He Yi, Meng Hen-Xing, Zhang Yu-Guang, Hou Shi-Fang, Li Qian, Han Jun-Ling, Qiu Lu-Gui, Han Zhong-Chao
Union Stem Cell & Gene Engineering Company LTD, Tianjin 300384, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2008 Oct;16(5):1121-5.
This study was purposed to investigate the biological characteristics and immunogenicity changes of ex vivo expanded megakaryocyte progenitors from human umbilical cord blood CD34(+) cells in order to provide experimental basis for clinical application of ex vivo expanded umbilical cord blood megakaryocyte progenitor cells. Mononuclear cells (MNCs) were obtained from umbilical cord blood by Ficoll-Hyapaque density gradient separation. CD34(+) cells were enriched by magnetic cell sorting (MACS). The selected CD34(+) cells were seeded in serum-free medium stimulated with thrombopoietin (TPO, 50 ng/ml), interleukin 11 (IL-11, 50 ng/ml), and heparin (25 U/ml) for 14 days. The immunophenotyping (CD34(+), CD41a(+), CD61(+), CD34(+) CD41a(+) and CD34(+) CD61(+) cells) of amplificated products, matured megakaryocyte apoptosis, and expression of human leukocyte antigen (HLA) class I and class II molecules were measured by fluorescence-activated cell sorter (FACS). The number of colony-forming units-megakaryocyte (CFU-Mk) was also evaluated by CFU-Mk assay. The results showed that the umbilical cord blood CD34(+) mononuclear cells could be effectively differentiated into megakaryocytes. The peak expression ratios of CD41a(+) and CD61(+) cells were all at 14th days, while that of CD34(+) CD41(+) and CD34(+) CD61(+) cells were at 7th day [(3.41 +/- 2.80)% and (1.89 +/- 1.43)%, respectively]. The expansion times of large and small CFU-Mk reached peak at 7th day (20.66 +/- 32.79) and 10th day (435.62 +/- 482.65), respectively. The apoptotic rates of megakaryocytes at 7th, 10th, 14th day were (19.48 +/- 9.64)%, (26.87 +/- 9.03)%, and (52.46 +/- 11.74)%, respectively. The apoptotic rate of megakaryocytes had no significant difference in 7 and 10 days culture (p > 0.05), while that significantly increased in culture for 14 day culture, compared with culture for 7 and 10 days (p < 0.05, respectively). The expression of HLA class I and class II molecules on megakaryocytes decreased along with the prolongation of expansion time and sharply decreased in 0 to 10 days. It is concluded that the cytokines of TPO, IL-11, and heparin can promote the expansion of megakaryocyte progenitors from umbilical cord blood CD34(+) mononuclear cells effectively in vitro. The peaked expansion times of large CFU-Mk, the peaked expression ratios of CD34(+) CD41(+) and CD34(+) CD61(+) cells were all at 7th day. So the culture for 7 days appeared to be the optimal duration of expanding megakaryocyte progenitors.
本研究旨在探讨人脐带血CD34(+)细胞体外扩增的巨核细胞祖细胞的生物学特性及免疫原性变化，为体外扩增的脐带血巨核细胞祖细胞的临床应用提供实验依据。采用Ficoll-Hyapaque密度梯度分离法从脐带血中获取单个核细胞（MNCs）。通过磁珠细胞分选（MACS）富集CD34(+)细胞。将分选得到的CD34(+)细胞接种于无血清培养基中，用血小板生成素（TPO，50 ng/ml）、白细胞介素11（IL-11，50 ng/ml）和肝素（25 U/ml）刺激培养14天。用荧光激活细胞分选仪（FACS）检测扩增产物的免疫表型（CD34(+)、CD41a(+)、CD61(+)、CD34(+) CD41a(+)和CD34(+) CD61(+)细胞）、成熟巨核细胞凋亡情况以及人白细胞抗原（HLA）I类和II类分子的表达。同时通过巨核细胞集落形成单位（CFU-Mk）测定法评估CFU-Mk的数量。结果显示，脐带血CD34(+)单个核细胞可有效分化为巨核细胞。CD41a(+)和CD61(+)细胞的峰值表达率均在第14天，而CD34(+) CD41(+)和CD34(+) CD61(+)细胞的峰值表达率在第7天[分别为(3.41±2.80)%和(1.89±1.43)%]。大、小CFU-Mk的扩增倍数分别在第7天（20.66±32.79）和第10天（435.62±482.65）达到峰值。第7天、第10天、第14天巨核细胞的凋亡率分别为(19.48±9.64)%、(26.87±9.03)%和(52.46±11.74)%。巨核细胞凋亡率在培养7天和10天时无显著差异（p>0.05），但与培养7天和10天相比，培养14天时显著升高（分别为p<0.05）。巨核细胞上HLA I类和II类分子的表达随扩增时间延长而降低，在0至10天内急剧下降。结论：TPO、IL-11和肝素细胞因子能在体外有效促进脐带血CD34(+)单个核细胞来源的巨核细胞祖细胞的扩增。大CFU-Mk的扩增倍数峰值、CD34(+) CD41(+)和CD34(+) CD61(+)细胞的峰值表达率均在第7天。因此，培养7天似乎是扩增巨核细胞祖细胞的最佳时间。
