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Subsets of undifferentiated human ES cells have been propose
Subsets of undifferentiated human ES cells have been proposed based upon studies of surface antigen markers, such as SSEA3 (Enver et al., 2005) or CD9 and GCTM2 (Laslett et al., 2007). SSEA3 is a cell surface antigen that is rapidly down-regulated as human ES cells differentiate (Draper et al., 2002a; Shevinsky et al., 1982), but when artificially removed from the cell surface, human ES cells still retain a pluripotent stem cell phenotype (Brimble et al., 2007; Fenderson et al., 1993). The carbohydrate based surface antigens SSEA3 and SSEA4 are expressed by red blood cells, but the red cells of a small percentage of the human population (pp and pk individuals) lack the capacity to synthesise these globoseries antigens, suggesting that SSEA3 is not necessary for human development (Tippett et al., 1986). Nevertheless, SSEA3 expression is closely associated with the undifferentiated phenotype of human ES cells, and in a comparative analysis of early passage diploid human ES cells, and later passage, culture adapted variants, Enver et al.(2005) suggested that undifferentiated human ES cells can exist in two substates, SSEA3Positive and SSEA3Negative. In the early passage diploid cells, the SSEA3Positive stem cell state was posited to be particularly unstable, so that very few undifferentiated SSEA3Negative stem cells could be detected by clonogenic assays. However, culture pitavastatin Supplier appeared to ‘trap’ the stem cells in the stem cell compartment so that clonogenic SSEA3Negative undifferentiated stem cells could be readily detected in the variant later passage lines. It was suggested that, like the Nanog−/− mouse ES cells, the SSEA3Negative substate of human ES cells is less stable and closer to commitment to differentiation than cells in the SSEA3Positive substate (Enver et al., 2005).
On the basis of findings described above we hypothesised that the pluripotent human ES cell compartment consists of cells that possess different characteristics with regard to differentiation potential. Differentiation of human ES cells is commonly performed on a population of cells, which does not enable investigation of behavioural disparities between individual ES cells. We have examined the functional heterogeneity of ES cells by testing the capacity of single cells to choose between a neural or non-neural fate. Human ES cells require intercellular cues for survival and retention of the undifferentiated phenotype (Fox et al., 2008) and consequently many human ES cell lines are not amenable to functional analysis at the single cell level. H14-BJ1 is a subline of culture adapted H14 human ES cells which permits functional analysis at the single cell level. We have used both culture adapted human ES cells and their malignant counterparts from teratocarcinomas, embryonal carcinoma cells, to investigate whether the SSEA3 substates are functionally different with regard to differentiation potential.
Results
Conclusions
Our observations are consistent with a model in which the undifferentiated stem cell compartment consists of a range of functionally distinct substates, which are characterised by graduated loss of SSEA3 expression. The observed accelerated differentiation of SSEA3Low cells to a neural phenotype is possibly due to SSEA3High and SSEA3Low stem cells representing distinct substates in the stem cell compartment, whereby SSEA3 expression level is a function of a cell\'s proximity to the ‘differentiation threshold’ (Fig. 4). The ‘differentiation threshold’ signifies the point at which an ES cell irreversibly crosses to the differentiated state. Our results indicate that the differentiation threshold resides within the SSEA3Negative zone, since a fraction of SSEA3Negative cells are capable of generating undifferentiated colonies in standard growth conditions, but unlike the SSEA3High or SSEA3Low cells, they do not generate OCT4 positive or neural rosette containing colonies in neural permissive conditions. Thus, human ES cells in close proximity to the ‘differentiation threshold’ respond differently to differentiation stimuli.