Molecularly Characterized Factors Governing the Growth of Murine Multipotent Stem Cells in Serum-Depleted Marrow Cultures

  • Francis C. Monette
  • George Siqounas
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 34)


The growth requirements of normal murine marrow-derived multipotent stem cells (CFU-GEMM) in a simple clonal cell culture system substantially devoid of exogenous serum proteins was assessed. The ability of murine interleukin-3 (I1–3), recombinant human erythropoietin (rEPO), and a crystalline preparation of the protoporphyrin hemin to support colony growth in “serum-free” cultures was examined by titration. The results suggest that both I1–3 and hemin are limiting for multipotential colony growth in “serum-free” cultures, but that EPO is not. In addition, the ‘sensitivity’ of CFU-GEMM to each growth factor appeared to increase in the “serum-free” environment as evidenced by a “shift-to-the-left” in all the titration curves. Nearly half of the GEMM colonies grew to full maturity in the absence of exogenous EPO. Given the optimal concentration of each growth factor, high colony growth was consistently observed in the “serum-free” cultures, with a range from 65% to 119% of the serum control level. It is therefore concluded that supplementation of murine marrow cultures with I1–3 and hemin alone may provide the necessary setting for studying the factors which modulate the growth of multipotent stem cells in a serum- free environment.


Colony Growth Multipotent Stem Cell Hematopoietic Growth Factor Colony Development Murine Bone Marrow Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Ihle, J.N., J. Keller, S. Oroszlan, L.E. Henderson, T.D. Copeland, F. Fitch, M.B. Prystowsky, E. Goldwasser, J.W. Schrader, E. Palaszynski, M. Dy, and B. Lebel. 1983. Biologic properties of homogeneous interleukin 3. I. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, P. Cell stimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity. J Immunol. 131: 282–287.PubMedGoogle Scholar
  2. 2.
    Clark-Lewis, I., S.B.H. Kent, and J.W. Schrader. 1984. Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells. J. Biol. Chem. 259: 7488–7494.PubMedGoogle Scholar
  3. 3.
    Jacobs, K., C. Shoemaker, R. Rudersdorf, S.D. Neill, R.J. Kaufman, A. Mufson, J. Seehra, S.S. Jones, R. Hewick, E.F. Rritsch, M. Kawakita, T. Shimizu, and T. Miyake. 1985. Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 313: 806–810.PubMedCrossRefGoogle Scholar
  4. 4.
    Cutler, R.L., D. Metcalf, N.A. Nicola, and G.R. Johnson. 1985. Purification of a multipotential colony-stimulating factor from pokeweed mitogen-stimulated mouse speen cell conditioned medium. J. Biol. Chem. 260: 6579–6587.PubMedGoogle Scholar
  5. 5.
    Hapel, A.J., M.C. Fung, R.M. Johnson, I.G. Young, G. Johnson, and D. Metcalf. 1985. Biologic properties of molecularly cloned and expressed murine interleukin-3. Blood. 65: 1453–1459.PubMedGoogle Scholar
  6. 6.
    Suda, J., T. Suda, K. Kubota, J.N. Ihle, M. Saito, and Y. Miura. 1986. Purified interleukin-3 and erythropoietin support the terminal differentiation of hemopoietic progenitors in serum-free culture. Blood 67: 1002–1006.PubMedGoogle Scholar
  7. 7.
    Iscove, N.N., L.J. Guilbert, and C. Weyman. 1980. Complete replacement of serum in primary cultures of erythropoietin- dependent red cell precursors (CFU-E) by albumin, transferrin, iron, unsaturated fafcfcy acid, lecithin and cholesterol. Exptl. Cell Res. 126: 121–126.PubMedCrossRefGoogle Scholar
  8. 8.
    Stewart, S., B. Zhu, and A. Axelrad. 1984. A “serum-free” medium for the production of erythropoietic bursts by murine bone marrow cells. Exptl. Hematol. 12: 309–318.Google Scholar
  9. 9.
