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    • In relation to SSEA expression the differentiation

    2018-10-22

    In relation to SSEA3 expression, the ‘differentiation threshold’ may lie at different points along the linear path of differentiation (Fig. 4) depending on the cell line and the culture conditions used to propagate undifferentiated ES cells. In comparison to normal human ES cells, the adapted human ES 5-ht receptors exhibit increased proliferation rates, high clonal efficiency and reduced spontaneous differentiation (Enver et al., 2005). Within normal ES cell cultures the ‘differentiation threshold’ may be positioned within the SSEA3 expressing zone, effectively condensing the size of the stem cell compartment in relation to SSEA3 expression. The adapted ES subline (H14 BJ1) used in this study possesses a stable pluripotent state that spontaneously differentiates at a reduced frequency in comparison to non-adapted cell lines yet generates differentiated teratocarcinomas when grown as xenograft tumours. Use of the culture adapted cell line allows for the ‘trapping’ of transient (but nevertheless important) states that would normally be inaccessible in ‘normal’ euploid cell lines. In this view, the adapted cell lines provide a means with which to analyse cell states that exist transitorily in the normal cells and are consequently difficult to observe. Thus, the use of an adapted cell line facilitated the analysis of single cell behaviour, however, further improvements in cell culture conditions will enable the analysis of single cell dissociated non-adapted hES cells. The observation that SSEA3Negative ES cells were capable of generating undifferentiated colonies in standard ES conditions yet displayed atypical differentiation in comparison to SSEA3Low and SSEA3High cells is possibly due to lineage priming. It has been reported that in routine ES culture the colony size of undifferentiated (OCT4 positive) human ES cells influences the expression levels of early differentiation markers (PAX6 and GATA6) which consequently bias differentiation upon embryoid body formation (Bauwens et al., 2008). The expression of early lineage markers in human ES colonies is reminiscent of the lineage priming which has been reported in mouse haematopoietic progenitor cells (Delassus et al., 1999). Haematopoietic progenitor subsets, isolated on the basis of Sca-1 expression, possess discrete gene expression profiles, which manifest in biassed erythroid or the myeloid differentiation capabilities (Chang et al., 2008). Notably the haematopoietic Sca-1 progenitor subsets are reported to be interconvertible. Likewise, SSEA3High and SSEA3Negative human ES cells are interconvertible. SSEA3 expression levels are informative of an ES cell\'s position within the stem cell compartment and its diminished expression may indicate the expression of early differentiation markers in cells. Isolation of lineage primed hES cells could help define the early stages of cell differentiation with a deeper knowledge of such lineage primed cells facilitating the directed differentiation of human ES cells. The pluripotent NTERA2 cell line has enabled us to track and model the expression of SSEA3 in human pluripotent stem cells. Undifferentiated cells were found to fluctuate between SSEA3High SSEA3Low SSEA3Negative states, whereby SSEA3Negative cells regained SSEA3 expression at high frequency. Although pluripotent cells often lost SSEA3 expression, they remained undifferentiated and the expression of SSEA3 was reacquired within days. Notably, the expression of SSEA3 is closely associated with the cloning efficiency of human ES and EC cells, suggesting that it remains a sensitive marker for assessing the health of a pluripotent stem cell population.
    Materials and methods
    Acknowledgments
    Introduction The potentially inexhaustible supply of self-renewing pluripotent human embryonic stem (hES) cells holds promise as a target source of transplantation material for the treatment of a myriad of debilitating and degenerative diseases. To date, the vast majority of hES cell lines have been derived on Mouse Embryonic Feeder (MEF) cells but these lines are inappropriate for clinical use because of the necessity for xenofree processing. There have been numerous reports describing the maintenance of hES colonies on primary human feeder layers from adult, neonatal and foetal sources including term and pre-term placenta (Deleu et al., 2009; Ilic et al., 2008; Liu et al., 2010; Tecirlioglu et al., 2010). Alternatives to primary cells include autogeneic cultures of human feeders derived from hES cells (Choo et al., 2008; Li et al., 2011), feeder-free (Hernandez et al., 2010; Rodin et al., 2010; Thomas et al., 2009), feeder-conditioned (Escobedo-Lucea & Stojkovic, 2010) and most recently suspension culture (Larijani et al., 2011). Although clinical grade, xenofree hES cell lines have recently been successfully derived on primary human feeders (Unger et al., 2008) such feeders are subject to donor supply and variability.