Ex vivo large-scale generation of human platelets from cord blood CD34+ cells
Ex vivo large-scale generation of human platelets from cord blood CD34+ cells
复制标题
DOI：
10.1634/stemcells.2006-0309
复制
发表时间：
2006-12-01
期刊：
影响因子：
5.2
通讯作者：
Niitsu, Yoshiro
中科院分区：
文献类型：
--
作者：
Matsunaga, Takuya;Tanaka, Ikuta;Niitsu, Yoshiro
在本研究中，我们使用三相培养系统从脐带血（CB）CD 34（+）细胞中产生血小板（PLT）。我们首先在补充有干细胞因子（SCF）、Flt-3/ Flk-2配体（FL）和血小板生成素（TPO）的无血清培养基中，在端粒酶基因转导的人基质细胞（hTERT基质）上培养500个CB CD 34（+）细胞14天。然后，我们将细胞转移到hTERT基质中，并在含有白细胞介素-11（IL- 11）以及原始细胞因子混合物的新鲜培养基中再培养14天。随后，我们将细胞在含有SCF、FL、TPO和IL- 11的液体培养基中再培养5天以从上清液中回收PLT级分，然后将其凝胶过滤以纯化PLT。1.0单位CB（5 x 106个CD 34（+）细胞）的PLT计算产量为1.26 x 10（11）- 1.68 x 10（11）PLT。这些PLT的数量相当于2.5 - 3.4单位的随机供体来源的PLT或2/ 5 - 6/ 10的单采血小板。CB衍生的PLT在形态学上表现出与外周血非常相似的特征，如电子显微镜照片所示，在功能上表现出与纤维蛋白原/ ADP聚集所示非常相似的特征，具有β-选择素和活化的糖蛋白IIb- IIIa抗原的外观。因此，该培养系统可能适用于大规模生产PLT以供未来临床使用。
In the present investigation, we generated platelets (PLTs) from cord blood ( CB) CD34(+) cells using a three- phase culture system. We first cultured 500 CB CD34(+) cells on telomerase gene- transduced human stromal cells ( hTERT stroma) in serum-free medium supplemented with stem cell factor ( SCF), Flt-3/ Flk-2 ligand ( FL), and thrombopoietin ( TPO) for 14 days. We then transferred the cells to hTERT stroma and cultured for another 14 days with fresh medium containing interleukin-11 (IL- 11) in addition to the original cytokine cocktail. Subsequently, we cultured the cells in a liquid culture medium containing SCF, FL, TPO, and IL- 11 for another 5 days to recover PLT fractions from the supernatant, which were then gel- filtered to purify the PLTs. The calculated yield of PLTs from 1.0 unit of CB ( 5 x 106 CD34(+) cells) was 1.26 x 10(11) - 1.68 x 10(11) PLTs. These numbers of PLTs are equivalent to 2.5 - 3.4 units of random donorderived PLTs or 2/ 5 - 6/ 10 of single- apheresis PLTs. The CB- derived PLTs exhibited features quite similar to those from peripheral blood in morphology, as revealed by electron micrographs, and in function, as revealed by fibrinogen/ ADP aggregation, with the appearance of beta-selectin and activated glycoprotein IIb- IIIa antigens. Thus, this culture system may be applicable for large- scale generation of PLTs for future clinical use.