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. 1989 May;83(5):1701–1709. doi: 10.1172/JCI114070

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.

K Sawada 1, S B Krantz 1, E N Dessypris 1, S T Koury 1, S T Sawyer 1
PMCID: PMC303879  PMID: 2651478

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akahane K., Tojo A., Urabe A., Takaku F. Pure erythropoietic colony and burst formations in serum-free culture and their enhancement by insulin-like growth factor I. Exp Hematol. 1987 Aug;15(7):797–802. [PubMed] [Google Scholar]
  2. Bersch N., Groopman J. E., Golde D. W. Natural and biosynthetic insulin stimulates the growth of human erythroid progenitors in vitro. J Clin Endocrinol Metab. 1982 Dec;55(6):1209–1211. doi: 10.1210/jcem-55-6-1209. [DOI] [PubMed] [Google Scholar]
  3. Casadevall N., Vainchenker W., Lacombe C., Vinci G., Chapman J., Breton-Gorius J., Varet B. Erythroid progenitors in polycythemia vera: demonstration of their hypersensitivity to erythropoietin using serum free cultures. Blood. 1982 Feb;59(2):447–451. [PubMed] [Google Scholar]
  4. Clarke B. J., Housman D. Characterization of an erythroid precursor cell of high proliferative capacity in normal human peripheral blood. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1105–1109. doi: 10.1073/pnas.74.3.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Claustres M., Chatelain P., Sultan C. Insulin-like growth factor I stimulates human erythroid colony formation in vitro. J Clin Endocrinol Metab. 1987 Jul;65(1):78–82. doi: 10.1210/jcem-65-1-78. [DOI] [PubMed] [Google Scholar]
  6. Dainiak N., Davies G., Kalmanti M., Lawler J., Kulkarni V. Platelet-derived growth factor promotes proliferation of erythropoietic progenitor cells in vitro. J Clin Invest. 1983 May;71(5):1206–1214. doi: 10.1172/JCI110869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dainiak N., Hoffman R., Ritchey A. K., Floyd V., Callahan M. In vitro steroid sensitivity testing: a possible means to predict response to therapy in primary hypoproliferative anemia. Am J Hematol. 1980;9(4):401–412. doi: 10.1002/ajh.2830090407. [DOI] [PubMed] [Google Scholar]
  8. Dainiak N., Kreczko S. Interactions of insulin, insulinlike growth factor II, and platelet-derived growth factor in erythropoietic culture. J Clin Invest. 1985 Sep;76(3):1237–1242. doi: 10.1172/JCI112079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dainiak N., Sutter D., Kreczko S. L-triiodothyronine augments erythropoietic growth factor release from peripheral blood and bone marrow leukocytes. Blood. 1986 Dec;68(6):1289–1297. [PubMed] [Google Scholar]
  10. Eaves A. C., Eaves C. J. Erythropoiesis in culture. Clin Haematol. 1984 Jun;13(2):371–391. [PubMed] [Google Scholar]
  11. GLICK S. M., ROTH J., YALOW R. S., BERSON S. A. IMMUNOASSAY OF HUMAN GROWTH HORMONE IN PLASMA. Nature. 1963 Aug 24;199:784–787. doi: 10.1038/199784a0. [DOI] [PubMed] [Google Scholar]
  12. Golde D. W., Bersch N., Chopra I. J., Cline M. J. Thyroid hormones stimulate erythropoiesis in vitro. Br J Haematol. 1977 Oct;37(2):173–177. doi: 10.1111/j.1365-2141.1977.tb06833.x. [DOI] [PubMed] [Google Scholar]
  13. Golde D. W., Bersch N., Li C. H. Growth hormone: species-specific stimulation of erythropoiesis in vitro. Science. 1977 Jun 3;196(4294):1112–1113. doi: 10.1126/science.870971. [DOI] [PubMed] [Google Scholar]
  14. Golde D. W., Cline M. J. Identification of the colony-stimulating cell in human peripheral blood. J Clin Invest. 1972 Nov;51(11):2981–2983. doi: 10.1172/JCI107124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Iscove N. N., Guilbert L. J., Weyman C. Complete replacement of serum in primary cultures of erythropoietin-dependent red cell precursors (CFU-E) by albumin, transferrin, iron, unsaturated fatty acid, lecithin and cholesterol. Exp Cell Res. 1980 Mar;126(1):121–126. doi: 10.1016/0014-4827(80)90476-0. [DOI] [PubMed] [Google Scholar]
  16. Iscove N. N., Sieber F., Winterhalter K. H. Erythroid colony formation in cultures of mouse and human bone marrow: analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agarose-concanavalin A. J Cell Physiol. 1974 Apr;83(2):309–320. doi: 10.1002/jcp.1040830218. [DOI] [PubMed] [Google Scholar]
  17. Kaushansky K., Lin N., Adamson J. W. Interleukin 1 stimulates fibroblasts to synthesize granulocyte-macrophage and granulocyte colony-stimulating factors. Mechanism for the hematopoietic response to inflammation. J Clin Invest. 1988 Jan;81(1):92–97. doi: 10.1172/JCI113316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. King D. J., Koekebakker M., Barr R. D. Modulation of human erythropoiesis by hydrocortisone in vitro. Eur J Haematol. 1987 Feb;38(2):137–140. doi: 10.1111/j.1600-0609.1987.tb01151.x. [DOI] [PubMed] [Google Scholar]
