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

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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.

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Acknowledgments

The authors thank Mr. Kevin Litton (Bachelor of arts in English) for editorial assistance.

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Authors and Affiliations

  1. 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan

    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 & Yoshiro Niitsu

  2. Department of Chemistry, Sapporo Medical University, Sapporo, Japan

    Shinsei Gasa

  3. Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan

    Hiromi Sakai & Eishun Tsuchida

  4. Department of Transfusion and Cell Therapy, Saitama Medical School Hospital, Saitama, Japan

    Kenji Ikebuchi

  5. Department of Molecular Medicine, Sapporo Medical University, Sapporo, Japan

    Hirofumi Hamada

Authors
  1. Akihito Fujimi
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  2. Takuya Matsunaga
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  3. Masayoshi Kobune
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  4. Yutaka Kawano
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  16. Shinsei Gasa
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  20. Hirofumi Hamada
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  21. Yoshiro Niitsu
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Correspondence to Takuya Matsunaga.

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Fujimi, A., Matsunaga, T., Kobune, M. et al. Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages. Int J Hematol 87, 339–350 (2008). https://doi.org/10.1007/s12185-008-0062-y

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  • DOI: https://doi.org/10.1007/s12185-008-0062-y

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