The derivation of human induced

The derivation of human induced pluripotent stem endopeptidase (hiPSCs) from fibroblasts (Takahashi et al., 2007) offers new opportunities to study XCI in human cells. For mouse fibroblasts, it has been shown that the Xi becomes reactivated during the reprogramming process, followed by random XCI (rXCI) upon differentiation of these miPSCs (Maherali et al., 2007; Stadtfeld et al., 2008). Similar to studies involving hESC lines, previous studies of XCI in hiPSCs have provided varying results. Systematic analysis of multiple female hiPSC lines derived from several fibroblast populations under different reprogramming strategies indicated that all hiPSC lines retained the Xi inherited from the starting fibroblasts (Amenduni et al., 2011; Ananiev et al., 2011; Cheung et al., 2011; Tchieu et al., 2010). In another study, it was found that in all hiPSC lines derived from one fibroblast population with established rXCI, one and the same X chromosome had become the Xi in all lines, indicating involvement of cell selection processes (Pomp et al., 2011). In contrast, other studies showed reactivation of the Xi, an apparent reversal of XCI that is herein referred to as X chromosome reactivation (XCR), in all or a limited number of hiPSC lines, but XCI was reinitiated upon differentiation of these hiPSC lines (Bruck and Benvenisty, 2011; Kim et al., 2011; Marchetto et al., 2010). XCR followed by reinitiation of XCI and stable establishment of the Xi upon hiPSC differentiation is a crucial step that needs to take place for hiPSCs to be applied for various purposes. If hiPSC lines do not pass through this series of events, they show signs of stochastic reactivation of the Xi inherited from the founder fibroblasts (Mekhoubad et al., 2012). This erosion of XCI is detrimental for studies involving cell types generated from female hiPSCs, as it can be expected that many of these cell types will be prone to gene dosage inequalities. Therefore, the availability of such hiPSC lines with stable XCR, having two active X chromosomes as in mESCs, would greatly advance research on modeling of X-linked human diseases and studies on regulatory mechanisms of human XCI. The varying results regarding XCR and XCI obtained for hiPSCs may be explained by different reprogramming techniques and the growth conditions in which hiPSCs are generated and maintained. In a recent study, it was found that growth of hESCs and hiPSCs in defined conditions (naive human stem cell medium [NHSM]) results in more naive iPSCs and leads to efficient loss of Xi specific markers, including XIST RNA and Xi-specific histone modifications, which are re-established upon differentiation (Gafni et al., 2013). Although these NHSM-cultured hESCs and hiPSCs resemble mESCs and miPSCs in several aspects, it is unclear whether the described loss of Xi-specific markers under these growth conditions has resulted in XCR, as expression of X-linked loci was not assessed (Gafni et al., 2013).
Results
Discussion Here, we have investigated the dynamics of XCI in hiPSCs derived from human female fibroblast cultures with completely skewed rXCI. This approach allowed us to include analysis of XCR and XCI at the single-cell level. The present results show that, at the single cell level, reprogramming of human cells into hiPSCs results in XCR, even at early passages. Biallelic expression of different X-linked loci indicates that the Xi is reactivated in a large part of the cell population of our hiPSC lines, but is variable between iPSC lines. In several hiPSC lines, we also detected XIST expression and expression of several X-linked genes from the allele that was not active in the starting fibroblast culture. Human iPSC lines share a clonal origin, and therefore, this switch in expression from one allele to the other, or biallelic expression, indicates that during the reprogramming process XCR must have occurred in a high proportion of cells. Differentiation of hiPSCs was found to result in completely skewed rXCI, similar to that of the starting fibroblast population, indicating a high selective pressure against cells that have inactivated the intact X chromosome.