Differential modeling of fragile X syndrome by human embryonic stem cells and induced pluripotent stem cells

In embryonic stem (ES) cell lines generated from human embryos determined through preimplantation genetic diagnosis to carry the fragile X mutation, the FMR1 gene is expressed in undifferentiated cells but undergoes transcriptional silencing following ES cell differentiation (Eiges et al., 2007). Here, we generated induced pluripotent stem (iPS) cell lines from fibroblasts of individuals carrying the fragile X mutation. Despite successful reprogramming of the somatic cells to pluripotency, the FMR1 gene remained inactive, and carried DNA methylation and histone modifications indicative of inactive heterochromatin. These data highlight critical differences between ES and iPS cells in modeling Fragile X disorder. Pluripotent stem cells are potentially an important tool to model human genetic disorders. Human embryonic stem cells can recapitulate early stages of human development, and they can also differentiate into cells from the three embryonic germ layers (Schuldiner et al., 2000; Thomson et al., 1998). Thus, human pluripotent stem cells can be used to analyze the effect of specific mutations on the differentiation of various cell types and on early developmental processes that are otherwise inaccessible for research. In the past few years several diseases have been modeled in pluripotent stem cells, either by direct gene mutagenesis or by deriving ES cells from embryos determined by preimplantation genetic diagnosis (PGD) to carry genetic mutation (Eiges et al., 2007; Urbach et al., 2004 reviewed in Lengerke and Daley, 2009). Recently, human pluripotent stem cells have been derived from somatic cells by introduction of defined factors (Lowry et al., 2008; Park et al., 2008c; Takahashi et al., 2007; Yu et al., 2007). These induced pluripotent stem (iPS) cells show remarkable similarity to human ES cells (Lowry et al., 2008; Park et al., 2008c; Takahashi et al., 2007; Yu et al., 2007). By reprogramming somatic cells from patients, one may isolate pluripotent cells that harbor disease-specific mutations (Park et al., 2008a); The reprogramming of somatic cells into pluripotent cells raised the question whether iPS cells will be able to replace human ES cells in basic research as well as in clinical applications (Belmonte et al., 2009). We are now in a unique position to compare disease phenotypes manifest in ES cells to those seen in iPS cells. Fragile X (FX) syndrome is the most common form of inherited mental retardation (Crawford et al., 2001; Rousseau et al., 1992). It is caused by the absence of expression of the fragile X mental retardation 1 (FMR1) gene (O'Donnell and Warren, 2002). The vast majority of FX patients do not express FMR1 due to CGG triplet repeat expansion in the 5' untranslated region of the gene (Pearson et al., 2005; Verkerk et al., 1991). Full expansion of the CGG repeat usually coincides with hypermethylation of the repeat region and its upstream promoter (Oberle et al., 1991), and with chromatin modifications such as histone H3 tail deacetylation, histone H3K9 methylation and histone H3K4 demethylation (Coffee et al., 1999). Until recently, early events in FMR1 silencing could not be characterized due to the lack of an appropriate animal model (Bontekoe et al., 1997; Lavedan et al., 1997), but human ES cells have now been derived from FX blastocysts determined through pre-implantation genetic diagnosis (PGD) (Eiges et al., 2007). In the undifferentiated FX-ES cells, the full expansion of the CGG triplet repeat is not sufficient to inactivate the expression of the FMR1 gene and gene silencing occurs only upon differentiation (Eiges et al., 2007). Evidence from chorionic villus samples supports a similar conclusion that transcriptional silencing of the FMR1 gene occurs with human development (Sutcliffe et al., 1992; Willemsen et al., 2002). Current data suggest that upon cell differentiation the mutated FMR1 gene recruits specific histone modifications followed by DNA methylation, which silence its transcription (Eiges et al., 2007; Pietrobono et al., 2005). In the current study we have isolated iPS cell lines from three FX affected males, and compared the regulation of FMR1 transcription to that of human FX-ES cells. Fibroblasts from four-year old and 28-year old individuals, as well as fetal-lung fibroblasts from a 22 week-old affected fetus with FX syndrome were reprogrammed in culture according to published protocols (Park et al., 2008b; Takahashi et al., 2007). The efficiency of reprogramming of the FX-fibroblasts was similar to that of the wt-fibroblasts (0.0056% and 0.0024% respectively), as determined by counting the number of Tra-1-60 positive colonies. Multiple FX-iPS cell clones were analyzed (seven from the first, two from the second and two from the third patient). The iPS cell clones demonstrated typical characteristics of pluripotent stem cells: morphology similar to that of ES cells and expression of alkaline phosphatase, Tra-1-60, OCT4, SOX2, NANOG, SSEA3, SSEA4, and Tra-1-81 (Supplementary Figure 1A); silencing of retroviral transgenes (Supplementary Figure 1B, and data not shown); reactivation of genes indicative of pluripotency (Supplementary Figure 1C,D and data not shown); and maintenance of a normal diploid karyotype (Supplementary Figure 1E). By hierarchical clustering and scatter plot analysis of DNA microarray results we observed that the iPS cells cluster together with human ES cells and apart from their cell of origin (Supplementary Figure 2A,B). The cells generated embryoid bodies (Supplementary Figure 2C-I) that expressed markers of endoderm (Supplementary Figure 2C-II&III), mesoderm (Supplementary Figure 2A-IV&V) and ectoderm (Supplementary Figure 2C-VI) as demonstrated by immunostaining, and also by RT-PCR (data not shown). The iPS cell lines also differentiated in vivo into teratomas that manifest elements of all embryonic germ layers (Supplementary Figure 2D). Thus, these human iPS cell lines met stringent criteria for pluripotency (Chan et al., 2009; Daley et al., 2009). The FMR1 gene is expressed in FX-ES cells, wt-ES cells, wild type skin fibroblasts and iPS cells derived from them (Figure 1A, see also supplementary figures 1 and 2). The FMR1 gene is also expressed in wild type lung fibroblasts (MRC5), and iPS cells derived from them (data not shown). In marked contrast to all of these cells, the FMR1 gene remained transcriptionally silent in all FX-iPS cell clones derived from skin or lung FX-fibroblasts (Figure 1A). These results suggest that the differences in the expression of FMR1 between the WT and FX-iPS cells are due to the FMR1 mutation and not due to the tissue source of the original fibroblasts. The absence of FMR1 gene expression in the FX-iPS cells was observed both in early passage (P5) and at higher passages (up to P18; Figure 1B), indicating a stable phenotype. The FX-iPS cells also lacked expression of the FMR1 protein by immunostaining (Figure 1C).