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Expansion of Primitive Human Hematopoietic Progenitors in a Perfusion Bioreactor System with IL-3, IL-6, and Stem Cell Factor

Bio/Technologyvolume 11pages358363 (1993) | Download Citation

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Abstract

Present methods for long-term hematopoietic culture (LTHC) employ a static culture environment which is not well-characterized. Primitive long-term culture-initiating cell (LTC-IC) numbers have been shown to decline in conventional static human LTHC, even with exogenous cytokine combinations. We have expanded human hematopoietic cells from umbilical cord blood on a preformed marrow stroma with synergistic cytokine combinations in a novel perfusion bioreactor system, which continuously maintained culture conditions within desired ranges. Interleukin-3 (IL-3) and interIeukin-6 (IL-6) in perfusion culture resulted in rapid 7-day expansion of granulocyte-macrophage colony forming units (CFU-GM, 11-fold), erythroid burst-forming units (BFU-E, 2.5-fold), and granulocyte-erythroid-macrophage colony forming units (CFU-Mix, 24-fold), compared to 6-fold, 1.4-fold, and no expansion, respectively, in static cultures. Addition of stem cell factor (SCF) to IL-3/IL-6 in static culture increased the extent of CFU-GM expansion (to 9-fold), but did not result in BFU-E or CFU-Mix expansion. In perfusion cultures with IL-3/IL-6/SCF, much greater expansions of CFU-GM (18-fold) and CFU-Mix (5.3-fold) were obtained. More importantly, expansion of LTC-IC (nearly 3-fold in two of three experiments) was only obtained with IL-3/IL-6/SCF and perfusion. The ability to expand hematopoietic cells while maintaining or expanding primitive progenitors has potential clinical applications in bone marrow transplantation and gene therapy.

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References

  1. 1

    Barlogie, B. and Gahrton, G. 1991. Bone marrow transplantation in multiple myeloma. Bone Marrow Transplant. 7: 71–79.

  2. 2

    Gale, R.P., Armitage, J.O. and Dicke, K.A. 1991. Autotransplants: Now and in the future. Bone Marrow Transplant. 7: 153–157.

  3. 3

    Chang, J., Morgenstern, G.R., Coutinho, L.H., Scarffe, J.H., Carr, T., Deakin, D.P., Testa, N.G. and Dexter, T.M. 1989. The use of bone marrow cells grown in long-term culture for autologous bone marrow transplantation in acute myeloid leukaemia: An update. Bone Marrow Transplant. 4: 5–9.

  4. 4

    Santos, G.W. and Colvin, O.M. 1986. Pharmacological purging of bone marrow with reference to autografting. Clin. Haematol. 15: 67–83.

  5. 5

    Dexter, T.M., Spooncer, E., Simmons, P. and Allen, T.D. 1984. Long-term marrow culture: An overview of techniques and experience, p. 57–96. In: Long Term Bone Marrow Culture. Wright, D. G. and Greenberger, J. S. (Eds.). Liss, New York.

  6. 6

    Sutherland, H.J., Eaves, C.J., Lansdorp, P.M., Thacker, J.D. and Hogge, D.E. 1991. Differential regulation of primitive human hematopoietic stem vells in long-term cultures maintained on genetically engineered murine stromal cells. Blood 78: 666–672.

  7. 7

    Schwartz, R.M., Palsson, B.O. and Emerson, S.G. 1991. Rapid medium perfusion rate significantly increases the productivity and longevity of human bone marrow cultures. Proc. Natl. Acad. Sci. USA 88: 6760–6764.

  8. 8

    Koller, M.R., Bender, J.G., Papoutsakis, E.T. and Miller, W.M. 1992. Beneficial effects of reduced oxygen tension and perfusion in long-term hematopoietic cultures. Ann. N. Y. Acad. Sci. 665: 105–116.

  9. 9

    Koller, M.R., Bender, J.G., Papoutsakis, E.T. and Miller, W.M. 1992. Effects of synergistic cytokine combinations, low oxygen, and irradiated stroma on the expansion of human cord blood progenitors. Blood 80: 403–411.

  10. 10

    Bernstein, I.D., Andrews, R.G. and Zsebo, K.M. 1991. Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+ lin-cells, and the generation of colony-forming cell progeny from CD34+lin-cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor. Blood 77: 2316–2321.

  11. 11

    Cashman, J., Eaves, A.C. and Eaves, C.J. 1985. Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures. Blood 66: 1002–1005.

  12. 12

    Cashman, J.D., Eaves, A.C., Raines, E.W., Ross, R. and Eaves, C.J. 1990. Mechanisms that regulate the cell cycle status of very primitive hematopoietic Cells in long-term human marrow cultures. I. Stimulatory role of a variety of mesenchymal cell activators and inhibitory role of TGF-β. Blood 75: 96–101.

