Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages
Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages
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DOI：
10.1007/s12185-008-0062-y
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发表时间：
2008-05-01
影响因子：
2.1
通讯作者：
Niitsu, Yoshiro
中科院分区：
文献类型：
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作者：
Fujimi, Akihito;Matsunaga, Takuya;Niitsu, Yoshiro
我们使用四相培养系统从脐带血（CB）CD 34（+）细胞产生红细胞（RBC）。我们首先在含有干细胞因子（SCF）、Flt-3/Flk-2配体和血小板生成素的无血清培养基中，在端粒酶基因转导的人基质细胞上培养CB CD 34（+）细胞，以扩增CD 34（+）细胞（980倍）和总细胞（10，400倍）（第一阶段）。将来自第一阶段的扩增的细胞与SCF、白细胞介素-3（IL-3）和促红细胞生成素（EPO）液体培养以扩增（113倍）并将它们分化成红细胞（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CD 34（+）细胞。将来自第一阶段的扩增细胞与粒细胞-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液体培养以产生单核细胞/巨噬细胞（75倍），将其与AB型血清和M-CSF孵育以使其完全分化为巨噬细胞。然后在EPO存在下将成红细胞与巨噬细胞共培养以扩增（三倍）并使其完全分化（61%正染性成红细胞加39%RBC）（第三阶段）。通过去红细胞过滤器从成红细胞和碎片中纯化RBC，以从1.0个CB单位（3.0个可用单位）中产生6.0 x 10（12）RBC。定性地，这些RBC显示出与成人外周RBC相似的血红蛋白含量、血红蛋白氧合和体内清除率。最后，在不存在巨噬细胞和细胞因子的情况下，通过Miharada等人最近描述的培养方法实现了几乎完全的正染性成红细胞去核（99.4%）（第四阶段）。通过去红细胞过滤器从残余成红细胞和碎片中纯化RBC，从1.0个CB单位（8.8个可用单位）中产生1.76 x 10（13）RBC，这是迄今报道的最高产量。因此，该方法可用于产生用于输血的替代RBC供应，研究靶向红系细胞的感染因子，以及作为一般的体外造血模型系统。
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 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.