International Journal of Hematology

, Volume 78, Issue 2, pp 126–132 | Cite as

Ex Vivo Expansion of Human Umbilical Cord Blood CD34+ Cells in a Collagen Bead—Containing 3-Dimensional Culture System

  • Han-Soo Kim
  • Jong Baeck Lim
  • Yoo Hong Min
  • Seung Tae Lee
  • Chuhl Joo Lyu
  • Eun Seok Kim
  • Hyun Ok Kim


Self-renewal of stem cells depends on several critical factors, including the hematopoietic microenvironment, interactions with supporting stromal cells, features of the extracellular matrix, hematopoietic growth factors, and cytokines. Our study investigated the role of artificial 3-dimensional microenvironments as a means of replicating a more physiologic milieu in expansion of cord blood CD34+ cells. In the 3-dimensional model, hematopoietic cells inside collagen beads are exposed to cytokines added to a culture medium. We found that amplification of CD34+ cells with a clonogenic assay, fluorescenceactivated cell sorter analysis, and bone marrow repopulation of NOD/SCID mice showed greater clonogenic ability of cells cultured by the 3-dimensional method compared with the 2-dimensional method. The present study demonstrated that 3-dimensional matrix support may be useful for extended periods in expansion and preservation of stem cells or progenitor cells in vitro.

