Human embryonic stem cells are a unique and critical resource to study epigenetics during early human development and have enormous clinical potential, but the varying epigenetic status of these different hESC lines may have a great impact on their future utility (Humpherys et al., 2001
; Li et al., 2005
; Mitalipov, 2006
; Allegrucci et al., 2007
). X-inactivation represents the early formation of facultative heterochromatin, that distinguishes one cell from another. From mouse it is widely believed that this commitment occurs with the onset of the earliest lineage commitments. Thus, a cell that has not already formed the cell specific and chromosome wide
facultative heterochromatin of the Xi can be reasonably considered more “epigenetically naive” than one that has taken this step, even if both populations express similar ES markers.
The ability to study the human
X-inactivation process requires the availability of female hES cell lines that are capable of initiating X-inactivation in culture. The only other systems available to study X-inactivation in human cells are: karyotypically abnormal male embryonal carcinoma (EC) cells (e.g., Looijenga et al., 1997
; Chow et al., 2003
), mouse/human somatic cell hybrids induced to express XIST (e.g., Clemson et al., 1998
; Anderson and Brown, 2002
), or transgenic somatic human cancer cell lines or mouse ES lines (e.g. Migeon et al., 1999
; Chow et al., 2002
; Hall et al., 2002
). For the mouse, it took the derivation of many female mouse ES lines before a few were found that maintained two X-chromosomes and initiated X-inactivation appropriately (Zvetkova et al., 2005
). Our findings indicate that this may also be the case for the human ES cell system. Here we find that subcultures of the majority of NIH approved female hES lines we have screened have undergone precocious X-inactivation while in an undifferentiated state.
Importantly, however, through detailed analysis of “sublines” of H9, we were able to identify two H9 sublines, one karyotypically normal (UW-H9), and one trisomy X (UW-H9+X), that retained most cells which had not yet inactivated their Xi, and which largely did so upon differentiation. We suggest that this delicate epigenetic balance is unstable and that there is a propensity for hESC maintained in culture to undergo “precocious” or “premature” X-inactivation. A recent report used PCR to examine the expression of many genes including XIST in a large number of hESC lines, but did not assess the X-inactivation status of these lines (Adewumi et al., 2007
). Few of the lines examined were NIH approved, and their competence to initiate X-inactivation upon differentiation was not studied. In addition, the detection of XIST expression in undifferentiated ES03 and ES02 cells is consistent with our demonstration of precocious inactivation. Adewumi et al. (2007)
also found that undifferentiated BG03 and HES01 had low XIST, similar to male cells, but it remains to be determined if these cells are capable of initiating inactivation. Additionally, as further demonstrated in our study, this status can vary significantly between sublines of the same line. Thus, to effectively use any NIH approved sublines to study human X-inactivation, and to propagate the most “epigenetically naïve” lines available, it will be valuable to expand and preserve sublines that retain competence to initiate X-inactivation upon differentiation.
Interestingly, these findings bear on the timing of the human X-inactivation process, which cannot be assumed to be the same as in the mouse, where neither the ICM of the blastocyst, nor the ES cells derived from it, show random X-inactivation. While the presence of Xi in so many apparently undifferentiated hESC cultures might suggest that random Xi occurs earlier in the human ICM at the blastocyst stage of normal (or IVF) human embryogenesis, we pursued detailed analysis of sublines of one line in attempt to determine if more epigenetically naïve cells were present in the original line established from the ICM. Our findings of certain subcultures that are a “tableau rasseau” with respect to Xi suggests that at least for the H9 isolate most likely follows a mouse-like pattern, and that the “precocious” inactivation is likely due to adaptation to culture. This could also be true for the other lines as well.
It is possible that in human ES cells, as in mouse, there may be selection against the presence of two active X chromosomes. Some evidence in mouse indicates that this is due to abnormal methylation levels (Zvetkova et al., 2005
). It may be very difficult and unnatural to induce ES cells of the blastocyst ICM to propagate indefinitely with two active X-chromosomes, which they were never biologically programmed to do. In fact, there is a very small window in mammalian development in which the two X-chromosomes remain active. Prior to the mouse blastocyst stage (<day 3.5), the paternal X remains inactive due to parental imprinting, and after this stage (>day 5.5) random X-inactivation has taken place, thus, leaving just 2 days during which two X-chromosomes may be active (Heard and Disteche, 2006
). However, in contrast to many female mouse ES lines, human cells do not eliminate
an X-chromosome during propagation, but tend to inactivate it. Since human X-chromosomes contain numerous genes that escape
inactivation and are required in two doses, human XX ES cells may need to find other ways to eliminate the presumed growth suppression associated with having two X-chromosomes active during ESC propagation. Our data and others suggest that the most common route is for hESC lines to initiate inactivation early.
In this screen we also found three very interesting hESC phenotypes which may provide valuable systems to study certain aspects of X-inactivation. The UW-H9 line often acquires an additional X-chromosome (UW-H9+X) (Ware et al., 2005
), but we find that only one
of the three X-chromosomes is inactivated in this subline whereas we saw two Xi in our previously described H9 trisomy-X line (Hoffman et al., 2005
). While we have not investigated the precise mechanism to account for this, this UW-H9 line could potentially provide a valuable model for defective X-chromosome “counting.”
Here we have surveyed the female human embryonic cell lines that were already established and approved for NIH funding when federal funding of hESC research was first authorized in August of 2001. While undifferentiated cultures of most sublines already contain an Xi (Enver et al., 2005
; Hoffman et al., 2005
; Allegrucci et al., 2007
), scrutiny of numerous sources of one line (H9) identified some sublines for which most cells “appropriately” initiate inactivation upon differentiation. These H9 sublines are thus the most promising source for cells that retain competence to initiate X-inactivation, and potentially other developmental commitments. As cultures of two hES lines from Technion also showed a mosaic X-inactivation phenotype, we believe it likely that careful analysis and cultivation of these and other hES lines, may identify additional sublines that retain X-inactivation competence. While this NIH-funded study does not address whether non-NIH approved hES lines show a similar diversity of phenotypes, our prior collaborative study with the Carpenter lab (Hoffman et al., 2005
) and a recent broad survey of gene expression by the International Stem Cell Initiative (Adewumi et al., 2007
) suggest that this may also appear to be the case for non-NIH approved lines as well (also see note below). The very act of establishing and forcing the expansion of “non-differentiated” hES cells may change the epigenetic properties present in the native ICM, including a strong propensity to inactivate one of the two X chromosomes. Consistent with our findings of differences between lines, other studies have shown distinct gene expression profiles between lines and cultures that do not seem to be explained by differentiation status (Doherty et al., 2000
; Bibikova et al., 2006
). Although a recent report did not directly determine if hESC lines displaying precocious X-inactivation also exhibited abnormal status for other imprinted genes (Adewumi et al., 2007
), it may be that in future efforts to establish developmentally naïve cells, either from ICM or from reversing the programming of more advanced cells, X-inactivation status may provide a valuable tool to evaluate the epigenetic status and competence of those cells.