  13. 13

    Caldwell, J., Palsson, B.O., Locey, B. and Emerson, S.G. 1991. Culture perfusion schedules influence the metabolic activity and granulocyte-macrophage colony-stimulating factor production rates of human bone marrow stromal cells. J. Cell. Physiol. 147: 344–353.

  14. 14

    Bjornson, B.H., Pincus, S.H., DiNapoli, A.M. and Desforges, J.F. 1984. Inhibition of CFU-NM and CFU-EOS by mature granulocytes. Blood 63: 376–379.

  15. 15

    Schwartz, R.M., Emerson, S.G., Clarke, M.F. and Palsson, B.O. 1991. In vitro myelopoiesis stimulated by rapid medium exchange and supplementation with hematopoietic growth factors. Blood 78: 3155–3161.

  16. 16

    Koller, M.R., Bender, J.G., Miller, W.M. and Papoutsakis, E.T. 1992. Reduced oxygen tension increases hematopoiesis in long-term culture of human stem and progenitor cells from cord blood and bone marrow. Exp. Hematol. 20: 264–270.

  17. 17

    Verfaillie, C., Blakolmer, K. and McGlave, P. 1990. Purified primitive human hematopoietic progenitor cells with long-term in vitro repopulating capacity adhere selectively to irradiated bone marrow stroma. J. Exp. Med. 172: 509–520.

  18. 18

    Gordon, M.Y., Clarke, D., Atkinson, J. and Greaves, M.F. 1990. Hemopoietic progenitor cell binding to the stromal microenvironment in vitro. Exp. Hematol. 18: 837–842.

  19. 19

    Liesveld, J.L., Abboud, C.N., Duesrst, R.E., Ryan, D.H., Brennan, J.K. and Lichtman, M.A. 1989. Characterization of human marrow stromal cells:Role in progenitor cell binding and granulopoiesis. Blood 73: 1794–1800.

  20. 20

    Sutherland, H.J., Lansdorp, P.M., Henkelman, D.H., Eaves, A.C. and Eaves, C.J. 1990. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc. Natl. Acad. Sci. USA 87: 3584–3588.

  21. 21

    Moore, M.A.S. 1991. Clinical implications of positive and negative hematopoietic stem cell regulators. Blood 78: 1–19.

  22. 22

    Eaves, C.J., Cashman, J.D. and Eaves, A.C. 1991. Methodology of long-term culture of human hemopoietic cells. J. Tiss. Cult. Meth. 13: 55–62.

  23. 23

    Ploemacher, R.E., vander Sluijs, J.P., van Beurden, C.A.J., Baert, M.R.M. and Chan, P.L. 1991. Use of limiting dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse. Blood 78: 2527–2533.

  24. 24

    Dexter, T.M., Allen, T.D. and Lajtha, L.G. 1976. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J. Cell Physiol. 91: 335–344.

  25. 25

    Bodine, D.M., Karlsson, S. and Nienhuis, A.W. 1989. Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 86: 8897–8901.

  26. 26

    Carow, C.E., Hangoc, G., Cooper, S.H., Williams, D.E. and Broxmeyer, H.E. 1991. Mast cell growth factor (c-kit ligand) supports the growth of human multipotential progenitor cells with a high replating potential. Blood 78: 2216–2221.

  27. 27

    Koller, M.R. and Palsson, B.O. 1993. Tissue engineering: Reconstitution of human hematopoiesis ex vivo. Biotechnol. Bioeng. In press

  28. 28

    Cherry, R.S. and Papoutsakis, E.T. 1990. Understanding and controlling fluid-mechanical injury of animal cells in bioreactors, p. 71–121. In: Animal cell Biotechnology, Vol. 4. Spier, R. and Griffiths, J.B. (Eds.). Academic Press, San Diego, CA.

  29. 29

    Zsebo, K.M., Wypych, J., McNiece, I.K, Lu, H.S., Smith, K.A., Karkare, S.B., Sachdev, R.K., Yuschenkoff, V.N., Birkett, N.C., Williams, L.R., Satyagal, V.N., Tung, W., Bosselman, R.A., Mendiaz, E.A. and Langley, K.E. 1990. Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver-conditioned medium. Cell 63: 195–201.

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Author information

Author notes

    • Manfred R. Koller

    Present address: Aastrom Biosciences, Inc., P.O. Box 376, Ann Arbor, MI, 48106

  1. Eleftherios T. Papoutsakis: Corresponding author.

Affiliations

  1. Department of Chemical Engineering, Northwestern University, Evanston, IL, 60208-3120

    • Manfred R. Koller
    • , William M. Miller
    •  & Eleftherios T. Papoutsakis
  2. Applied Sciences, Baxter Healthcare Corporation, Round Lake, IL, 60073

    • Manfred R. Koller
    •  & James G. Bender

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DOI

https://doi.org/10.1038/nbt0393-358

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