    Goodman, J.W., E.A. Hall, K.L. Miller, and S.G. Shinpock. 1985. Interleukin 3 promotes erythroid burst formation in “serum-free” cultures without detectable erythropoietin. Nat. Acad. Sci. Proced. USA 82: 3291–3295.CrossRefGoogle Scholar
  10. 10.
    Cormier, F., P. Baines, N. Lucien, and G.A. Boffa. 1985. Complete replacement of serum in cultures of murine primitive erythroid and multipotential progenitor cells: absolute requirement for spleen conditioned medium. Cell Differen. 17: 261–269.CrossRefGoogle Scholar
  11. 11.
    Dainiak, N., S. Kreczko, A. Cohen, R. Pannel, and J. Lawler. 1985. Primary human marrow cultures for erythroid bursts in a serum-substituted system. Exptl. Hematol. 13: 1073–1079.Google Scholar
  12. 12.
    Mliason, J.F., and N. Odartchenko. 1985. Colony formation by primitive hemopoietic progenitor cells in serum-free medium. Natl. Acad. Sci. Proced.USA 82: 775–779.CrossRefGoogle Scholar
  13. 13.
    Koike, K., E.R. Stanley, J.N. Ihle, and M. Ogawa. 1986. Macrophage colony formation supported by purified CSF-1 and/or interleukin 3 in serum-free culture: Evidence for hierarchical difference in macrophage colony-forming cells. Blood. 67: 859–864.PubMedGoogle Scholar
  14. 14.
    Konwalinka, G., D. Geissler, C. Peschel, C. Breier, K. Grunewald, R. Odavic, and H. Braunsteiner. 1986. Human erythropoiesis in vitro and the source of burst-promoting activity in a serum — free system. Exptl. Hematol. 14: 899–903.Google Scholar
  15. 15.
    Dainiak, N. 1985. Role of defined and undefined serum additives to hematopoietic stem cell culture. Cronkite, E.P., N. Dainiak, R.P. McCaffery, P.J. Quesenberry (eds.), Alan R. Liss Inc., Hematopoietic Stem Cell Physiology, New York, p. 59–76.Google Scholar
  16. 16.
    Metcalf, D., G.R. Johnson, and T.E. Mandel. 1979. Colony formation in agar by multipotential hemopoietic cells. J. Cell. Physiol. 98: 401–420.PubMedCrossRefGoogle Scholar
  17. 17.
    Fauser, A.A., and H.A. Messner. 1979. Identification of megakaryocytes macrophages, and eosinophils in colonies of human bone marrow containing neutrophilic granulocytes and erythroblasts. Blood. 53: 1023–1027.PubMedGoogle Scholar
  18. 18.
    Hara, H., and K. Noguchi. 1981. Clonal Nature of pluripotent hemopoietic precursors in vitro. Stem. Cells. 1: 53–60.PubMedGoogle Scholar
  19. 19.
    Spivak, J.L., R.R.L. Smith, and J.N. Ihle. 1985. Interleukin 3 promotes the in vitro proliferation of murine pluripotent hematopoietic stem cells. J. Clin. Invest. 76: 1613–1621.PubMedCrossRefGoogle Scholar
  20. 20.
    Migliaccio, A.R., and J.W.M. Visser. 1986. Proliferation of purified murine hemopoietic stem cells in serum-free cultures stimulated with purified stem cell-activating factor. Exptl. Hematol. 14: 1043–1048.Google Scholar
  21. 21.
    Suda, T., J. Suda, M. Ogawa, and J.N. Ihle. 1985. Permissive role of interleukin 3 (IL-3) in proliferation and differentiation of multipotential hemopoietic progenitors in culture. J. Cell. Physiol. 124: 182–190.PubMedCrossRefGoogle Scholar
  22. 22.
    Iscove, N.N., C.A. Roitsch, N. Williams, and L.J. Guilbert. 1982. Molecules stimulating early red cell, granulocyte, macrophage, and megakaryocyte precursors in culture: Similarity in size, hydrophobicity and charge. J. Cell. Physiol. (Suppl. 1 ): 65–78.CrossRefGoogle Scholar
  23. 23.
    Metcalf, D. 1985. The granulocyte-macrophage colony-stimulating factors. Science. 229: 16–22.PubMedCrossRefGoogle Scholar
  24. 24.
    Broxmeyer, H.E., D.E. Williams, G. Hangoc, S. Cooper, S. Gillis, R.K. Shadduck, and D.C. Bicknell. 1987. Synergistic myelopoietic actions in vivo after administration to mice of combinations of purified natural murine colony-stimulating factor 1, recombinant murine interleukin 3, and recombinant murine granulocyte/macrophage colony-stimulating factor. Proc. Natl. Acad. Sci. USA 84: 3871–3875.PubMedCrossRefGoogle Scholar
