Korean Chemical Engineering Research

The Korean Institute of Chemical Engineers (한국화학공학회)
권호 표지

Ex vivo Expansion of CD34+ Hematopoietic Cells from Cord Blood in Various Culture Environments

다양한 배양 조건에서 제대혈 유래 CD34+ 조혈 세포의 체외 증식

  • Choi, Yong Woon (Department of Bioscience and Biotechnology, Sejong University) ;
  • Oh, Duk Jae (Department of Bioscience and Biotechnology, Sejong University)
  • 최용운 (세종대학교 공과대학 생명공학과) ;
  • 오덕재 (세종대학교 공과대학 생명공학과)
  • Received : 2005.12.08
  • Accepted : 2006.01.06
  • Published : 2006.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

This study compared cell expansion and colony forming ability in human cord blood stem cells cultured ex vivo with two kinds of cytokine combinations, two kinds of media, presence or absence of fetal bovine serum (FBS) and two or three dimensional (2D or 3D) culture environments. Purified $CD34^+$ cells were cultured in the IMDM (Iscove's Modified Dulbecco's Medium) and SFM (Serum Free Medium) containing a cytokine cocktail-I (coc-I) (EPO, GMCSF, SCF, and IL-3) or a cytokine cocktail-II (coc-II) (TPO, G-CSF, SCF, IL-6, and Flt3/Flk-2 ligand) with or without FBS. Generally, higher cellular and clonogenic expansion were observed in the coc-I cytokine condition, compared to coc-II cytokine condition. 3D (Methocult) and 2D (IMDM + coc-I + FBS) conditions gave the greatest cell ($2,258{\pm}456$ fold) and CFU (BFU-E: $652{\pm}19$, CFU-GM: $520{\pm}58$, CFU-GEMM: $339{\pm}100$ fold) expansions, respectively. In aspect of medium, IMDM was better than SFM, except for coc-II condition without FBS. In conclusion, 'IMDM + coc-I + FBS' and 'IMDM + coc-I' were the best CFU expansions on the occasion of all culture conditions. FBS and 2D conditions had affirmative effect on CFU expansion, generally. These data might provide a variety of notions about ex vivo expansion of hematopoietic stem cells.

본 연구에서는 제대혈 유래의 조혈줄기세포를 효과적으로 배양하기 위한 선행 연구로서 세포 배양 환경에 따른 조혈줄기세포 증식능의 변화를 관찰하였다. 제대혈의 단핵구 세포에서 분리한 $CD34^+$ 세포를 성장인자 조성-I(이하, coc-I) (EPO, GM-CSF, SCF, IL-3) 및 성장인자 조성-II(이하, coc-II) (TPO, G-CSF, SCF, IL-6, Flt3/Flk-2 ligand)가 포함되어 있는 IMDM(Iscove's modified Dulbecco's medium) 및 무혈청 배지(serum free media, SFM)에서 배양하였으며, 우태아혈청(FBS)의 첨가 영향, 2차원 및 3차원 배양 후 각 조건에서의 세포 증식 및 콜로니 형성능을 비교하였다. 일반적으로 coc-I에서의 세포 증식 및 콜로니 증식이 coc-II에서보다 높았다. 3차원 배양(methocult)에서는 가장 높은 세포 증식($2,258{\pm}456$배)을 나타냈으며, 같은 조성의 2차원 배양(IMDM + coc-I + FBS)에서는 가장 높은 콜로니 증식(BFU-E: $652{\pm}19$, CFU-GM: $520{\pm}58$, CFU-GEMM: $339{\pm}100$배)이 나타났다. 배지를 기준으로 보면, coc-II 조성에 우태아혈청이 포함되지 않은 경우를 제외한 모든 경우에서 세포 증식 및 콜로니 증식이 무혈청 배지에서보다 IMDM에서 높았다. 결론적으로, 모든 배양 조건 중에서 'IMDM + coc-I + FBS' 및 'IMDM + coc-I'에서 가장 좋은 콜로니 증식을 보였으며, 우태아혈청의 첨가 및 2차원 배양 조건이 콜로니 증식에 더 효과적인 것으로 확인되었다. 본 연구 결과는 앞으로 조혈줄기세포의 체외 증식에 필요한 공정개발이나 생물반응기 설계에 유용한 정보를 제공할 수 있을 것으로 사료된다.

