International Journal of Hematology

, Volume 87, Issue 4, pp 339–350

Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages

  • Akihito Fujimi
  • Takuya Matsunaga
  • Masayoshi Kobune
  • Yutaka Kawano
  • Taiko Nagaya
  • Ikuta Tanaka
  • Satoshi Iyama
  • Tsuyoshi Hayashi
  • Tsutomu Sato
  • Koji Miyanishi
  • Tamotsu Sagawa
  • Yasushi Sato
  • Rishu Takimoto
  • Tetsuji Takayama
  • Junji Kato
  • Shinsei Gasa
  • Hiromi Sakai
  • Eishun Tsuchida
  • Kenji Ikebuchi
  • Hirofumi Hamada
  • Yoshiro Niitsu
Original Article

Abstract

We generated red blood cells (RBC) from cord blood (CB) CD34+ cells using a four-phase culture system. We first cultured CB CD34+ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34+ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34+ cells from a different donor using the same co-culture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 × 1012 RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 × 1013 RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.

Keywords

Red blood cell Cord blood CD34+ cell Stromal cell 

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Copyright information

© The Japanese Society of Hematology 2008

Authors and Affiliations

  • Akihito Fujimi
    • 1
  • Takuya Matsunaga
    • 1
  • Masayoshi Kobune
    • 1
  • Yutaka Kawano
    • 1
  • Taiko Nagaya
    • 1
  • Ikuta Tanaka
    • 1
  • Satoshi Iyama
    • 1
  • Tsuyoshi Hayashi
    • 1
  • Tsutomu Sato
    • 1
  • Koji Miyanishi
    • 1
  • Tamotsu Sagawa
    • 1
  • Yasushi Sato
    • 1
  • Rishu Takimoto
    • 1
  • Tetsuji Takayama
    • 1
  • Junji Kato
    • 1
  • Shinsei Gasa
    • 2
  • Hiromi Sakai
    • 3
  • Eishun Tsuchida
    • 3
  • Kenji Ikebuchi
    • 4
  • Hirofumi Hamada
    • 5
  • Yoshiro Niitsu
    • 1
  1. 1.4th Department of Internal Medicine, Sapporo Medical UniversitySchool of MedicineSapporoJapan
  2. 2.Department of ChemistrySapporo Medical UniversitySapporoJapan
  3. 3.Advanced Research Institute for Science and EngineeringWaseda UniversityTokyoJapan
  4. 4.Department of Transfusion and Cell TherapySaitama Medical School HospitalSaitamaJapan
  5. 5.Department of Molecular MedicineSapporo Medical UniversitySapporoJapan

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