Terminal neural differentiation was achieved by plating expanded cells plated at a seeding density of 40,000 cells per cm2 on polyornthine/laminin plates as above in expansion medium lacking both EGF and bFGF, with medium replacement every 3�C5 days until the indicated endpoint time. Two wells were imaged on an IX-Micro automated microscope , and 9 images per well were taken . Banked fibroblast cell lines were obtained from three clinically typical male FXS patients and two unaffected males and BJ1 . As expected, all three FXS patient fibroblast lines had CGG repeat sizes in the full mutation range in the FMR1 59UTR on the X chromosome . One of the FXS cell lines, GM05131, however, was shown to be from a mosaic donor, having two predominant bands corresponding to 800 and 166 CGG repeats. Mosaicism in FXS patients has been documented before, and is estimated to occur in 20�C40% of patients, possibly due to instability of the repeat Rapamycin length during in utero somatic cell development . In the case of fibroblasts derived from GM05131, the fibroblast population was a mixture of cells that have the full mutation and cells with the permutation CGG-repeat lengths. Upon increasing CGG-repeat length in the FMR1 gene beyond the normal range of 6�C50, an increase in the methylation of CpG sites in the promoter region leads to epigenetic silencing of the gene . To quantitatively compare the levels of methylation within the promoter region of FMR1, bisulfite pyrosequencing was used to query methylation status at 22 CpG sites. Both full mutation fibroblast lines, GM05848 and GM05185 had highly methylated promoter regions, with a mean of 1433953-83-3 approximately 85% of the CpG sites methylated, consistent with the expected hypermethylation of this region in the FXS . In contrast, the control BJ-1 fibroblast lines had barely detectable levels of CpG methylation in the same region. The mean CpG methylation level of this region in the mosaic GM05131 fibroblast cell line was approximately 60%, most likely because of the presence of both cells with a hypermthylated CpG full mutation as well as premutation fibroblasts with unmethylated FMR1 promotor regions. The combination of an expanded 59-UTR CGG trinucleotide repeat along with the high degree of CpG site methylation of this region would be expected to result in the silencing of the expression of the FMR1 gene. In order to test this directly, we performed quantitative RT-PCR analysis to assess the relative FMR1 expression levels in the cell lines . While the control lines expressed the FMR1 gene, the full mutation fibroblast lines had undetectable levels of FMR1 mRNA expression. Interestingly, the expression level of the FMR1 gene in the mosaic GM05131 line was approximately four-fold higher than the control. This is consistent with reports that the presence of the premutation causes an increase in gene expression from the FMR1 promoter from two- to ten-fold over unaffected controls . FMRP protein expression in the patient fibroblasts was determined by Western blot analysis .