its general anabolic effect or by activation of somatomedin receptors (8,10).We recently developed a culture system in which sparsely seeded normal chicken heart mesenchymal cells are proliferatively quiescent in the presence of an interstitial concentration of plasma (10%) but their Rous sarcoma virus (RSV)-infected counterparts proliferate at a maximal rate (11,12). Quiescence of the normal heart mesenchymal cells is achieved by heparinization and heat-inactivation of the plasma, a treatment that appears to paralyze a mitogen-generating system present in the plasma (12). The quiescent state achieved by heparinization and inactivation of the plasma component of the culture medium may be quite different from the lack ofproliferation that results from the use of restricted serum concentrations or limiting cell culture densities: The latter experimental tactic involves an element of limitation of nutrient and supportive factors and therefore may not be altogether physiological (13-17). Because sparsely seeded normal chicken heart mesenchymal cells are quiescent in culture medium containing an interstitial concentration of heparinized, inactivated plasma, we have used these cells to study the mitogenic activity of EGF and of insulin. We have used RSV-infected chicken heart mesenchymal cells as a maximal proliferation standard for these studies (11,12). MATERIALS AND METHODSOur basic materials an...

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