Human colony-forming units-erythroid do not require accessory cells, but do require direct interaction with insulin-like growth factor I and/or insulin for erythroid development.
The presence of heterogeneous erythroid progenitor cells, contaminant cells, or serum may alter erythroid colony development in vitro. To obtain highly purified colony-forming units-erythroid (CFU-E), we cultured partially purified human blood burst-forming units-erythroid (BFU-E) in methylcellulose with recombinant human erythropoietin (rHuEPO) for 7 d and generated cells that consisted of 30-60% CFU-E, but no BFU-E. A serum-free medium was used that allowed development of the same number of erythroid colonies as serum containing medium, but with a greater percentage of larger colonies. This medium consisted of delipidated crystalline bovine serum albumin, iron saturated transferrin, lipid suspension, fibrinogen, thrombin, Iscove's modified Dulbecco's medium/F-12[HAM], and insulin plus rHuEPO. When CFU-E were cultured in a limiting dilution assay and the percentage of nonresponder wells was plotted against cell concentration, both serum-free cultures and serum-containing cultures yielded overlapping straight lines through the origin indicating that CFU-E development did not depend on accessory cells and that insulin acted directly on the CFU-E. Human recombinant interleukin 3 (IL-3) and/or granulocyte-macrophage colony-stimulating factor had no effect on CFU-E growth, while they markedly enhanced BFU-E growth. Physiological concentrations of recombinant human insulin-like growth factor I (IGF-I) enhanced CFU-E growth in the absence of insulin and, together with rHuEPO in serum-free medium, provided a plating efficiency equal to that of serum-containing medium. Limiting dilution analysis in serum-free medium with IGF-I showed a straight line through the origin indicating that IGF-I also acted directly on the CFU-E and not through an effect on accessory cells. These data demonstrate that CFU-E do not require accessory cells, but do require IGF-I and/or insulin which act directly on the CFU-E.
Sawada
K
K
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Krantz
S B
SB
Dessypris
E N
EN
Koury
S T
ST
Sawyer
S T
ST
D000328:Adult; D001790:Blood Physiological Phenomena; D002455:Cell Division; D003114:Colony-Forming Units Assay; D003115:Colony-Stimulating Factors / Q000494:pharmacology; D003470:Culture Media; D004906:Erythrocyte Count; D004912:Erythrocytes / Q000187:drug effects / Q000502:physiology*; D004920:Erythropoiesis / Q000187:drug effects*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000502:physiology*; D006728:Hormones / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000502:physiology*; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D007377:Interleukin-3 / Q000494:pharmacology; D013002:Somatomedins / Q000502:physiology*
D003115:Colony-Stimulating Factors; D003470:Culture Media; D006133:Growth Substances; D006728:Hormones; D007328:Insulin; D007377:Interleukin-3; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D016178:Granulocyte-Macrophage Colony-Stimulating Factor