title: Separation of SSEA-4 and TRA-1-60 labelled undifferentiated human embryonic stem cells from a heterogeneous cell population using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS).
abstract: 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.
authors.affiliation: Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Chui Yee
authors.initials: CY
authors.lastname: Fong
authors.forename: Gary S L
authors.initials: GS
authors.lastname: Peh
authors.forename: Kalamegam
authors.initials: K
authors.lastname: Gauthaman
authors.forename: Ariff
authors.initials: A
authors.lastname: Bongso
mesh_terms: D000954:Antigens, Surface / Q000032:analysis / Q000096:biosynthesis*; D014408:Biomarkers, Tumor; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods*; D003584:Cytological Techniques; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008280:Magnetics; D011509:Proteoglycans / Q000096:biosynthesis*; D055746:Stage-Specific Embryonic Antigens / Q000096:biosynthesis*; D013194:Staining and Labeling
chemical_list: D000954:Antigens, Surface; D014408:Biomarkers, Tumor; D011509:Proteoglycans; D055746:Stage-Specific Embryonic Antigens; C107064:TRA-1-60 antigen, human; C066584:stage-specific embryonic antigen-4