Human ESCs are the gold standard to which hIPS cells are measured. Previous investigators have documented the epigenetic state of the X chromosome in hESCs after line establishment and on cells that have been exposed to at least one cryopreservation event. The general consensus is that established lines of hESCs have undergone XCI, and this result is often used as epigenetic evidence (together with culture conditions) that hESCs are more closely related to murine EpiSCs, which also exhibit XCI (reviewed in 25
). EpiSCs are derived from dissected epiblasts at either day 5.75 post-coitum (34
), or egg cylinder stage embryos at day 5.5 post-coitum (35
). In the mouse, this stage is when major changes to the embryo are underway, including the loss of the zonapellucida, implantation in the uterine epithelium and specialization of the extra embryonic lineages. In our study, human embryos were derived from blastocysts at days 6–7 post-fertilization where the zonapellucida was still mostly encapsulating a simple blastocyst (Supplementary Material, Figure S5
). This stage of human development has been elegantly analyzed by Okamoto et al
), which revealed that the majority of blastomeres at day 7 exhibit biallelic X-linked gene expression, and no enrichment of H3K27me3 on an X chromosome. In our study, we show that the new UCLA hESC lines derived from days 6–7 post-fertilization human embryos contained a significant fraction of nuclei that had not undergone XCI by passages 20–24 under normoxic conditions based on the nuclear exclusion assay. Although it is difficult to relate hESCs to any stage of development, our data would lend strong support to the theory that hESCs and EpiSCs are not derived from an equivalent developmental stage using XCI and embryo morphology as an indicator. Instead, our data strongly support the hypothesis that XCI of hESCs is driven by in vitro
culture, and that the timing of XCI after hESC derivation and the accumulation of XIST-independent class III nuclei is line-dependent.
In order to address the stability of class I, II and III nuclei, we cryopreserved the four female hESC lines at a time when the proportion of class I nuclei were >50% (UCLA1), >70% (UCLA5) and >90% (UCLA3 and UCLA4). Our data demonstrate that unlike continuous culture from the point of derivation where class I nuclei are relatively stable up to passage 24, a single cryopreservation event followed by thawing and re-analysis again at passages 20–24 results in conversion or selection of class III nuclei in three out of four hESC lines. These data illustrate that a given hESC line can significantly change after a major event such as cryopreservation. From a practical perspective, our data indicate that sharing hESC lines between laboratories or initiating new experiments from cryopreserved stocks does not guarantee preservation of class I or II nuclei. The subline experiment using HSF-6 also indicates that expanding hESCs from single cells in the presence of ROCK inhibitor promotes the class III state. However, given that the parental line also progressed to 100% class III during the same time frame suggests that increased ‘fitness’ of class III cells resulting in out-competition cannot be the sole cause for line progression to class III. Instead, our data argue that a combination of increased survival and epigenetic progression from class II to class III is responsible for increasing proportions of class III nuclei in a given hESC line with time.
One of the critical parameters in our study was the obvious heterogeneity at a single cell level within and between independently derived hESC lines with regard to the proportion of class I, II and III nuclear states. Under normoxic conditions, this epigenetic heterogeneity is ultimately resolved to an apparent irreversible class III XIST-independent state. In our studies, B and D were beneficial for improving the odds of retaining class I nuclei from new hESC derivations over sequential passages, but could not completely prevent the emergence of increasing proportions of class III nuclei with continuous culture post-derivation. This was further illustrated in the two HSF-6 sublines, where only 30% of nuclei were reprogrammed to class I, and the majority still progressed to class III. Why specific class II nuclei are more susceptible to B and B + D reprogramming is unclear from the current study, but suggests that within the class II population there is additional heterogeneity. Single-cell heterogeneity is a feature of hESC culture and is proposed to represent a stable continuum of cell states (36
). Together, our data demonstrate that the effect of B and D are line-dependent, and we propose that this is due not to the stochastic effects of the chemicals, but instead to the proportion of class I, II and III nuclei at the time when the chemicals are first introduced. Put simply, the more class II and III nuclei in a given culture, the less effective the chemical treatment for acquiring or retaining class I.
Maintaining class I nuclei in the self-renewing state is critical for studies that involve examining mechanisms of XCI as well as X-linked diseases. However, there is evidence that class III cells differentiate poorly as EBs (17
), and after extensive passaging, class III cells exhibit compromised response to BMP4 in adherent culture and an inability to differentiate into trophoblast (18
). These studies have led to the hypothesis that cells in the class III state are less capable of differentiation and possibly no longer pluripotent in teratoma assays. Here, we show unequivocally that hESC lines composed of 100% class III nuclei are fully capable of losing OCT4 protein during differentiation in vitro
, and differentiate as teratomas in vivo
, thereby refuting the hypothesis that class III nuclei do not differentiate and are not pluripotent. It should be noted that the teratoma assay does not capture the nuances of lineage-directed in vitro
differentiation where line-specific differences have been observed (37
). In the current study, we do not know whether class III nuclei in vitro
exhibit preference towards certain lineages or cell types relative to class I or II and this will be critical to analyze in future studies. This is particularly important given that in vitro
differentiation is the clinically relevant starting point to cell therapy.
Prior to the current study, three independent methods had been developed to reverse or prevent XCI in hESCs. For example, derivation and maintenance in hypoxia improves the odds of retaining class I nuclei relative to normoxic conditions but cannot act to reprogram XCI cells to an active state (26
). Although we did not evaluate hypoxia in our studies, our work does demonstrate that normoxia is compatible with the survival and self-renewal of class I nuclei for at least 20 passages without cryopreservation. Media supplementation with B has also been used previously to induce loss of XIST
from H9 hESCs (28
). Our data support a role for B in reprogramming class II nuclei to a class I state across multiple lines, although having no effect on class III. In more recent studies, hESCs can be reprogrammed to a naïve state by ectopic expression of Oct4
and by growth in media used to culture murine ESCs (27
). This strategy facilitates the recovery of cells that are capable of undergoing random XCI upon lineage differentiation. However, stable expression of transgenes is not relevant for generating clinically relevant cell types.
Although B and D supplementation demonstrates proof of principle that hESC nuclei in normoxic conditions can be maintained in the class I state, our microarray analysis indicated that B and D activity is not restricted to the X chromosome with the identification of more than 100 autosomal genes that are also differentially expressed in treated cultures. Gene ontology analysis did not reveal enrichment in any specific functional group. However, it did not escape our notice that one of the most significantly up-regulated gene was the imprinted gene H19, which was increased 36-fold with B + D treatment. Loss of imprinting has been associated with tumorigenicity (38
). Furthermore, evidence exists that up-regulation of H19
is associated with human hepatocellular carcinoma (39
). Although we do not know the functional consequence of H19
up-regulation on hESC biology, the fact that B + D caused gene expression changes genome-wide including an imprinted locus would possibly argue against the use of B + D in routine hESC culture as a strategy for sustaining the class I state.
In summary, our data highlight the importance of maintaining hESC derivation efforts. Gold standard hESC lines should be the benchmark for human pluripotent stem cell research and we have shown this is achievable by preventing and reversing epigenetic progression of a major in vitro artifact. Developing new experimental approaches aimed at sustaining human pluripotent nuclei in an epigenetic state closer in identity to the days 6–7 human blastocyst is to work towards a more robust gold standard.