Key words

Cord blood CD34+ hematopoietic stem cell Ex vivo expansion Collagen beads NOD/SCID mouse 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Kogler G, Callejas J, Hakenberg P, et al. Hematopoietic transplant potential of unrelated cord blood: critical issues.J Hematother. 1996;5:105–116.CrossRefPubMedGoogle Scholar
  2. 2.
    Mayani H, Landsorp PM. Biology of human umbilical cord Blood—derived hematopoietic stem/progenitor cells.Stem Cells. 1998;16:153–165.CrossRefPubMedGoogle Scholar
  3. 3.
    Kobari L, Pflumio F, Giarratana M-C, et al. In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34+ cord blood cells.Exp Hematol. 2000;28:1470–1480.CrossRefPubMedGoogle Scholar
  4. 4.
    Noort WA, Willemze R, Falkenburg JHF. Comparison of repopulating ability of hematopoietic progenitor cells isolated from human umbilical cord blood or bone marrow cells in NOD/SCID mice.Bone Marrow Transplant. 1998;22(suppl 1):S58-S60.PubMedGoogle Scholar
  5. 5.
    Querol S, Capmany G, Cancelas JA, et al. Expansion of cord blood progenitor cells.Bone Marrow Transplant. 1998;21:S77-S80.PubMedGoogle Scholar
  6. 6.
    Wagner JE, Kerman NA, Steinbuch M, et al. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease.Lancet. 1995;346:214–219.CrossRefPubMedGoogle Scholar
  7. 7.
    Locatelli F, Maccario R, Comoli P, et al. Hematopoietic and immune recovery after transplantation of cord blood progenitor cells in children.Bone Marrow Transplant. 1996;18:1095–1101.PubMedGoogle Scholar
  8. 8.
    van Hennik PB, de Koning AE, Ploemacher RE. Seeding efficiency of primitive human hematopoietic cells in nonobese diabetic/ severe combined immune deficiency mice: implications for stem cell frequency assessment.Blood. 1999;94:3055–3061.PubMedGoogle Scholar
  9. 9.
    Broxmeyer HE, Hangoc G, Cooper S, et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults.Proc Natl Acad Sci U S A. 1992;89:4109–4113.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Piacibello W, Sanavio F, Garetto L, et al. Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplificationvs proliferation and differentiation.Leukemia. 1998;12:718–727.CrossRefPubMedGoogle Scholar
  11. 11.
    Kogler G, Callejas J, Sorg RV, et al. The effect of different thawing methods, growth factor combinations and media on the ex vivo expansion of umbilical cord blood primitive and committed progenitors.Bone Marrow Transplant. 1998;21:233–241.CrossRefPubMedGoogle Scholar
  12. 12.
    Petzer AL, Zandstra PW, Piret JM, et al. Differential cytokine effects on primitive (CD34+CD38-) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin.J Exp Med. 1996;183:2551–2558.CrossRefPubMedGoogle Scholar
  13. 13.
    Piacibello W, Sanavio F, Garetto L, et al. Extensive amplification and self-renewal of human primitive hematopoietic stem cell from cord blood.Blood. 1997;89:2644–2653.PubMedGoogle Scholar
  14. 14.
    Denning-Kendall PA, Nicol A, Horsley H, et al. Is in vitro expansion of human cord blood cells clinically relevant?Bone Marrow Transplant. 1998;21:225–232.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Li K, Liu J, Fok T, et al. Human neonatal blood: stem cell content, kinetics of CD34+ cell decline and ex vivo expansion capacity.Br J Haematol. 1999;104:178–185.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Xu R, Reems JA. Umbilical cord blood progeny cells that retain a CD34+ phenotype after ex vivo expansion have less engraftment potential than unexpanded CD34+ cells.Transfusion. 2001;41:213–218.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Lam AC, Li K, Zhang XB, et al. Preclinical ex vivo expansion of cord blood hematopoietic stem and progenitor cells: duration of culture; the media, serum supplements, and growth factors used; and engraftment in NOD/SCID mice.Transfusion. 2001;41:1567–1576.CrossRefPubMedGoogle Scholar
  18. 18.
    Kohler T, Plettig R, Schaffer B, et al. Defining optimum conditions for the ex vivo expansion of human umbilical cord blood cells: influences of progenitor enrichment, interference with feeder layers, early-acting cytokines and agitation of culture vessels.Stem Cells. 1999;17:19–24.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Tun T, Miyoshi H, Ema H, et al. New type of matrix for bone marrow cell cultures: in vitro culture and in vivo transplantation experiments.ASAIO J. 2000;46:522–526.CrossRefPubMedGoogle Scholar
  20. 20.
    Wang T-Y, Brennan JK, Wu JHD. Multilineal hematopoiesis in a three-dimensional murine long-term bone-marrow culture.Exp Hematol. 1995;23:26–32.PubMedGoogle Scholar
  21. 21.
    Li Y, Ma T, Kniss DA, Yang ST, Laske LC. Human cord cell hematopoiesis in three dimensional nonwoven fibrous matrices: in vitro simulation of the marrow microenvironment.J Hematother Stem Cell Res. 2001;10:355–368.CrossRefPubMedGoogle Scholar
  22. 22.
    Min YH, Lee ST, Choi KM, et al. Ex vivo generation of functional dendritic cells from mobilized CD34+ hematopoietic stem cells.Yonsei Med J. 1998;39:328–338.CrossRefPubMedGoogle Scholar
  23. 23.
    Kie J-H, Yang W-I, Lee M-K, et al. Decrease in apoptosis and increase in polyploidization of megakaryocytes by stem cell factor during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin.Stem Cells. 2002;20:73–79.CrossRefPubMedGoogle Scholar
  24. 24.
    Ueda T, Tsuji K, Yoshino H, et al. Expansion of human NOD/ SCID-repopulating cells by stem cell factor, Flk2/flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor.J Clin Invest. 2000;105:1013–1021.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    DiGiusto DL, Lee R, Moon J, et al. Hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood progenitor cells evaluated in vitro and in vivo.Blood. 1996;84:1261–1271.Google Scholar
  26. 26.
    Kusadasi N, van Soest PL, Mayen AE, et al. Successful short-term ex vivo expansion of NOD/SCID repopulating ability and CAFC week 6 from umbilical cord blood.Leukemia. 2000;14:1944–1953.CrossRefPubMedGoogle Scholar
  27. 27.
    Rossmanith T, Schroder B, Bug G, et al. Interleukin 3 improves the ex vivo expansion of primitive cord blood progenitor cells and maintains the engraftment potential of scid repopulating cells.Stem Cells. 2001;19:313–320.CrossRefPubMedGoogle Scholar
  28. 28.
    Varas F, Bernard A, Bueren JA. Restrictions in the stem cell function of murine bone marrow grafts after ex vivo expansion of short-term repopulating progenitors.Exp Hematol. 1998;26:100–109.PubMedGoogle Scholar
  29. 29.
    Holyoake T, Alcorn M, Richmond L, et al. CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens.Bone Marrow Transplant. 1997;19:1095–1101.CrossRefPubMedGoogle Scholar
  30. 30.
    Bentley SA. Close range cell:cell interaction required for stem cell maintenance in continuous bone marrow cultures.Exp Hematol. 1981;9:308–312.PubMedGoogle Scholar
  31. 31.
    Szilvassy SJ, Bass MJ, van Zant G, et al. Organ-specific homing defines engraftment kinetics of murine hematopoietic stem cells and is compromised by ex vivo expansion.Blood. 1999;93:1557–1566.PubMedGoogle Scholar
  32. 32.
    Koenigsmann MP, Koenigsmann M, Notter M, et al. Adhesion molecules on peripheral blood-derived CD34+ cells: effects of cryopreservation and short-term ex vivo incubation with serum and cytokines.Bone Marrow Transplant. 1998;22:1077–1085.CrossRefPubMedGoogle Scholar
  33. 33.
    Peled A, Petit I, Kollet O, et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.Science. 1999;283:845–848.CrossRefPubMedGoogle Scholar

Copyright information

© The Japanese Society of Hematology 2003

Authors and Affiliations

  • Han-Soo Kim
    • 1
    • 2
  • Jong Baeck Lim
    • 3
  • Yoo Hong Min
    • 2
  • Seung Tae Lee
    • 2
  • Chuhl Joo Lyu
    • 4
  • Eun Seok Kim
    • 5
  • Hyun Ok Kim
    • 3
  1. 1.Brain Korea 21 Project for Medical SciencesUSA
  2. 2.Departments of Internal MedicineUSA
  3. 3.Department of Laboratory MedicineYonsei University College of MedicineSeoulKorea
  4. 4.Pediatrics, Yonsei University, College of MedicineSeoul
  5. 5.Department of Oral and Maxillofacial SurgeryChungnam National University HospitalDaejeonKorea

Personalised recommendations