Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages.
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.
Fujimi
Akihito
A
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Matsunaga
Takuya
T
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan. matunaga@sapmed.ac.jp.
Kobune
Masayoshi
M
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Kawano
Yutaka
Y
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Nagaya
Taiko
T
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Tanaka
Ikuta
I
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Iyama
Satoshi
S
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Hayashi
Tsuyoshi
T
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Sato
Tsutomu
T
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Miyanishi
Koji
K
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Sagawa
Tamotsu
T
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Sato
Yasushi
Y
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Takimoto
Rishu
R
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Takayama
Tetsuji
T
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Kato
Junji
J
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Gasa
Shinsei
S
Department of Chemistry, Sapporo Medical University, Sapporo, Japan.
Sakai
Hiromi
H
Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan.
Tsuchida
Eishun
E
Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan.
Ikebuchi
Kenji
K
Department of Transfusion and Cell Therapy, Saitama Medical School Hospital, Saitama, Japan.
Hamada
Hirofumi
H
Department of Molecular Medicine, Sapporo Medical University, Sapporo, Japan.
Niitsu
Yoshiro
Y
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
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
D018952:Antigens, CD34; C509186:TERT protein, human; D019098:Telomerase