Keywords

References

  1. Zon, L. I., 'Developmental Biology of Hematopoiesis,' Blood, 86(8), 2876-2891(1995)
  2. Hann, I. M., Bodger, M. P. and Hoffbrand, A. V., 'Development of Pluripotent Hematopoietic Progenitor Cells in the Human Fetus,' Blood, 62(1), 118-123(1983)
  3. Linch, D. C., Knott, L. J., Rodeck, C. H. and Huehns, E. R., 'Studies of Circulating Hemopoietic Progenitor Cells in Human Fetal Blood,' Blood, 59(5), 976-979(1982)
  4. Gluckman, E., Broxmeyer, H. A., Auerbach, A. D., Friedman, H. S., Douglas, G. W., Devergie, A., Esperou, H., Thierry, D., Socie, G. and Lehn, P., 'Hematopoietic Reconstitution in a Patient with Fanconi's Anemia by Means of Umbilical-cord Blood from an HLA-identical Sibling,' N Engl J Med, 321(17), 1174- 1178(1989) https://doi.org/10.1056/NEJM198910263211707
  5. Kogler, G., Radke, T. F., Lefort, A., Sensken, S., Fischer, J., Sorg, R.V. and Wernet, P., 'Cytokine Production and Hematopoiesis Supporting Activity of Cord Blood-derived Unrestricted Somatic Stem Cells,' Exp Hematol, 33(5), 573-583(2005) https://doi.org/10.1016/j.exphem.2005.01.012
  6. Ende, M., 'Lymphangiosarcoma. Report of a Case,' Pac Med Surg, 74(2), 80-82(1966)
  7. Kubanek, B., Ferrari, L., Tyler, W. S., Howard, D., Jay, S. and Jr. Stohlman, F., 'Regulation of Erythropoiesis. 23. Dissociation Between Stem Cell and Erythroid Response to Hypoxia,' Blood, 32(4), 586-596(1968)
  8. Jr. Stohlman, F., 'Some Aspects of Erythrokinetics,' Semin Hematol, 4(4), 304-314(1967)
  9. Metcalf, D., 'Control of Granulocytes and Macrophages: Molecular, Cellular, and Clinical Aspects,' Science, 254(5031), 529-533 (1991) https://doi.org/10.1126/science.1948028
  10. Ihle, J. N., 'Interleukin-3 and Hematopoiesis,' Chem Immunol, 51, 65-106(1992) https://doi.org/10.1159/000319080
  11. Leary, A. G., Wong, G. G., Clark, S. C., Smith, A. G. and Ogawa, M., 'Leukemia Inhibitory Factor Differentiation-inhibiting Activity/ human Interleukin for DA Cells Augments Proliferation of Human Hematopoietic Stem Cells,' Blood, 75(10), 1960-1964(1990)
  12. Socinski, M. A., Cannistra, S. A., Elias, A., Antman, K. H., Schnipper, L. and Griffin, J. D., 'Granulocyte-Macrophage Colony Stimulating Factor Expands the Circulating Haemopoietic Progenitor cell Compartment in Man,' Lancet, 1(8596), 1194- 1198(1988)
  13. Migliaccio, G., Migliaccio, A. R. and Adamson, J. W., 'In Vitro Differentiation of Human Granulocyte/macrophage and eRythroid Progenitors: Comparative Analysis of the Influence of Recombinant Human Erythropoietin, G-CSF, GM-CSF, and IL-3 in Serum-supplemented and Serum-deprived Cultures,' Blood, 72(1), 248-256(1988)
  14. Tomonaga, M., Golde, D. W. and Gasson, J. C., 'Biosynthetic (recombinant) Human Granulocyte-macrophage Colony-stimulating Factor: Effect on Normal Bone Marrow and Leukemia Cell Lines,' Blood, 67(1), 31-36(1986)
  15. Clark, S. C. and Kamen, R., 'The Human Hematopoietic Colony- stimulating Factors,' Science, 236(4806), 1229-1237(1987) https://doi.org/10.1126/science.3296190
