Hao Mu, Li Si-Dan, Wu Tong, Meng Heng-Xin, Li Chang-Hong, Xu Yan, Qiu Lu-Gui
National Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2008 Dec;16(6):1403-7.
The aim of this study was to investigate the support effects of mesenchymal stem cells (MSCs) on umbilical cord blood (UCB) CD34+ cell (HSPC) expansion in vitro and its influence on cell characteristics including the surface marker of CD34+ cells, homing adhesion molecules and colony-forming ability. The mononucleated cells (MNCs) were isolated from UCB, then the CD34+ cells were isolated from freshly obtained MNCs by immunomagnetic beads, the MSC feeder cells exposed to gamma-ray of 137Cs were prepared by MSC feeder. The CD34+ cells were inoculated in different culture media. Experiment was divided into 3 groups: HSPC+CK group in which cytokines were added to medium (SCF, FL and TPO); HSPC+MSC group in which CD34+ cells were inoculated on MSC feeder; HSPC+MSC+CK group in which cytokines and MSC feeder cells were added to medium. After culture for 4, 7, 10, 14 days the MNC amount was counted and expansion ability of CD34+ cells was evaluated. The immunotypes of CD34+ cells and subsets, homing adhesion molecules and colony-forming ability in different groups detected by flow cytometry. The results showed that the amount of MNCs and CD34+ cells all obviously increased during culture for 14 days, the expansion levels of MNCs in 3 groups were HSPC+MSC+CK group>HSPC+CK group>HSPC+MSC group in proper order. Within 10 days of expansion in vitro amount of MNCs obtained significant expansion, meantime the expansion of CD34+ cells was higher also. The CD34+ count in 3 groups at day 4 of culture decreased significantly as compared with 0 day of culture (p<0.01). The CD34+ cells ratios in 3 groups after expansion were HSPC+MSC group>HSPC+MSC+CK group>HSPC+CK group in proper order (p<0.01), while CD34+ subset levels in 3 groups were different, the CD34+CD38- cells in HSPC+CK group at 4 days of culture increased transiently (62.71%), then quickly decreased, the CD34+CD38- cell ratio at day 7 was 0.05%, while the CD34+CD38- cell ratio in HSPC+MSC group at day 7 was 18.92%, difference was significant as compared with HSPC+CK group (p<0.05). The analysis of colony-forming units showed that the colony-forming ability at various time points after expansion all sustained in high level. It is concluded that in short-time (<7 day) culture of UCB CD34+ cells the combination of MSCs with cytokines can significantly expand the CD34+ cells and make the HSPCs to maintain original biologic characteristics.
本研究旨在探讨间充质干细胞（MSCs）对体外脐血（UCB）CD34+细胞（造血干细胞，HSPC）扩增的支持作用及其对细胞特性的影响，包括CD34+细胞的表面标志物、归巢黏附分子和集落形成能力。从UCB中分离单个核细胞（MNCs），然后通过免疫磁珠从新鲜获得的MNCs中分离CD34+细胞，用MSCs饲养层制备经137Csγ射线照射的MSC饲养细胞。将CD34+细胞接种于不同培养基中。实验分为3组：HSPC+CK组，向培养基中添加细胞因子（SCF、FL和TPO）；HSPC+MSC组，将CD34+细胞接种于MSC饲养层上；HSPC+MSC+CK组，向培养基中添加细胞因子和MSC饲养细胞。培养4、7、10、14天后，计数MNC数量并评估CD34+细胞的扩增能力。通过流式细胞术检测不同组中CD34+细胞及其亚群的免疫表型、归巢黏附分子和集落形成能力。结果显示，培养14天期间MNCs和CD34+细胞数量均明显增加，3组中MNCs的扩增水平依次为HSPC+MSC+CK组>HSPC+CK组>HSPC+MSC组。体外扩增10天内，MNCs数量获得显著扩增，同时CD34+细胞的扩增也较高。培养第4天时，3组中的CD34+细胞计数与培养第0天时相比显著降低（p<0.01）。扩增后3组中CD34+细胞比例依次为HSPC+MSC组>HSPC+MSC+CK组>HSPC+CK组（p<0.01），而3组中CD34+亚群水平不同，培养第4天时HSPC+CK组中CD34+CD38-细胞短暂增加（62.71%），然后迅速下降，第7天时CD34+CD38-细胞比例为0.05%，而HSPC+MSC组第7天时CD34+CD38-细胞比例为18.92%，与HSPC+CK组相比差异显著（p<0.05）。集落形成单位分析显示，扩增后各时间点的集落形成能力均维持在高水平。结论是，在UCB CD34+细胞的短期（<7天）培养中，MSCs与细胞因子联合可显著扩增CD34+细胞并使造血干细胞维持原始生物学特性。
