International Journal of Hematology

, Volume 81, Issue 1, pp 18–25

Expansion of CD34+ Cells on Telomerized Human Stromal Cells without Losing Erythroid-Differentiation Potential in a Serum-Free Condition

  • Masayoshi Kobune
  • Yutaka Kawano
  • Junji Kato
  • Yoshinori Ito
  • Hiroki Chiba
  • Kiminori Nakamura
  • Akihito Fujimi
  • Takuya Matsunaga
  • Hirofumi Hamada
  • Yoshiro Niitsu


Erythropoiesis progresses from stem cell expansion on stromal cells through the formation of an erythroblastic island. Our aim was to assess the feasibility of using human stromal cells for erythroid production and differentiation. When cord blood CD34+ cells were cocultured with telomerized human stromal cells (hTERT-stromal cells) for 2 weeks, the CD34+ cells and burst-forming units-erythroid (BFU-E) significantly expanded, and a few hematopoietic cells transmigrated below the stromal layer. When nonadherent hematopoietic progenitor cells that had expanded above the hTERT-stromal cells (group B) were collected and subjected to our erythroid-differentiation protocol, they differentiated into erythroblasts with a slight hemoglobin synthesis. When the few hematopoietic cells that had transmigrated below the stromal layer were expanded for an additional 2 to 6 weeks, they exhibited a cobblestone-like appearance, and a large amount of BFU-E clambered weekly from the underside of the stromal layer to above the stromal layer (group C).When the hematopoietic progenitor cells in group C were subjected to the erythroid-differentiation protocol, large numbers of mature erythroblasts (more than 300,000 times the initial CD34+ cell number) were produced. Our hTERT-stromal expansion protocol may contribute to the construction of a system for large-scale, long-term production of erythroid cells.