  16. Ohara, A., Suda, T., Saito, M., Miura, Y., Okabe, T. and Takaku, F., 'Effect of Recombinant Human Granulocyte Colony-stimulating Factor on Hemopoietic Cells in Serum-free Culture,' Exp Hematol, 15(6), 695-699(1987)
  17. de Sauvage, F. J., Hass, P. E., Spencer, S. D., Malloy, B. E., Gurney, A. L., Spencer, S. A., Darbonne, W. C., Henzel, W. J., Wong, S. C., Kuang, W. J., Karl, J. O., Bruce, H., Lawrence, A. S., Jr., David, V. G. and Dan, L. E., 'Stimulation of Megakaryocytopoiesis and Thrombopoiesis by the c-Mpl Ligand,' Nature, 369(6481), 533- 538(1994) https://doi.org/10.1038/369533a0
  18. Bartley, T. D., Bogenberger, J., Hunt, P., Li, Y. S., Lu, H. S., Martin, F., Chang, M. S., Samal, B., Nichol, J. L., Swift, S., Johnson, M. J., Hsu, R.-Y., Parker, V. P., Suggs, S., Skrine, J. D., Merewether, L. A., Clogston, C., Hsu, E., Hokom, M. M., Hornkohl, A., Choi, E., Pangelinan, M., Sun, Y., Mar, V., McNinch, J., Simonet, L., Jacobsen, F., Xie, C., Shutter, J., Chute, H., Basu, R., Selander, L., Trollinger, D., Sieu, L., Padilla, D., Trail, G., Elliott, G., Izumi, R., Covey, T., Crouse, J., Garcia, A., Xu, W., Castillo, J. D., Biron, J., Cole, S., Hu, M. C.-T., Pacifici, R., Ponting, I., Saris, C., Wen, D., Yung, Y. P., Lin, H. and Rosselman R. A., 'Identification and Cloning of a Megakaryocyte Growth and Development Factor that is a Ligand for the Cytokine Receptor Mpl,' Cell, 77(7), 1117-1124 (1994) https://doi.org/10.1016/0092-8674(94)90450-2
  19. Hannum, C., Culpepper, J., Campbell, D., McClanahan, T., Zurawski, S., Bazan, J. F., Kastelein, R., Hudak, S., Wagner, J., Mattson, J., Luh, J., Duda, G., Martina, N., Peterson, D., Menon, S., Shanafelt, A., Muench, M., Kelner, G., Namikawa, R., Rennick, D., Roncarolo, M.-G., Zlotnik, A., Rosnet, O., Dubreuil, P., Birnbaum, D. and Lee, F., 'Ligand for FLT3/FLK2 Receptor Tyrosine Kinase Regulates Growth of Haematopoietic Stem Cells and is Encoded by Variant RNAs,' Nature, 368(6472), 643-648 (1994) https://doi.org/10.1038/368643a0
  20. Lyman, S. D., James, L., Vanden Bos, T., de Vries, P., Brasel, K., Gliniak, B., Hollingsworth, L. T., Picha, K. S., McKenna, H. J., Splett, R. R., Fletcher, F. A., Maraskovsky, E., Farrah, T., Foxworthe, D., Williams, D. E. and Beckmann, M. P., 'Molecular Cloning of a Ligand for the flt3/flk-2 Tyrosine Kinase Receptor: a Proliferative Factor for Primitive Hematopoietic Cells,' Cell, 75(6), 1157-1167(1993) https://doi.org/10.1016/0092-8674(93)90325-K
  21. Rennick, D., Jackson, J., Yang, G., Wideman, J., Lee, F. and Hudak, S., 'Interleukin-6 Interacts with Interleukin-4 and Other Hematopoietic Growth Factors to Selectively Enhance the Growth of Megakaryocytic, Erythroid, Myeloid, and Multipotential Progenitor Cells,' Blood, 73(7), 1828-1835(1989)
  22. Hodgkin, P. D., Bond, M. W., O'Garra, A., Frank, G., Lee, F., Coffman, R. L., Zlotnik, A. and Howard, M., 'Identification of IL-6 as a T Cell-derived Factor that Enhances the Proliferative Response of Thymocytes to IL-4 and Phorbol Myristate Acetate,' J. Immunol, 141(1), 151-157(1988)
