We have previously shown that human ESC cultures maintained

We have previously shown that human ESC cultures maintained in serum-supplemented medium on feeder cell layer support consist of a hierarchy of cells defined by a continuum of levels of expression of stem cell surface alk inhibitor and pluripotency-specific genes (Laslett et al., 2007). Heterogeneity and lineage priming are most meaningfully evaluated at the single-cell level. Examination of the expression of a small panel of pluripotency and lineage-specific genes at the single-cell level provided evidence for heterogeneity in pluripotency gene expression, and for lineage priming in the stem cell population (Hough et al., 2009). Here, we extend this quantitative analysis of gene expression at the single-cell level to a much larger panel of genes, using more sensitive assays, and relate it to key biological features of ESCs. We further compare heterogeneity of human ESC cultures under different growth conditions. The results show that the capacity for self-renewal lies in a restricted subset of cells marked by expression of a key set of genes associated with cell-cell interactions. Using a selective combination of cell surface markers, we describe the prospective isolation of a minority cell population with high levels of self-renewal, high and uniform levels of expression of pluripotency-associated genes, and no lineage priming, features of the naive state.
Results
Discussion Many recent studies have documented heterogeneity of gene expression and lineage priming within PSC populations. One interpretation of these findings is that heterogeneity is an inherent feature of the pluripotent state. This is a feasible hypothesis, because pluripotent cells exist only within a transient time window of mammalian embryonic development, during which they are poised to undergo specification first toward extraembryonic, then embryonic cell lineages. Mammalian development is plastic and highly regulative, and pathways that affect ESC specification in vitro can regulate the size of the pluripotent cell population in vivo (Morris et al., 2012), suggesting that the pluripotent population in vivo can readily undergo state transitions. However, for a restricted point in development in vivo, or under specific conditions in vitro, pluripotent cells with prototypic patterns of gene expression may exist locked into a pure state. Single-cell analysis revealed that individual epiblast cells of the 64 cell stage mouse embryo expressed predominantly pluripotency genes only, in contrast to cells at the 16 and 32 cell stages, which coexpressed pluripotency genes along with genes of the extraembryonic lineages (Guo et al., 2010). Recent evidence suggests that mouse ESCs maintained under conditions that maximize self-renewal are far more homogenous in gene expression and epigenetic status than cells maintained under conditions that are permissive for spontaneous differentiation (Marks et al., 2012). Our data on hESCs indeed confirm that heterogeneity is certainly a function of stem cell microenvironment. Cells maintained in FCS show heterogeneity in expression of pluripotency genes and exist in a continuum between a primordial self-renewing cell population and cells that are primed toward neural differentiation. By contrast, the majority of the cells maintained in KSR/FGF and feeder layers are in the high and mid cell compartments, as are those grown in MTeSR. Nonetheless, a significant subset of cells grown in these conditions showed priming toward the endodermal lineage. That the cells expressing HNF4a, GATA6, and GATA4 most likely represent precursors of extraembryonic endoderm rather than definitive endoderm is suggested by their coexpression of LEFTY1, MIXL1, and CER1 along with pluripotency genes including CD9 (Cheng et al., 2012; Perea-Gomez et al., 2002). It is clear from our work and from many other published studies that human ESCs can undergo differentiation into cells resembling primitive endoderm (Darr and Benvenisty, 2009; Feng et al., 2012; Hyslop et al., 2005; Pera et al., 2004; Séguin et al., 2008; Sumi et al., 2007; Takayama et al., 2011). Recent studies have identified cells of the primitive endoderm lineage in the human conceptus (O’Leary et al., 2012; Yan et al., 2013). O’Leary et al. (2012) showed that the expression of GATA-4 and GATA-6 in the inner cell mass and epiblast followed the temporal patterns seen during mouse hypoblast development. Yan et al. (2013) performed RNA sequencing (RNA-seq) on single cells from late human blastocysts and classified cells as epiblast, primitive endoderm, and trophectoderm. This study showed considerable overlap in gene expression between epiblast and primitive endoderm but not trophoblast. In line with our results, GATA-4, GATA-6, HNF4A, NODAL, LEFTY, GDF3, and TDGF1 were all expressed in epiblast and primitive endoderm, which also expressed KRT8 at high levels. These findings strongly suggest that the lineage priming we have observed is a feature of normal human development and are in line conceptually with the results of Canham et al. (2010), who identified two subpopulations of mouse ESCs, one predisposed to extraembryonic differentiation, the other to somatic fates.