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. 1994 Dec 6;91(25):12140–12144. doi: 10.1073/pnas.91.25.12140

Establishment of an adherent cell feeder layer from human umbilical cord blood for support of long-term hematopoietic progenitor cell growth.

Z Q Ye 1, J K Burkholder 1, P Qiu 1, J C Schultz 1, N T Shahidi 1, N S Yang 1
PMCID: PMC45392  PMID: 7527553

Abstract

Previous attempts to establish a stromal cell feeder layer from human umbilical cord blood (HUCB) have met with very limited success. It has been suggested that there is an insufficient number of stromal precursor cells in HUCB to form a hematopoietic-supporting feeder layer in primary cultures. The present study shows that HUCB does contain a significant accessory cell population that routinely develops into a confluent, adherent cell layer under defined primary culture conditions. HUCB-derived adherent layers were shown to support long-term hematopoietic activity for an average of 4 months. This was achieved by using a customized coverslip with a modified surface structure as the cell attachment substratum and using a specialized culture feeding regime. We have characterized the various cell types (including fibroblasts, macrophages, and endothelial cells) and extracellular matrix proteins (including fibronectin, collagen III, and laminin) that were present in abundance in the HUCB-derived adherent cell layer. In contrast, oil red O-staining fat cells were rarely detected. ELISA and bioassays showed that stem cell factor and interleukin 6 were produced by the HUCB stromal cell cultures, but interleukin 3 or granulocyte/macrophage colony-stimulating factor was not detected. Application of this hematopoietic culture system to transgenic and gene therapy studies of stem cells is discussed.