  25. 25.
    Monette, F.C., and S.A. Holden. 1982. Hemin enhances the in vitro growth of primitive erythroid progenitor cells. Blood 60: 527–530.PubMedGoogle Scholar
  26. 26.
    Monette, F.C. and G. Sigounas. 1984. Factors affecting the proliferation and differentiation of clonogenic hematopoietic stem cells in vitro. Blood Cells 10: 261–286.PubMedGoogle Scholar
  27. 27.
    Lu, L., H.E. Broxmeyer, P.A. Meyers, M.A.S. Moore, and H.T. Thaler. 1983. Association of cell cycle expression of la-like antigenic determinants on normal human multipotential (CFU-GEMM) and erythroid (BFU-E) progenitor cells with regulation in vitro by acidic isoferritins. Blood. 61: 250–256.PubMedGoogle Scholar
  28. 28.
    Porter, P.N., R.H. Meintz, and K. Mesner. 1979. Enhancement of erythroid colony growth in culture by hemin. Exptl. Hematol. 7: 11–16.Google Scholar
  29. 29.
    Ibrahim, N.G., J.D. Lutton, and R.D. Levere. 1982. The role of haem biosynthetic and degradative enzymes in erythroid colony development: The effect of haemin. Brit. J. Haematol. 50: 17–28.CrossRefGoogle Scholar
  30. 30.
    Rothmann, J., C.F. Hertogs, Z. Malik, and D.H. Pluznik. 1983. Hemin stimulating effect on colony formation of leukemic and bone marrow cells. Exptl. Hematol. 11: 147–153.Google Scholar
  31. 31.
    Holden, S.A., H.N. Steinberg, E.A. Matzinger, and F.C. Monette. 1983. Further characterization of the hemin-induced enhancement of primitive erythroid progenitor cell growth in vitro. Exptl. Hematol. 11: 953–960.Google Scholar
  32. 32.
    Ross, J., and D. Sautner. 1978. Induction of globin mRNA accumulation by hemin in extracts of Friend erythroleukemia cells. J. Biol. Chem. 253: 7124–7126.Google Scholar
  33. 33.
    Mager, D., and A. Bernstein. 1979. The role of heme in the regulation of the late program of Friend cell erythroid differentiation. J. Cell. Physiol. 100: 467–479.PubMedCrossRefGoogle Scholar
  34. 34.
    Gross, M. 1979. Control of protein synthesis by hemin: Evidence that the hemin-controlled translational repressor inhibits formation of 80S initiation complexes from 48S intermediate initiation complexes. J. Biol. Chem. 254: 2370–2377.PubMedGoogle Scholar
  35. 35.
    Deutsch, J., and O.O. Blumenfield. 1977. Effect of hemin, sodium periodate and concanavalin A on in vitro biosynthesis of rabbit reticulocyte membrane proteins. Biochem. Biophys. Res. Commun. 79: 1224–1230.PubMedCrossRefGoogle Scholar
  36. 36.
    Fuhr, J.E., E.G. Bamberger, C.B. Lozzio, and B.B. Lozzio. 1981. Induction of hemoglobin synthesis in original K562 cell line. Blood Cells. 7: 389–395.PubMedGoogle Scholar
  37. 37.
    Charnay, P., and T. Maniatis. 1983. Transcriptional regulation of globin gene expression in the human erythroid cell line K562. Science 220: 1281–1283.PubMedCrossRefGoogle Scholar
  38. 38.
    Bonanou-Tzedaki, S.A., M. Sohi, and H.R.V. Arnstein. 1981. Regulation of erythroid Cell Differentiation by haemin. Cell Differen. 10: 267–279.CrossRefGoogle Scholar
  39. 39.
    Viola, L., R. Biagini, R. Barbieri, and R. Gambari. 1987. Inhibition of hemoglobin accumulation by monoclonal antibodies to the human transferrin receptor is reversed by hemin. Expt. Hematol. 15: 1145–1152.Google Scholar
  40. 40.
    Galbraith, R.A., S. Sassa, and A. Kappas. 1985. Heme binding to murine erythroleukemia cells. J. Biol. Chem. 260: 12198–12202.PubMedGoogle Scholar
  41. 41.
    Rothmann, J., C. F. Hertogs, and D.A. Pluznik. 1977. Replacement of serum by hemolysate as growth promoter for murine leukemic and normal hemopoietic progenitor cells in culture. Exptl. Hematol. 5: 117–124.Google Scholar
  42. 42.
    Chen, J.J. and I.M. London. 1981. Hemin enhances the differentiation of mouse 3T3 cells to adipocytes. Cell. 26: 117–122.PubMedCrossRefGoogle Scholar
