Fujimi Akihito, Matsunaga Takuya, Kobune Masayoshi, Kawano Yutaka, Nagaya Taiko, Tanaka Ikuta, Iyama Satoshi, Hayashi Tsuyoshi, Sato Tsutomu, Miyanishi Koji, Sagawa Tamotsu, Sato Yasushi, Takimoto Rishu, Takayama Tetsuji, Kato Junji, Gasa Shinsei, Sakai Hiromi, Tsuchida Eishun, Ikebuchi Kenji, Hamada Hirofumi, Niitsu Yoshiro
4th Department of Internal Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan.
Department of Chemistry, Sapporo Medical University, Sapporo, Japan.
Int J Hematol. 2008 May;87(4):339-350. doi: 10.1007/s12185-008-0062-y.
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.
我们使用四阶段培养系统从脐带血（CB）CD34+细胞生成红细胞（RBC）。我们首先将CB CD34+细胞培养在转导了端粒酶基因的人基质细胞上，置于含有干细胞因子（SCF）、Flt-3/Flk-2配体和血小板生成素的无血清培养基中，以扩增CD34+细胞（980倍）和总细胞（10,400倍）（第一阶段）。第一阶段扩增的细胞用SCF、白细胞介素-3（IL-3）和促红细胞生成素（EPO）进行液体培养，以扩增（113倍）并使其分化为成红细胞（第二阶段）。为了获得用于下一阶段的巨噬细胞，我们使用相同的共培养系统扩增来自不同供体的CD34+细胞。第一阶段扩增的细胞用粒细胞-巨噬细胞集落刺激因子、巨噬细胞集落刺激因子（M-CSF）、IL-3和SCF进行液体培养，以生成单核细胞/巨噬细胞（75倍），然后将其与AB型血清和M-CSF一起孵育，使其完全分化为巨噬细胞。然后将成红细胞与巨噬细胞在EPO存在下共培养，以扩增（3倍）并使其完全分化（61%正染红细胞加39% RBC）（第三阶段）。通过去白细胞滤器从成红细胞和碎片中纯化RBC，从1.0单位CB中生成6.0×10¹²个RBC（3.0个可输血单位）。定性地，这些RBC的血红蛋白含量、血红蛋白氧合和体内清除情况与成人外周RBC相似。最后，通过Miharada等人最近描述的培养方法，在没有巨噬细胞和细胞因子的情况下，实现了正染红细胞几乎完全去核（99.4%）（第四阶段）。通过去白细胞滤器从残留的成红细胞和碎片中纯化RBC，从1.0单位CB中生成1.76×10¹³个RBC（8.8个可输血单位），这是迄今报道的最高产量。因此，该方法可能有助于生成用于输血的替代RBC供应、研究靶向红系细胞的感染因子，并作为一般的体外造血模型系统。