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Selected References

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  1. Andreoni C., Moreau I., Rigal D. Long-term culture of human bone marrow. I. characterization of adherent cells in flow cytometry. Exp Hematol. 1990 Jun;18(5):431–437. [PubMed] [Google Scholar]
  2. Broxmeyer H. E., Douglas G. W., Hangoc G., Cooper S., Bard J., English D., Arny M., Thomas L., Boyse E. A. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci U S A. 1989 May;86(10):3828–3832. doi: 10.1073/pnas.86.10.3828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brugger W., Möcklin W., Heimfeld S., Berenson R. J., Mertelsmann R., Kanz L. Ex vivo expansion of enriched peripheral blood CD34+ progenitor cells by stem cell factor, interleukin-1 beta (IL-1 beta), IL-6, IL-3, interferon-gamma, and erythropoietin. Blood. 1993 May 15;81(10):2579–2584. [PubMed] [Google Scholar]
  4. Cardoso A. A., Li M. L., Batard P., Hatzfeld A., Brown E. L., Levesque J. P., Sookdeo H., Panterne B., Sansilvestri P., Clark S. C. Release from quiescence of CD34+ CD38- human umbilical cord blood cells reveals their potentiality to engraft adults. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8707–8711. doi: 10.1073/pnas.90.18.8707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dexter T. M. Stromal cell associated haemopoiesis. J Cell Physiol Suppl. 1982;1:87–94. doi: 10.1002/jcp.1041130414. [DOI] [PubMed] [Google Scholar]
  6. Ershler W. B., Sun W. H., Binkley N., Gravenstein S., Volk M. J., Kamoske G., Klopp R. G., Roecker E. B., Daynes R. A., Weindruch R. Interleukin-6 and aging: blood levels and mononuclear cell production increase with advancing age and in vitro production is modifiable by dietary restriction. Lymphokine Cytokine Res. 1993 Aug;12(4):225–230. [PubMed] [Google Scholar]
  7. Gartner S., Kaplan H. S. Long-term culture of human bone marrow cells. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4756–4759. doi: 10.1073/pnas.77.8.4756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Golde D. W. The stem cell. Sci Am. 1991 Dec;265(6):86–93. doi: 10.1038/scientificamerican1291-86. [DOI] [PubMed] [Google Scholar]
  9. Hows J. M., Bradley B. A., Marsh J. C., Luft T., Coutinho L., Testa N. G., Dexter T. M. Growth of human umbilical-cord blood in longterm haemopoietic cultures. Lancet. 1992 Jul 11;340(8811):73–76. doi: 10.1016/0140-6736(92)90396-k. [DOI] [PubMed] [Google Scholar]
  10. Issaad C., Croisille L., Katz A., Vainchenker W., Coulombel L. A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38- progenitor cells in long-term cultures and semisolid assays. Blood. 1993 Jun 1;81(11):2916–2924. [PubMed] [Google Scholar]
  11. Keller D. C., Du X. X., Srour E. F., Hoffman R., Williams D. A. Interleukin-11 inhibits adipogenesis and stimulates myelopoiesis in human long-term marrow cultures. Blood. 1993 Sep 1;82(5):1428–1435. [PubMed] [Google Scholar]
  12. Koller M. R., Bender J. G., Papoutsakis E. T., Miller W. M. Effects of synergistic cytokine combinations, low oxygen, and irradiated stroma on the expansion of human cord blood progenitors. Blood. 1992 Jul 15;80(2):403–411. [PubMed] [Google Scholar]
  13. Li C. L., Johnson G. R. Stimulation of multipotential, erythroid and other murine haematopoietic progenitor cells by adherent cell lines in the absence of detectable multi-CSF (IL-3). Nature. 1985 Aug 15;316(6029):633–636. doi: 10.1038/316633a0. [DOI] [PubMed] [Google Scholar]
  14. Migliaccio G., Migliaccio A. R., Druzin M. L., Giardina P. J., Zsebo K. M., Adamson J. W. Long-term generation of colony-forming cells in liquid culture of CD34+ cord blood cells in the presence of recombinant human stem cell factor. Blood. 1992 May 15;79(10):2620–2627. [PubMed] [Google Scholar]
  15. Moritz T., Keller D. C., Williams D. A. Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease. J Exp Med. 1993 Aug 1;178(2):529–536. doi: 10.1084/jem.178.2.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Novotny J. R., Duehrsen U., Welch K., Layton J. E., Cebon J. S., Boyd A. W. Cloned stromal cell lines derived from human Whitlock/Witte-type long-term bone marrow cultures. Exp Hematol. 1990 Aug;18(7):775–784. [PubMed] [Google Scholar]
  17. Rowley S. D., Brashem-Stein C., Andrews R., Bernstein I. D. Hematopoietic precursors resistant to treatment with 4-hydroperoxycyclophosphamide: requirement for an interaction with marrow stroma in addition to hematopoietic growth factors for maximal generation of colony-forming activity. Blood. 1993 Jul 1;82(1):60–65. [PubMed] [Google Scholar]
  18. Strobel E. S., Gay R. E., Greenberg P. L. Characterization of the in vitro stromal microenvironment of human bone marrow. Int J Cell Cloning. 1986 Sep;4(5):341–356. doi: 10.1002/stem.5530040506. [DOI] [PubMed] [Google Scholar]
  19. Sutherland H. J., Eaves C. J., Eaves A. C., Dragowska W., Lansdorp P. M. Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood. 1989 Oct;74(5):1563–1570. [PubMed] [Google Scholar]
  20. Sutherland H. J., Hogge D. E., Cook D., Eaves C. J. Alternative mechanisms with and without steel factor support primitive human hematopoiesis. Blood. 1993 Mar 15;81(6):1465–1470. [PubMed] [Google Scholar]
  21. Sutherland H. J., Lansdorp P. M., Henkelman D. H., Eaves A. C., Eaves C. J. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Natl Acad Sci U S A. 1990 May;87(9):3584–3588. doi: 10.1073/pnas.87.9.3584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Touw I., Löwenberg B. No stimulative effect of adipocytes on hematopoiesis in long-term human bone marrow cultures. Blood. 1983 Apr;61(4):770–774. [PubMed] [Google Scholar]
  23. Verfaillie C., Blakolmer K., McGlave P. Purified primitive human hematopoietic progenitor cells with long-term in vitro repopulating capacity adhere selectively to irradiated bone marrow stroma. J Exp Med. 1990 Aug 1;172(2):509–502. doi: 10.1084/jem.172.2.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vilmer E., Sterkers G., Rahimy C., Denamur E., Elion J., Broyart A., Lescoeur B., Tiercy J. M., Gerota J., Blot P. HLA-mismatched cord-blood transplantation in a patient with advanced leukemia. Transplantation. 1992 May;53(5):1155–1157. [PubMed] [Google Scholar]
  25. Williams D. A. Ex vivo expansion of hematopoietic stem and progenitor cells--robbing Peter to pay Paul? Blood. 1993 Jun 15;81(12):3169–3172. [PubMed] [Google Scholar]

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