Aberrant Patterns of X Chromosome Inactivation in a New Line of Human Embryonic Stem Cells Established in Physiological Oxygen Concentrations

Abstract

One of the differences between murine and human embryonic stem cells (ESCs) is the epigenetic state of the X chromosomes in female lines. Murine ESCs (mESCs) present two transcriptionally active Xs that will undergo the dosage compensation process of XCI upon differentiation, whereas most human ESCs (hESCs) spontaneously inactivate one X while keeping their pluripotency. Whether this reflects differences in embryonic development of mice and humans, or distinct culture requirements for the two kinds of pluripotent cells is not known. Recently it has been shown that hESCs established in physiological oxygen levels are in a stable pre-XCI state equivalent to that of mESCs, suggesting that culture in low oxygen concentration is enough to preserve that epigenetic state of the X chromosomes. Here we describe the establishment of two new lines of hESCs under physiological oxygen level and the characterization of the XCI state in the 46,XX line BR-5. We show that a fraction of undifferentiated cells present XIST RNA accumulation and single H3K27me foci, characteristic of the inactive X. Moreover, analysis of allele specific gene expression suggests that pluripotent BR-5 cells present completely skewed XCI. Our data indicate that physiological levels of oxygen are not sufficient for the stabilization of the pre-XCI state in hESCs.

Preventing erosion of X-chromosome inactivation in human embryonic stem cells

“…2 ; Source Data file). These results led us to conclude that culturing primed pluripotent hESCs in atmospheric vs. physiological O 2 concentration does not alter the loss of XIST RNA expression and coating in hESCs, in agreement with other reports 29 , 35 , 43 , 45 , 68 – 70 . Furthermore, some X-linked genes become derepressed when XIST RNA is lost in cultured hESCs, also in agreement with previously published results 31 , 34 , 35 , 38 , 42 , 43 , 58 .…”

“…Much prior work has demonstrated that prolonged culture of female hPSCs results in loss of XIST RNA coating and erosion of X-inactivation 29 , 31 , 35 , 36 , 38 , 39 , 42 , 43 , 45 , 57 , 59 . Consistent with previous findings, our data rule out atmospheric O 2 concentration as a cause of XIST RNA loss in hESCs 35 , 43 , 45 , 68 – 70 . Instead, we found that a primary source of XIST RNA loss in hESCs is the composition of the hESC culture medium.…”

“…To investigate the underlying causes of XIST RNA coating loss in hESCs, we next cultured hESCs in atmospheric (20%) and physiological (5%) O 2 concentrations. Atmospheric O 2 has been reported to cause loss of XIST RNA expression in cultured female hESCs in some studies 34 , 42 , 47 , 67 but not others 29 , 35 , 43 , 45 , 68 – 70 . We derived two new female hESC lines on HFFs, UM63-1 and UM77-2, and passaged them from P5 to P14 onto Matrigel-coated surfaces under atmospheric and physiological O 2 concentrations in parallel.…”

A Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,-15,-18,-21 Embryo

“…From these, despite its complex aneuploidy, only embryo 1 gave rise to a new line of hESC, named BR-6 ( Fig 1 ). This rate of derivation, although relatively low, was similar to those obtained by our group with euploid embryos [ 24 , 25 ].…”

The transcriptomes of novel marmoset monkey embryonic stem cell lines reflect distinct genomic features

“…This allows the conclusion that X-inactivation in genotypically female ES cells of the common marmoset monkey occurs in a XIST -dependent manner. However, X inactivation does not seem to be an essential inherent characteristic for pluripotency in vitro 66 . Our data suggest that marmoset ES cells, with regard to X-inactivation, in general rather resemble human than mouse ES cells.…”