Key words

Erythropoiesis Human stromal cells Erythroblast 


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  1. 1.
    Coste I, Gauchat JF, Wilson A, et al. Unavailability of CD147 leads to selective erythrocyte trapping in the spleen. Blood. 2001;97:3984–3988.PubMedCrossRefGoogle Scholar
  2. 2.
    Kapur R, Cooper R, Zhang L, Williams DA. Cross-talk between α4β1/α5β1 and c-Kit results in opposing effect on growth and survival of hematopoietic cells via the activation of focal adhesion kinase, mitogen-activated protein kinase, and Akt signaling pathways. Blood. 2001;97:1975–1981.PubMedCrossRefGoogle Scholar
  3. 3.
    Yanai N, Sekine C, Yagita H, Obinata M. Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis. Blood. 1994;83:2844–2850.PubMedGoogle Scholar
  4. 4.
    Suenobu S, Takakura N, Inada T, et al. A role of EphB4 receptor and its ligand, ephrin-B2, in erythropoiesis. Biochem Biophys Res Commun. 2002;293:1124–1131.PubMedCrossRefGoogle Scholar
  5. 5.
    Arroyo AG, Yang JT, Rayburn H, Hynes RO. Alpha4 integrins regulate the proliferation/differentiation balance of multilineage hematopoietic progenitors in vivo. Immunity. 1999;11:555–566.PubMedCrossRefGoogle Scholar
  6. 6.
    Hamamura K, Matsuda H, Takeuchi Y, Habu S, Yagita H, Okumura K. A critical role of VLA-4 in erythropoiesis in vivo. Blood. 1996;87:2513–2517.PubMedGoogle Scholar
  7. 7.
    Wang Z, Miura N, Bonelli A, et al. Receptor tyrosine kinase, EphB4 (HTK), accelerates differentiation of select human hematopoietic cells. Blood. 2002;99:2740–2747.PubMedCrossRefGoogle Scholar
  8. 8.
    Gibellini D, Bassini A, Re MC, et al. Stroma-derived factor 1α induces a selective inhibition of human erythroid development via the functional upregulation of Fas/CD95 ligand. Br J Haematol. 2000;111:432–440.PubMedCrossRefGoogle Scholar
  9. 9.
    Sadahira Y, Yoshino T, Monobe Y. Very late activation antigen 4-vascular cell adhesion molecule 1 interaction is involved in the formation of erythroblastic islands. J Exp Med. 1995;181:411–415.PubMedCrossRefGoogle Scholar
  10. 10.
    Hanspal M, Smockova Y, Uong Q. Molecular identification and functional characterization of a novel protein that mediates the attachment of erythroblasts to macrophages. Blood. 1998;92:2940–2950.PubMedGoogle Scholar
  11. 11.
    Roy V, Verfaillie CM. Soluble factor(s) produced by adult bone marrow stroma inhibit in vitro proliferation and differentiation of fetal liver BFU-E by inducing apoptosis. J Clin Invest. 1997;100:912–920.PubMedCrossRefGoogle Scholar
  12. 12.
    Schuetze S, Stenberg PE, Kabat D. The Ets-related transcription factor PU.1 immortalizes erythroblasts. Mol Cell Biol. 1993;13:5670–5678.PubMedGoogle Scholar
  13. 13.
    Wada H, Suda T, Miura Y, Kajii E, Ikemoto S, Yawata Y. Expression of major blood group antigens on human erythroid cells in a two phase liquid culture system. Blood. 1990;75:505–511.PubMedGoogle Scholar
  14. 14.
    Okumura N, Tsuji K, Nakahata T. Changes in cell surface antigen expressions during proliferation and differentiation of human erythroid progenitors. Blood. 1992;80:642–650.PubMedGoogle Scholar
  15. 15.
    Shintani N, Kohgo Y, Kato J, et al. Expression and extracellular release of transferrin receptors during peripheral erythroid progenitor cell differentiation in liquid culture. Blood. 1994;83:1209–1215.PubMedGoogle Scholar
  16. 16.
    Panzenbock B, Bartunek P, Mapara MY, Zenke M. Growth and differentiation of human stem cell factor/erythropoietin-dependent erythroid progenitor cells in vitro. Blood. 1998;92:3658–3668.PubMedGoogle Scholar
  17. 17.
    Neildez-Nguyen TM, Wajcman H, Marden MC, et al. Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo. Nat Biotechnol. 2002;20:467–472.PubMedCrossRefGoogle Scholar
  18. 18.
    Kapur R, Majumdar M, Xiao X, McAndrews-Hill M, Schindler K, Williams DA. Signaling through the interaction of membranerestricted stem cell factor and c-kit receptor tyrosine kinase: genetic evidence for a differential role in erythropoiesis. Blood. 1998;91:879–889.PubMedGoogle Scholar
  19. 19.
    Furusawa T, Yanai N, Hara T, Miyajima A, Obinata M. Integrinassociated protein (IAP, also termed CD47) is involved in stromasupported erythropoiesis. J Biochem (Tokyo). 1998;123:101–106.Google Scholar
  20. 20.
    Sato Y, Hong HN, Yanai N, Obinata M. Involvement of stromal membrane-associated protein (SMAP-1) in erythropoietic microenvironment. J Biochem (Tokyo). 1998;124:209–216.Google Scholar
  21. 21.
    Hanspal M. Importance of cell-cell interactions in regulation of erythropoiesis. Curr Opin Hematol. 1997;4:142–147.PubMedCrossRefGoogle Scholar
  22. 22.
    Lee G, Spring FA, Parsons SF, et al. Novel secreted isoform of adhesion molecule ICAM-4: potential regulator of membraneassociated ICAM-4 interactions. Blood. 2003;101:1790–1797.PubMedCrossRefGoogle Scholar
  23. 23.
    Spring FA, Parsons SF, Ortlepp S, et al. Intercellular adhesion molecule- 4 binds α4β1 and αV-family integrins through novel integrinbinding mechanisms. Blood. 2001;98:458–466.PubMedCrossRefGoogle Scholar
  24. 24.
