FIG. 2.

FXS induced pluripotent stem cells (iPSCs) preserve characteristic genetic and epigenetic marks of FMR1 silencing. iPSCs were generated from individual fibroblast lines. (A) Phase-contrast images and immunofluorescence of selected iPSC lines show expression of pluripotency transcription factors OCT4 and SOX2 and cell surface markers Tra1-81 and SSEA4, but not the neuronal transcription factor PAX6. Scale bars=100 μm. (B) Gel electrophoresis for CGG repeat length confirms that repeat length in iPSCs from FXS individuals is preserved after reprogramming. The FX11-7 fibroblasts had two populations of cells with different CGG repeat lengths (mosaic) and isogenic full-mutation and premutation iPSCs were generated from these cells. (C) Average level of methylation at each CpG site in the FMR1 promoter region as determined by bisulfate sequencing. The FXS lines have significant methylation of the FMR1 promoter, while control iPSCs do not. (D) Quantitative RT-PCR for the FMR1 gene shows that the full-mutation FXS iPSCs (FX08-1, FX11-7, and FX13-2) express almost no FMR1 compared with control. The FX11-9U premutation clone expresses normal to high levels of FMR1, whereas its isogenic full-mutation counterpart, FX11-7, expresses no detectable FMR1. Expression is normalized to control C603-4 line. Error bars=SE, n=3. (E) Immunoblot for FMRP in the fibroblasts shows that control cells express FMRP while the FXS cells do not. Color images available online at www.liebertpub.com/scd