Fong Chui Yee, Peh Gary S L, Gauthaman Kalamegam, Bongso Ariff
Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Stem Cell Rev Rep. 2009 Mar;5(1):72-80. doi: 10.1007/s12015-009-9054-4. Epub 2009 Jan 31.
A major concern in human embryonic stem cell (hESC)-derived cell replacement therapy is the risk of tumorigenesis from undifferentiated hESCs residing in the population of hESC-derived cells. Separation of these undifferentiated hESCs from the differentiated derivatives using cell sorting methods may be a plausible approach in overcoming this problem. We therefore explored magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) to separate labelled undifferentiated hESCs from a heterogeneous population of hESCs and hepatocellular carcinoma cells (HepG2) deliberately mixed respectively at different ratios (10:90, 20:80, 30:70, 40:60 and 50:50) to mimic a standard in vitro differentiation protocol, instead of using a hESC-differentiated cell population, so that we could be sure of the actual number of cells separated. HES-3 and HES-4 cells were labelled in separate experiments for the stem cell markers SSEA-4 and TRA-1-60 using primary antibodies. Anti-PE magnetic microbeads that recognize the PE-conjugated SSEA-4 labelled hESCs was added to the heterogeneous cell mixture and passed through the MACS column. The cells that passed through the column ('flow-through' fraction) and those retained ('labelled' fraction') were subsequently analysed using FACS. The maximum efficacy of hESCs retention using MACS was 81.0 +/- 2.9% (HES-3) and 83.6 +/- 4.2% (HES-4). Using FACS, all the undifferentiated hESCs labelled with the two cell-surface markers could be removed by selective gating. Both hESCs and HepG2 cells in the 'flow-through' fraction following MACS separation were viable in culture whereas by FACS separation only the HepG2 cells were viable. FACS efficiently helps to eliminate the undifferentiated hESCs based on their cell-surface antigens expressed.
人类胚胎干细胞（hESC）衍生细胞替代疗法中的一个主要问题是，hESC衍生细胞群体中存在的未分化hESC有发生肿瘤的风险。使用细胞分选方法将这些未分化的hESC与分化衍生物分离，可能是克服这一问题的一种可行方法。因此，我们探索了磁性激活细胞分选（MACS）和荧光激活细胞分选（FACS），以分别从故意按不同比例（10:90、20:80、30:70、40:60和50:50）混合的hESC和肝癌细胞（HepG2）异质群体中分离标记的未分化hESC，以模拟标准的体外分化方案，而不是使用hESC分化的细胞群体，以便我们能够确定分离的实际细胞数量。在单独的实验中，使用一抗对HES-3和HES-4细胞进行干细胞标志物SSEA-4和TRA-1-60的标记。将识别与PE偶联的SSEA-4标记的hESC的抗PE磁性微珠添加到异质细胞混合物中，并通过MACS柱。随后使用FACS分析通过柱子的细胞（“流通”部分）和保留的细胞（“标记”部分）。使用MACS保留hESC的最大效率为81.0±2.9%（HES-3）和83.6±4.2%（HES-4）。使用FACS，可以通过选择性门控去除所有用两种细胞表面标志物标记的未分化hESC。MACS分离后“流通”部分中的hESC和HepG2细胞在培养中均存活，而通过FACS分离，只有HepG2细胞存活。FACS基于表达的细胞表面抗原有效地帮助消除未分化的hESC。
