title: Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages.
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 coculture 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 x 10(12) 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 x 10(13) 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.
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Akihito
authors: A
authors: Fujimi
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan. matunaga@sapmed.ac.jp.
authors: Takuya
authors: T
authors: Matsunaga
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Masayoshi
authors: M
authors: Kobune
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Yutaka
authors: Y
authors: Kawano
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Taiko
authors: T
authors: Nagaya
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Ikuta
authors: I
authors: Tanaka
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Satoshi
authors: S
authors: Iyama
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Tsuyoshi
authors: T
authors: Hayashi
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Tsutomu
authors: T
authors: Sato
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Koji
authors: K
authors: Miyanishi
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Tamotsu
authors: T
authors: Sagawa
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Yasushi
authors: Y
authors: Sato
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Rishu
authors: R
authors: Takimoto
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Tetsuji
authors: T
authors: Takayama
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Junji
authors: J
authors: Kato
authors: Department of Chemistry, Sapporo Medical University, Sapporo, Japan.
authors: Shinsei
authors: S
authors: Gasa
authors: Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan.
authors: Hiromi
authors: H
authors: Sakai
authors: Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan.
authors: Eishun
authors: E
authors: Tsuchida
authors: Department of Transfusion and Cell Therapy, Saitama Medical School Hospital, Saitama, Japan.
authors: Kenji
authors: K
authors: Ikebuchi
authors: Department of Molecular Medicine, Sapporo Medical University, Sapporo, Japan.
authors: Hirofumi
authors: H
authors: Hamada
authors: 4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
authors: Yoshiro
authors: Y
authors: Niitsu
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism*; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D018920:Coculture Techniques; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D019098:Telomerase / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; C509186:TERT protein, human; D019098:Telomerase