Luo Fang, Yang Yu-jia, Wang Xia, Yu Xiao-he, Wang Qing-hong, Song Jian-hui, Liu Chen-tao, Huang Rong
Department of Pediatrics, Xiangya Hospital, Central South University, Changsha 410008, China.
Zhonghua Er Ke Za Zhi. 2006 Jul;44(7):509-12.
Emerging evidences suggest that mesenchymal stem cells (MSCs) can be isolated from human umbilical cord blood (HUCB) and cultured in vitro, the same as the MSCs derived from bone marrow. However previous attempts to isolate MSCs from UCB showed a low rate of success (less than 30%). The present study was conducted to clarify the factors that influence the yields of MSCs from HUCB of different gestational age deliveries and to observe the bioactivity of MSCs derived from UCB.
HUCB units were divided into three groups: gestational age (GA) 40 w group (n = 11); GA 36 w group (n = 6); GA 32 w or less than 32 w group (n = 5), cultured with optimal culture conditions. The relationship of the yields of MSCs derived from HUCB with several factors such as GA, the collected volume of HUCB and the mononuclear cells (MNCs) count of UCB, and the relationship among these factors were investigated. The bioactivity was observed by drawing the growth curve, calculating the population doubling, counting the fibroblast colony forming units (CFU-F) and detecting the surface antigen expression of MSCs by flow cytometry.
The success rate of generating MSCs cells was up to 54.5%. There were some correlations between the success rate and such factors as the MNCs count, the GA and the volume of UCB. The rate could be enhanced to 83.3% when the MNCs count was more than 1.25 x 10(8)/L. There was a negative correlation between the MNCs count in the same HUCB volume and the gestational age. The count of CFU-F varied with gestational age, the count of CFU-F was higher in smaller gestational age than the older. In the primary culture some cells displayed a fibroblast-like morphology and expressed MSCs-related antigens CD29, CD105, and the expression rate of these antigens were enhanced from 62.1% to 85.0% in one passage. The hematopoietic cells antigens CD34 and CD45 were less than 3% all the time.
The success rate could be increased when the MNCs count was more then 1.25 x 10(8)/L. There was a negative correlation between the MNCs count of the same UCB volume and the gestational age, the activity to form the CFU-F of UCB varied with gestational age; isolation of MSCs from UCB of pre-term deliveries may be relatively easier as compared to those from full term deliveries.
新出现的证据表明，间充质干细胞（MSCs）可从人脐带血（HUCB）中分离并在体外培养，与源自骨髓的MSCs相同。然而，先前从脐带血中分离MSCs的尝试成功率较低（低于30%）。本研究旨在阐明影响不同孕周分娩的HUCB中MSCs产量的因素，并观察源自脐带血的MSCs的生物活性。
将HUCB样本分为三组：孕周（GA）40周组（n = 11）；GA 36周组（n = 6）；GA 32周或小于32周组（n = 5），在最佳培养条件下培养。研究了源自HUCB的MSCs产量与GA、HUCB采集量和脐带血单个核细胞（MNCs）计数等因素的关系，以及这些因素之间的关系。通过绘制生长曲线、计算群体倍增、计数成纤维细胞集落形成单位（CFU-F）和用流式细胞术检测MSCs的表面抗原表达来观察生物活性。
产生MSCs细胞的成功率高达54.5%。成功率与MNCs计数、GA和脐带血体积等因素之间存在一些相关性。当MNCs计数超过1.25×10⁸/L时，成功率可提高到83.3%。相同脐带血体积中的MNCs计数与孕周呈负相关。CFU-F计数随孕周而变化，孕周较小者的CFU-F计数高于孕周较大者。在原代培养中，一些细胞呈现成纤维细胞样形态并表达与MSCs相关的抗原CD29、CD105，这些抗原的表达率在传代一次后从62.1%提高到85.0%。造血细胞抗原CD34和CD45一直低于3%。
当MNCs计数超过1.25×10⁸/L时，成功率可提高。相同脐带血体积中的MNCs计数与孕周呈负相关，脐带血形成CFU-F的活性随孕周而变化；与足月分娩的脐带血相比，从早产脐带血中分离MSCs可能相对更容易。
