Hao Si-Guo, Sun Guan-Lin, Wu Wei-Li, Wu Ying-Li
Stem Cell Laboratory, Shanghai Institute of Hematolgy, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2003 Dec;11(6):569-75.
This study was to investigate dynamics of biological properties of CD133(+) cells from human umbilical cord blood (UCB) during short-term culture containing the combination of hematopoietic growth factors and the feasibility of in vitro expansion of CD133(+) cells. The biology activities including analysis of cell cycle, immunophenotype, telomerase activity, expression of adhesion molecules and expansion potential of CD133(+) cells were monitored during ex-vivo expansion, and compared with those of CD34(+) cells. The results showed that the contents of CD133(+) and CD34(+) cells in fresh UCB were (1.05 +/- 0.73)% and (1.40 +/- 0.56)% respectively. About 79.62% of CD34(+) cells expressed CD133, and more than 97% of CD133(+) cells were CD133(+)CD34(+), markedly higher than that in CD34(+) fraction (P < 0.01). No significant differences were observed in content of cells expressing CD38, CD13, CD14, CD61 and glycophorin-A between the two fractions. Expansion of CD133(+), CD133(+)CD34(+) and CD34(+)CD38(-) cells at 10 days and those of CFU-mix, HPP-CFC and CD34(+)CD38(-) cells at 6 days from CD133(+) cells group were significantly higher than those from the CD34(+) cell group (P < 0.05). Analysis of immunophenotype showed that CD133(+)CD34(+) cells declined gradually while CD133(-)CD34(+) and CD133(-)CD34(-) cells increased during ex-vivo expansion; basal telomerase activities of fresh UCB CD133(+) and CD34(+) cells were low but significantly exceeded that of CD34(-) fraction (P < 0.05). At first week of expansion, telomerase activity was significantly upregulated, after two weeks, telomerase activity remarkably declined, and decreased to baseline or below the limits of detection in day 20. More than 90% of CD133(+) cells expressed CD49d and CD11a, and, more than 85% of the cells expressed CD54, about 50% of cells expressed CD62L. At the early stage of expansion, expression of CD49d was upregulated, expression of CD11a remaining no change, while as expression of CD54 and CD62L was downregulated. Expression of all adhesion molecules was decreased gradually with extend of culture. But expression of these adhesion molecules on CD34(+) subsets were not affected significantly during expansion. It is concluded that CD133(+) population may be a more primitive hematopoietic stem/progenitor cells (HSPC) than CD34(+) cells, CD133(+) cells have great expansion potential for ex-vivo expansion and is a suitable target cell for ex-vivo expansion of HSPC. Downregulation of adhesion molecules and telomerase activity may be one of the reasons for delayed engraftment of expanded products.
本研究旨在探讨在含有造血生长因子组合的短期培养过程中，人脐带血（UCB）中CD133(+)细胞生物学特性的动态变化以及CD133(+)细胞体外扩增的可行性。在体外扩增过程中监测CD133(+)细胞的生物学活性，包括细胞周期分析、免疫表型、端粒酶活性、黏附分子表达及扩增潜能，并与CD34(+)细胞进行比较。结果显示，新鲜UCB中CD133(+)和CD34(+)细胞含量分别为(1.05±0.73)%和(1.40±0.56)%。约79.62%的CD34(+)细胞表达CD133，且超过97%的CD133(+)细胞为CD133(+)CD34(+)，显著高于CD34(+)组分中的比例（P<0.01）。两组分中表达CD38、CD13、CD14、CD61和血型糖蛋白A的细胞含量无显著差异。CD133(+)细胞组在第10天的CD133(+)、CD133(+)CD34(+)和CD34(+)CD38(-)细胞扩增以及在第6天的CFU-mix、HPP-CFC和CD34(+)CD38(-)细胞扩增均显著高于CD34(+)细胞组（P<0.05）。免疫表型分析显示，在体外扩增过程中CD133(+)CD34(+)细胞逐渐减少，而CD133(-)CD34(+)和CD133(-)CD34(-)细胞增加；新鲜UCB中CD133(+)和CD34(+)细胞的基础端粒酶活性较低，但显著超过CD34(-)组分（P<0.05）。在扩增的第一周，端粒酶活性显著上调，两周后，端粒酶活性显著下降，并在第20天降至基线或低于检测限。超过90%的CD133(+)细胞表达CD49d和CD11a，超过85%的细胞表达CD54，约50%的细胞表达CD62L。在扩增早期，CD49d表达上调，CD11a表达无变化，而CD54和CD62L表达下调。随着培养时间延长，所有黏附分子的表达均逐渐降低。但在扩增过程中，这些黏附分子在CD34(+)亚群上的表达未受到显著影响。结论是，CD133(+)群体可能是比CD34(+)细胞更原始的造血干/祖细胞（HSPC），CD133(+)细胞具有很大的体外扩增潜能，是HSPC体外扩增的合适靶细胞。黏附分子下调和端粒酶活性降低可能是扩增产物植入延迟的原因之一。
