Wang S Y, Hsu M L, Huang M Z, Hsu H C, Tzeng C H, Hung J H, Ho C K
Department of Medical Research, Veterans General Hospital-Taipei, 201 Shih-pai Road, Taipei 11217, Taiwan, ROC.
Acta Haematol. 2001;105(1):38-44. doi: 10.1159/000046531.
A total of 50 human umbilical cord blood (UCB) samples were studied. The hematopoietic stem/progenitor (CD34+) populations were isolated from UCB mononuclear cells (MNC) by means of immunomagnetic separation. Double immunofluorescent staining of UCB CD34+ cells revealed that there was a high proportion (82.33 +/- 4.47%) of CD34+ cells co-expressing CD13, while the percentage of CD34+ CD33+ cells was much lower (22.17 +/- 3.35%). In contrast, for co-expressing lymphoid differentiation antigens, the proportion of CD34+CD38+ cells (38.34 +/- 6.09%) was relatively higher than that of CD34+CD10+ cells (11.52 +/- 1.24%) or CD34+CD2+ cells (9.84 +/- 2.30%). For stimulating the ex vivo expansion of UCB progenitor cells, no single hematopoietic growth factor (HGF) was efficacious when used alone, while combination of 4 HGFs, such as GM-CSF, G-CSF, IL-3, and SCF could induce a 55-fold increase in the myeloid progenitor cells, day-14 CFU-GM, in a short term of 7 days' liquid culture. Cryopreservation of UCB as MNC preparations at -196 degrees C could satisfactorily retain the number and activity of CD34+ cells. After thawing, a high recovery rate of about 80% CD34+ cells was obtained. When suspended in liquid cultures containing a combination of 4 HGFs, as shown above, the frozen cord blood progenitor cells could be well expanded, reaching a >50-fold increase in day-14 CFU-GM, which was very similar to that of the fresh UCB samples. In addition, a similar result was also seen in CFU-GEMM, indicating that after cryopreservation the recovered UCB progenitor cells retain an intact clonogeneic ability capable of efficiently responding to hematopoietic growth factors for ex vivo expansion.
共研究了50份人脐带血（UCB）样本。通过免疫磁珠分离法从UCB单个核细胞（MNC）中分离出造血干/祖细胞（CD34+）群体。对UCB CD34+细胞进行双重免疫荧光染色显示，共表达CD13的CD34+细胞比例较高（82.33±4.47%），而CD34+CD33+细胞的百分比则低得多（22.17±3.35%）。相比之下，对于共表达淋巴样分化抗原，CD34+CD38+细胞的比例（38.34±6.09%）相对高于CD34+CD10+细胞（11.52±1.24%）或CD34+CD2+细胞（9.84±2.30%）。为了刺激UCB祖细胞在体外的扩增，单独使用单一造血生长因子（HGF）无效，而GM-CSF、G-CSF、IL-3和SCF这4种HGF联合使用可在7天的短期液体培养中使髓系祖细胞（第14天的CFU-GM）增加55倍。将UCB作为MNC制剂在-196℃下冷冻保存可令人满意地保留CD34+细胞的数量和活性。解冻后，获得了约80%的CD34+细胞的高回收率。当悬浮于含有上述4种HGF组合的液体培养物中时，冷冻的脐血祖细胞可得到良好的扩增，第14天的CFU-GM增加>50倍，这与新鲜UCB样本非常相似。此外，在CFU-GEMM中也观察到了类似的结果，表明冷冻保存后回收的UCB祖细胞保留了完整的克隆形成能力，能够有效响应造血生长因子进行体外扩增。