  43. 43.
    Stenzel, K.H., A.L. Rubin, and A. Novogradsky. 1981. Mitogenic and comitogenic properties of hemin. J. Immunol. 127: 2469–2473.PubMedGoogle Scholar
  44. 44.
    Mslik, Z., D. Creter, A. Cohen, and M. Djaldetti. 1983. Haemin affects platelet aggregation and lymphocyte mitogenicity in whole blood incubations. Cytobios. 38: 33–38.Google Scholar
  45. 45.
    Honette, F.C., and G. Sigounas. 1987. Sensitivity of murine multipotential stem cell colony (CFU-GEMM) growth to interleukin- 3, erythropoietin, and hemin. Exptl. Hematol. 15: 729–734.Google Scholar
  46. 46.
    Monette, F.C., and G. Sigounas. 1988. Growth of murine multipotent stem cells in a simple “serum-free” culture system: Role of interleukin-3, erythropoietin, and hemin. Exptl. Hematol. 16: (In Press).Google Scholar
  47. 47.
    McLeod, D.L., M.M. Shreeve, and A.A. Axelrad. 1974. Improved plasma culture system for production of erythrocytic colonies in vitro: quantitative assay method for CFUe. Blood. 44: 517–534.PubMedGoogle Scholar
  48. 48.
    Iscove, N.N., and F. Sieber. 1975. Erythroid progenitors in mouse bone marrow detected by macroscopic colony formation in culture. Exptl. Hematol. 3: 32–43.Google Scholar
  49. 49.
    Monette, F.C., and G. Sigounas. Hemin Acts synergistically with interleukin-3 to promote the growth of multipotent stem cells (CFU-GEMM) in “serum-free” cultures of normal murine bone marrow. (Submitted)Google Scholar
  50. 50.
    Broxmeyer, H.E., D.E. Williams, G. Hangoc, S. Cooper, S. Gillis, R.K. Shadduck, and D.C. Bicknell. 1987. Synergistic myelopoietic action in vivo after administration to mice of combinations of purified natural murine colony-stimulating factor 1, recombinant murine interleukin 3, and recombinant murine granulocyte/macro-phage colony-stimulating factor. Proc. Natl. Acad. Sci. (USA). 84: 3871–3875.CrossRefGoogle Scholar
  51. 51.
    Chen, B.E.-M., and C.R. Clark. 1986. Interleukin 3 (11–3) regulates the in vitro proliferation of both blood monocytes and peritoneal exudate macrophages: synergism between a macrophage lineage-specific colony-stimulating factor (CSF-1) and II 3. J. Immunol. 137: 563–570.PubMedGoogle Scholar
  52. 52.
    Schmitt, E., B. Fassbender, K. Beyreuther, E. Spaeth, R. Schwarzkopf, and E. Rude. 1987. Characterization of a T-cell-derived lymphokine that acts synergistically with IL 3 on the growth of murine mast cells and is identical with IL 4. Immunobio1. 174: 406–419.CrossRefGoogle Scholar
  53. 53.
    Nicola, N.A., and D. Metcalf. 1986. Binding of iodinated multipotential colony-stimulating factor (Interleukin-3) to murine bone marrow cells. J. Cell Physiol. 128: 180–188.PubMedCrossRefGoogle Scholar
  54. 54.
    Majuri, R., and R. Grasbeck. 1987. A rosette receptor assay with haem-microbeads. Demonstration of a haem receptor on K562 cells. Eur. J. Haematol. 38: 21–25.PubMedCrossRefGoogle Scholar
  55. 55.
    Taketani, S., H. Kohno, and R. Tokunaga. 1987. Cell surface receptor for hemopexin in human leukemia HL60 cells. Specific binding, affinity labeling, and fate of heme. J. Biol. Chem. 262: 4639–4643.PubMedGoogle Scholar
  56. 56.
    Monette, F.C., S.A. Holden, M.J. Sheehy, and E.A. Matzinger. 1984. Specificity of hemin action in vivo at early stages of hematopoietic Cell Differentiation. Exptl. Hematol. 12: 782–787.Google Scholar
  57. 57.
    Monette, F.C., and S.A. Holden. 1982. Hemin enhancement of primitive erythroid progenitors in vitro: Relationship to burst-- promoting activity (BPA). Exptl. Hematol. 10: (Suppl. 12): 281–294.Google Scholar

Copyright information

© Plenum Press, New York 1988

Authors and Affiliations

  • Francis C. Monette
    • 1
  • George Siqounas
    • 1
  1. 1.Department of BiologyBoston UniversityBostonUSA

Personalised recommendations