X (Chromosome) Marks the Plot

In female mammalian embryos, the X chromosome represents a problem. Since mammalian females have two X chromosomes, the embryo contains twice as much of the gene products of the X chromosome as opposed to male mammalian embryos, which only have one copy of the X chromosome. How is this problem solved? X chromosome inactivation (XCI). XCI occurs very early during female mammalian development, and it occurs on a cell-by-cell basis, and occurs randomly. The embryo has some cells that have one copy of the X chromosome inactivated and all the other cells have the other copy of the X chromosome inactivated. This is the reason the bodies of mammalian females are mosaics in which some cells have one copy of the X chromosome inactivated and yet other cells in which the other copy of the X chromosome is inactivated. Thus genetic diseases that map to the X chromosome will affect the entire body of the mammalian male but only a portion of the mammalian female’s body.

What does this mean for stem cells? Quite a bit. Embryonic stem cells are derived from the inner cell mass of the blastocyst-stage embryo. This is precisely the time when the cells of the embryo begin to randomly select a copy of the X chromosome to inactivate. The timing of XCI differs slightly from one species to another. In mice, for example, both copies of the X chromosome are active in mouse embryonic stem cells (ESCs) (Fan and Tran, Hum Genet 130 (2011):217-22; Chaumeil, et al., Cytogenet Genome Res 99 (2002):75-84), and XCI occurs when the cells differentiate (Murakami, et al., Development 138 (2011):197-202). Human ESCs, however, vary tremendously (Dvash and Fan, Epigenetics 4 (2009):19-22), with a few hESC lines showing activation of both copies of the X chromosome and many others showing inactivation of one or the other copy of the X chromosome. Human induced pluripotent stem cells (iPSCs) are derived from adult cells that already have one copy of the X chromosome inactivated. Therefore, de-differentiation of adult cells into iPSCs undoes XCI and activates both copies of the X chromosome (Maherali, et al., Cell Stem Cell 1 (2007):55-70 & Hanna, et al., PNAS 107 (2010):9222-7).

XCI is a process that is linked to pluripotency. The genes necessary for the maintenance of pluripotency (OCT4, Sox2, Nanog) all repress genes necessary for XCI (Xist) and activate genes that repress XCI (Tsix). Therefore, XCI seems to be a factor in the down-regulation of pluripotency in early embryonic cells.

There is a new study that underscores this link between XCI and pluripotency. Researchers at the Gladstone Institutes at the University of California, San Francisco have expanded upon the so-called Kyoto method for making iPSCs. The Kyoto method uses an animal cell line that grows in the culture dish and makes a protein called LIF (leukemia inhibitory factor). LIF activates the growth of cultured iPSCs and allows them to grow and establish an iPSC line.

According to Kiichiro Tomoda from the Gladstone Institute, iPSC derivation on LIF-making feeder cells always produces IPSCs that have two active copies of the X chromosome. However, if iPSCs are derived on feeder cells that do not make LIF, the result is very poor iPSCs derivation and the resultant iPSCs only have one active copy of the X chromosome. Furthermore, by passaging iPSCs that were derived from non-LIF-making feeder cells on LIF-making feeder cells, the inactivated X chromosome became active. This shows that iPSC derivation is highly sensitive to the environment in which the cells are derived. If also shows how to make iPSCs that more closely resemble early embryonic cells.