  19. Kurtz A., Jelkmann W., Bauer C. A new candidate for the regulation of erythropoiesis. Insulin-like growth factor I. FEBS Lett. 1982 Nov 22;149(1):105–108. doi: 10.1016/0014-5793(82)81081-8. [DOI] [PubMed] [Google Scholar]
  20. Kurtz A., Jelkmann W., Bauer C. Insulin stimulates erythroid colony formation independently of erythropoietin. Br J Haematol. 1983 Feb;53(2):311–316. doi: 10.1111/j.1365-2141.1983.tb02025.x. [DOI] [PubMed] [Google Scholar]
  21. Lindemann A., Riedel D., Oster W., Meuer S. C., Blohm D., Mertelsmann R. H., Herrmann F. Granulocyte/macrophage colony-stimulating factor induces interleukin 1 production by human polymorphonuclear neutrophils. J Immunol. 1988 Feb 1;140(3):837–839. [PubMed] [Google Scholar]
  22. McLeod D. L., Shreeve M. M., Axelrad A. A. Improved plasma culture system for production of erythrocytic colonies in vitro: quantitative assay method for CFU-E. Blood. 1974 Oct;44(4):517–534. [PubMed] [Google Scholar]
  23. Morell B., Froesch E. R. Fibroblasts as an experimental tool in metabolic and hormone studies. II. Effects of insulin and nonsuppressible insulin-like activity (NSILA-S) on fibroblasts in culture. Eur J Clin Invest. 1973 Mar;3(2):119–123. doi: 10.1111/j.1365-2362.1973.tb00338.x. [DOI] [PubMed] [Google Scholar]
  24. Nathan D. G., Chess L., Hillman D. G., Clarke B., Breard J., Merler E., Housman D. E. Human erythroid burst-forming unit: T-cell requirement for proliferation in vitro. J Exp Med. 1978 Feb 1;147(2):324–339. doi: 10.1084/jem.147.2.324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ogawa M., MacEachern M. D., Avila L. Human marrow erythropoiesis in culture: II. Heterogeneity in the morphology, time course of colony formation, and sedimentation velocities of the colony-forming cells. Am J Hematol. 1977;3:29–36. doi: 10.1002/ajh.2830030104. [DOI] [PubMed] [Google Scholar]
  26. Perrine S. P., Greene M. F., Lee P. D., Cohen R. A., Faller D. V. Insulin stimulates cord blood erythroid progenitor growth: evidence for an aetiological role in neonatal polycythaemia. Br J Haematol. 1986 Nov;64(3):503–511. doi: 10.1111/j.1365-2141.1986.tb02206.x. [DOI] [PubMed] [Google Scholar]
  27. Rechler M. M., Zapf J., Nissley S. P., Froesch E. R., Moses A. C., Podskalny J. M., Schilling E. E., Humbel R. E. Interactions of insulin-like growth factors I and II and multiplication-stimulating activity with receptors and serum carrier proteins. Endocrinology. 1980 Nov;107(5):1451–1459. doi: 10.1210/endo-107-5-1451. [DOI] [PubMed] [Google Scholar]
  28. Sawada K., Krantz S. B., Kans J. S., Dessypris E. N., Sawyer S., Glick A. D., Civin C. I. Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin. J Clin Invest. 1987 Aug;80(2):357–366. doi: 10.1172/JCI113080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schmid C., Steiner T., Froesch E. R. Insulin-like growth factors stimulate synthesis of nucleic acids and glycogen in cultured calvaria cells. Calcif Tissue Int. 1983 Jul;35(4-5):578–585. doi: 10.1007/BF02405097. [DOI] [PubMed] [Google Scholar]
  30. Schmid C., Steiner T., Froesch E. R. Preferential enhancement of myoblast differentiation by insulin-like growth factors (IGF I and IGF II) in primary cultures of chicken embryonic cells. FEBS Lett. 1983 Sep 5;161(1):117–121. doi: 10.1016/0014-5793(83)80742-x. [DOI] [PubMed] [Google Scholar]
  31. Schoenle E., Zapf J., Humbel R. E., Froesch E. R. Insulin-like growth factor I stimulates growth in hypophysectomized rats. Nature. 1982 Mar 18;296(5854):252–253. doi: 10.1038/296252a0. [DOI] [PubMed] [Google Scholar]
  32. Stewart S., Zhu B., Axelrad A. A "serum-free" medium for the production of erythropoietic bursts by murine bone marrow cells. Exp Hematol. 1984 Jun;12(5):309–318. [PubMed] [Google Scholar]
  33. Zapf J., Froesch E. R., Humbel R. E. The insulin-like growth factors (IGF) of human serum: chemical and biological characterization and aspects of their possible physiological role. Curr Top Cell Regul. 1981;19:257–309. doi: 10.1016/b978-0-12-152819-5.50024-5. [DOI] [PubMed] [Google Scholar]
  34. Zapf J., Schoenle E., Froesch E. R. Insulin-like growth factors I and II: some biological actions and receptor binding characteristics of two purified constituents of nonsuppressible insulin-like activity of human serum. Eur J Biochem. 1978 Jun 15;87(2):285–296. doi: 10.1111/j.1432-1033.1978.tb12377.x. [DOI] [PubMed] [Google Scholar]
  35. Zapf J., Walter H., Froesch E. R. Radioimmunological determination of insulinlike growth factors I and II in normal subjects and in patients with growth disorders and extrapancreatic tumor hypoglycemia. J Clin Invest. 1981 Nov;68(5):1321–1330. doi: 10.1172/JCI110379. [DOI] [PMC free article] [PubMed] [Google Scholar]

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