title: Vitamin C and thiol reagents promote the in vitro growth of murine granulocyte/macrophage progenitor cells by neutralizing endogenous inhibitor(s).
abstract: Growth of murine hemopoietic cells in culture requires the presence of a stimulator of stem cell proliferation, "colony stimulating factor" (CSF). A widely used source of CSF is lung conditioned medium (LCM). We have earlier shown that the great variability of CSF activities in different batches of LCM is due to varying amounts of inhibitor(s). The present study expands the observation that the addition of ascorbic acid to the murine bone marrow soft agar assay system removes the inhibitory activity. The vitamin probably acts as an antioxidant or free radical scavenger, since addition of reduced (but not oxidized) glutathione, cysteine, dithiothreitol or 2-mercaptoethanol to the cultures also inactivates the endogeneous inhibitor. Cysteine and glutathione gave the highest colony numbers, were active at concentrations present in body fluids and did not inhibit colony growth even at concentrations ten times higher than optimum. No synergistic effects could be observed between the different antioxidants. At optimum concentration (usually 0.45 mmol/l) the otherwise bell-shaped dose-response curve for conditioned medium changed to a sigmoid curve. Antioxidants had no growth promoting effect in the absence of CSF. The presence of cysteine or vitamin C revealed CSF-like activity in conditioned media of tissues not considered to be potent producers of such factors. It has been reported that individual batches of foetal calf serum contain different levels of reduced glutathione, and we suggest that one of the batch variable growth regulators in foetal calf serum may be reduced glutathione. The results indicate a possible physiological role of antioxidants in granulopoiesis and suggest that cysteine or reduced glutathione should be freshly added to culture systems assaying CSF and/or granulocyte macrophage progenitor cells.
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology*; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003115:Colony-Stimulating Factors / Q000032:analysis; D003470:Culture Media; D003545:Cysteine / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D005609:Free Radicals; D005978:Glutathione / Q000494:pharmacology*; D006098:Granulocytes; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000187:drug effects*; D008264:Macrophages; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D011506:Proteins / Q000032:analysis
chemical_list: D000975:Antioxidants; D003115:Colony-Stimulating Factors; D003470:Culture Media; D005609:Free Radicals; D011506:Proteins; D008623:Mercaptoethanol; C016313:colony inhibiting factor; D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid; D004229:Dithiothreitol