  23. Caracciolo, D., Clark, S. C. and Rovera, G., 'Human Interleukin- 6 Supports Granulocytic Differentiation of Hematopoietic Progenitor Cells and Acts Synergistically with GM-CSF,' Blood, 73(3), 666-670(1989)
  24. Brandt, J., Briddell, R. A., Srour, E. F., Leemhuis, T. B. and Hoffman, R., 'Role of c-kit Ligand in the Expansion of Human Hematopoietic Progenitor Cells,' Blood, 79(3), 634-641(1992)
  25. Brandt, J. E., Bhalla, K. and Hoffman, R., 'Effects of Interleukin- 3 and c-kit Ligand on the Survival of Various Classes of Human Hematopoietic Progenitor Cells,' Blood, 83(6), 1507-1514(1994)
  26. Lebkowski, J. S., Schain, L. R. and Okarma, T. B., 'Serum-free Culture of Hematopoietic Stem Cells: a Review,' Stem Cells, 13(6), 607-612(1995) https://doi.org/10.1002/stem.5530130605
  27. Daley, J. P., Daley, B. M., Wysocki, M. G., Caligiuri, M. A. and Biddle, W. C., 1996. Ex Vivo Expansion of Human Hematopoietic Progenitor Cells in Serum-free STEMPRO$^{TM}$-34 Medium, pp. 62-67. vol. 18. Life Technologies, Gaithersburg, U. S. A
  28. Piacibello, W., Sanavio, F., Garetto, L., Severino, A., Dane, A., Gammaitoni, L. and Aglietta, M., 'Differential Growth Factor Requirement of Primitive Cord Blood Hematopoietic Stem Cell for Self-renewal and Amplification vs Proliferation and Differentiation,' Leukemia, 12(5), 718-727(1998) https://doi.org/10.1038/sj.leu.2401003
  29. Balducci, E., Azzarello, G., Valenti, M. T., Capuzzo, G. M., Pappagallo, G. L., Pilotti, I., Ausoni, S., Bari, M., Rosetti, F., Sartori, D., Ciappa, A., Porcellini, A. and Vinante, O., 'The Impact of Progenitor Enrichment, Serum, and Cytokines on the Ex Vivo Expansion of Mobilized Peripheral Blood Stem Cells: a Controlled Trial,' Stem Cells, 21(1), 33-40(2003) https://doi.org/10.1634/stemcells.21-1-33
  30. Mobest, D., Mertelsmann, R. and Henschler, R., 'Serum-free Ex Vivo Expansion of CD34(+) Hematopoietic Progenitor Cells,' Biotechnol Bioeng, 60(3), 341-347(1998) https://doi.org/10.1002/(SICI)1097-0290(19981106)60:3<341::AID-BIT10>3.0.CO;2-P
  31. Poloni, A., Giarratana, M. C., Firat, H., Kobari, L., Gorin, N. C. and Douay, L., 'The Ex Vivo Expansion Capacity of Normal Human Bone Marrow Cells is Dependent on Experimental Conditions: Role of the Cell Concentration, Serum and CD34+ Cell Selection in Stroma-free Cultures,' Hematol Cell Ther, 39(2), 49- 58(1997) https://doi.org/10.1007/s00282-997-0049-9
  32. Gilmore, G. L., DePasquale, D. K., Lister, J. and Shadduck, R. K., 'Ex vivo Expansion of Human Umbilical Cord Blood and Peripheral Blood CD34(+) Hematopoietic Stem Cells,' Exp Hematol, 28(11), 1297-1305(2000) https://doi.org/10.1016/S0301-472X(00)00531-2
  33. Shadduck, R. K., Gilmore, G. L. and Lister, J., 'Role of Serumfree Medium in the Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells,' Stem Cells, 18(2), 154-155(2000) https://doi.org/10.1634/stemcells.18-2-154