Vascular tissue engineering by cord blood stem cell

在获得知情同意后，在正常足月分娩期间获得人类脐带血。单核细胞采用Ficoll-Hypaque密度梯度离心分离，CD34^+细胞采用磁珠分离法纯化。95%以上的细胞为CD34^+。纯化的CD34^+细胞在各种细胞因子组合的无血清悬浮培养中孵育，并在第14天和第21天使用单克隆抗体面板流式细胞术进行分析。采用IL6、SCF、TPO、IL3、EPO、FLT3和G-CSF联合检测悬浮培养中各时间点产生的各种祖细胞。部分实验将含有端粒酶全长cDNA的质粒（hTERT）和模拟载体转染到具有白血病干细胞特征的K562细胞中，以期为端粒酶基因治疗的可能途径提供数据。巨核细胞分数以CD41^+和/或CD42^+细胞的百分比来确定。TPO对巨核细胞分化具有生长优势，而SCF对巨核细胞分化具有抑制作用。在含TPO作为单一生长因子的培养中，CD41^+细胞在第14天达到最大增殖，当培养基中添加ingenol时，CD41^+细胞的总百分比稳步增加。hTERT在k562细胞中的异位表达在缺乏血清的情况下显示出生存优势。转导后的细胞保留表型特征、分化能力和对TPA的信号转导反应。这些数据表明，正常造血干细胞异位表达hTERT可能在不改变先天生物学特性的情况下赋予生存优势。

Human umbilical cord blood was obtained during normal full-term deliveries after obtaining informed consent. Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation and CD34^+ cells were purified by a magnetic bead separation method. More than 95% of recovered cell was CD34^+.Purified CD34^+ cells were incubated in serum-free suspension culture in the presence of various combination of cytokines and analyzed by flowcytometry using a panel of monoclonal antibodies at day 14 and 21. A combination of IL6, SCF, TPO, IL3, EPO, FLT3 and G-CSF was used for the determination of various progenitors generated in suspension culture at each time points. In some experiments, the plasmid containing the full-length cDNA of telomerase(hTERT) and mock vector were transfected to K562 cells with the characteristics of leukemic stem cells in order to provide data on a possible approach to telomerase gene therapy.The megakaryocytic fraction was determined as the percentage of CD41^+ and/or CD42^+ cells. TPO always resulted in growth advantage for megakaryocytic differentiation, whereas SCF suppressed the differentiation. In cultures containing TPO as a single growth factor, maximal expansion of CD41^+ cells was achieved at day 14 and total percentage of CD41^+ cells steadily increased when ingenol was added in culture medium. Ectopic expression of hTERT in k562 cells showed a survival advantage in the absence of serum. Transduced cells retained phenotypic characteristics, differentiation ability, and the signal transduction response to TPA. These data suggest that ectopic expression of hTERT by normal hematopoietic stem cells may confer a survival advantage without changing innate biological characteristics.