Matsunaga Takuya, Tanaka Ikuta, Kobune Masayoshi, Kawano Yutaka, Tanaka Maki, Kuribayashi Kageaki, Iyama Satoshi, Sato Tsutomu, Sato Yasushi, Takimoto Rishu, Takayama Tetsuji, Kato Junji, Ninomiya Takafumi, Hamada Hirofumi, Niitsu Yoshiro
Fourth Department of Internal Medicine, Sapporo Medical University, School of Medicine, S1W17, Sapporo, Japan.
Stem Cells. 2006 Dec;24(12):2877-87. doi: 10.1634/stemcells.2006-0309. Epub 2006 Sep 7.
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 10(6) 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 donor-derived 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 P-selectin and activated glycoprotein IIb-IIIa antigens. Thus, this culture system may be applicable for large-scale generation of PLTs for future clinical use.
在本研究中，我们使用三相培养系统从脐带血（CB）CD34(+)细胞生成血小板（PLT）。我们首先在转导了端粒酶基因的人基质细胞（hTERT基质）上培养500个CB CD34(+)细胞，培养基为无血清培养基，添加干细胞因子（SCF）、Flt-3/Flk-2配体（FL）和血小板生成素（TPO），培养14天。然后将细胞转移至hTERT基质，用除原始细胞因子混合物外还含有白细胞介素-11（IL-11）的新鲜培养基再培养14天。随后，我们在含有SCF、FL、TPO和IL-11的液体培养基中再培养细胞5天，以从上清液中回收PLT组分，然后进行凝胶过滤以纯化PLT。从1.0单位CB（5×10(6)个CD34(+)细胞）计算得出的PLT产量为1.26×10(11)-1.68×10(11)个PLT。这些PLT数量相当于2.5-3.4单位随机供体来源的PLT或单采血小板的2/5-6/10。电子显微镜照片显示，CB来源的PLT在形态上与外周血来源的PLT非常相似，纤维蛋白原/ADP聚集试验显示其在功能上也相似，出现了P-选择素和活化的糖蛋白IIb-IIIa抗原。因此，这种培养系统可能适用于大规模生成PLT以供未来临床使用。