    Yanai N, Satoh T, Obinata M. Endothelial cells create a hematopoietic inductive microenvironment preferential to erythropoiesis in the mouse spleen. Cell Struct Funct. 1991;16:87–93.PubMedCrossRefGoogle Scholar
  25. 25.
    Inada T, Iwama A, Sakano S, Ohno M, Sawada K, Suda T. Selective expression of the receptor tyrosine kinase, HTK, on human erythroid progenitor cells. Blood. 1997;89:2757–2765.PubMedGoogle Scholar
  26. 26.
    Kuwahara I, Ikebuchi K, Hamada H, et al. Changes in N-glycosylation of human stromal cells by telomerase expression. Biochem Biophys Res Commun. 2003;301:293–297.PubMedCrossRefGoogle Scholar
  27. 27.
    Kobune M, Kawano Y, Ito Y, et al. Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells. Exp Hematol. 2003;31:715–722.PubMedCrossRefGoogle Scholar
  28. 28.
    Kawano Y, Kobune M, Yamaguchi M, et al. Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT)- transfected human stromal cells. Blood. 2003;101:532–540.PubMedCrossRefGoogle Scholar
  29. 29.
    Kobune M, Ito Y, Kawano Y, et al. Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID-2mβ/repopulating cells. Blood. 2004;104:1002–1009.PubMedCrossRefGoogle Scholar
  30. 30.
    Kobune M, Xu Y, Baum C, Kelley MR, Williams DA. Retrovirusmediated expression of the base excision repair proteins, formamidopyrimidine DNA glycosylase or human oxoguanine DNA glycosylase, protects hematopoietic cells from N,Nβ,Nα- triethylenethiophosphoramide (thioTEPA)-induced toxicity in vitro and in vivo. Cancer Res. 2001;61:5116–5125.PubMedGoogle Scholar
  31. 31.
    Yoshida M, Tsuji K, Ebihara Y, et al. Thrombopoietin alone stimulates the early proliferation and survival of human erythroid, myeloid and multipotential progenitors in serum-free culture. Br J Haematol. 1997;98:254–264.PubMedCrossRefGoogle Scholar
  32. 32.
    Dai MS, Heinrich MC, Broxmeyer HE, Lu L. Enhancing effects of co-transduction of both human erythropoietin receptor and c-kit cDNAs into hematopoietic stem/progenitor cells from cord blood on proliferation and differentiation of erythroid progenitors. Cytokines Cell Mol Ther. 2000;6:1–8.PubMedCrossRefGoogle Scholar
  33. 33.
    Ueda T, Tsuji K, Yoshino H, et al. Expansion of human NOD/SCIDrepopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. J Clin Invest. 2000;105:1013–1021.PubMedCrossRefGoogle Scholar
  34. 34.
    Zermati Y, Fichelson S, Valensi F, et al. Transforming growth factor inhibits erythropoiesis by blocking proliferation and accelerating differentiation of erythroid progenitors. Exp Hematol. 2000;28:885–894.PubMedCrossRefGoogle Scholar
  35. 35.
    Telen MJ. Red blood cell surface adhesion molecules: their possible roles in normal human physiology and disease. Semin Hematol. 2000;37:130–142.PubMedCrossRefGoogle Scholar
  36. 36.
    Wojda U, Noel P, Miller JL. Fetal and adult hemoglobin production during adult erythropoiesis: coordinate expression correlates with cell proliferation. Blood. 2002;99:3005–3013.PubMedGoogle Scholar
  37. 37.
    Papadaki HA, Kritikos HD, Valatas V, Boumpas DT, Eliopoulos GD. Anemia of chronic disease in rheumatoid arthritis is associated with increased apoptosis of bone marrow erythroid cells: improvement following anti-tumor necrosis factor-_ antibody therapy. Blood. 2002;100:474–482.PubMedCrossRefGoogle Scholar
  38. 38.
    Tsuji T, Waga I, Tezuka K, Kamada M, Yatsunami K, Kodama H. Integrin _2 (CD18)-mediated cell proliferation of HEL cells on a hematopoietic-supportive bone marrow stromal cell line, HESS-5 cells. Blood. 1998;91:1263–1271.PubMedGoogle Scholar
  39. 39.
    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;81:2916–2924.PubMedGoogle Scholar
  40. 40.
    Okamoto T, Aoyama T, Nakayama T, et al. Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells. Biochem Biophys Res Commun. 2002;295:354–361.PubMedCrossRefGoogle Scholar
  41. 41.
    Serakinci N, Guldberg P, Burns JS, et al. Adult human mesenchymal stem cell as a target for neoplastic transformation. Oncogene. 2004;23:5095–5098.PubMedCrossRefGoogle Scholar
  42. 42.
    Keith WN. From stem cells to cancer: balancing immortality and neoplasia. Oncogene. 2004;23:5092–5094.PubMedCrossRefGoogle Scholar
  43. 43.
    Xiaoxue Y, Zhongqiang C, Zhaoqing G, Gengting D, Qingjun M, Shenwu W. Immortalization of human osteoblasts by transferring human telomerase reverse transcriptase gene. Biochem Biophys Res Commun. 2004;315:643–651.PubMedCrossRefGoogle Scholar

Copyright information

© The Japanese Society of Hematology 2005

Authors and Affiliations

  • Masayoshi Kobune
    • 1
    • 2
  • Yutaka Kawano
    • 1
  • Junji Kato
    • 1
  • Yoshinori Ito
    • 2
    • 3
  • Hiroki Chiba
    • 1
  • Kiminori Nakamura
    • 2
  • Akihito Fujimi
    • 1
  • Takuya Matsunaga
    • 1
  • Hirofumi Hamada
    • 2
    • 3
  • Yoshiro Niitsu
    • 1
  1. 1.Fourth Department of Internal MedicineSapporo Medical University School of MedicineSapporoJapan
  2. 2.Department of Molecular MedicineSapporo Medical University School of MedicineSapporoJapan
  3. 3.Division of Gene TherapySapporo Medical University School of MedicineSapporoJapan

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