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. 2011;6(10):e26203.
doi: 10.1371/journal.pone.0026203. Epub 2011 Oct 12.

Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome

Steven D Sheridan  1 , Kraig M Theriault, Surya A Reis, Fen Zhou, Jon M Madison, Laurence Daheron, Jeanne F Loring, Stephen J Haggarty
Affiliations

Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome

Steven D Sheridan et al. PLoS One. 2011.

Abstract

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. In addition to cognitive deficits, FXS patients exhibit hyperactivity, attention deficits, social difficulties, anxiety, and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 5' untranslated region of the Fragile X Mental Retardation (FMR1) gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP). Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations, we report on the generation of induced pluripotent stem cell (iPSC) lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons, these iPSC models will aid in the discovery of novel therapeutics for FXS and other autism-spectrum disorders sharing common pathophysiology.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Analysis Fragile X Patient Fibroblasts: FMR1 CGG-repeat Length, CpG Methylation, and Expression
. (A) Predominant CGG-repeat size in the FMR1 promoter as determined by Southern blot analysis. (B) Bisulphite pyrosequencing analysis of the FMR1 promoter reported as relative methylation level at indicated CpG positions (FMR1 promoter CpG site schematic not to scale). (C) FMR1 transcript expression levels as determined by qRT-PCR shown as fold increase over BJ1 control (ND - non-detectable). (D) Western blot analysis of FMRP protein levels in indicated fibroblast lines, β-actin is shown as a loading control.
Figure 2
Figure 2. Derivation and Characterization of FXS Induced Pluripotent Stem Cells.
(A) Alkaline phosphatase enzymatic and pluripotent marker (OCT4, NANOG and SSEA-4) immunocytochemical analysis of FXS patient-derived iPSC clones. (B) Endogenous OCT4, NANOG and REX1 pluripotency-associated transcript expression as analyzed by RT-PCR in indicated iPSC lines and fibroblasts (NTC - non-template containing control). (C) Bisulphite pyrosequencing analysis of the endogenous OCT4/POU5F1 promoter in indicated lines (open circles, unmethylated (<50%) CpGs; black circles, methylated CpGs). (D) Embryoid body pathological evaluation of H&E stained sections (clone 848-iPS1 shown) indicating representative ectoderm (neural epithelium, left), endoderm (respiratory epithelium, center) and mesoderm (connective tissue, right) germ layers.
Figure 3
Figure 3. Effects of iPSC Generation on FMR1 CGG-repeat Length, CpG Methylation and Expression.
(A) Predominant CGG-repeat length in the FMR1 promoter as determined by Southern blot analysis. (B) Pyrosequencing analysis of the FMR1 promoter reported as relative methylation level at indicated CpG positions (FMR1 promoter CpG site schematic not to scale). (C) FMR1 transcript expression levels as determined by qRT-PCR shown as fold increase over BJ1 control (ND - non-detectable). (D) Western blot analysis of FMRP protein levels in indicated iPSC lines, β-actin was used as a loading control.
Figure 4
Figure 4. Isolation and Characterization of Expandable Neuronal Progenitor Cells from iPSC Clones.
(A) Immunocytochemical analysis of NESTIN and SOX1 (red, nuclei DNA staining overlaid in blue) expression in expanded neural cells from indicated iPSC lines expanded in the presence of mitogens EGF and bFGF. (B) Bisulphite pyrosequencing analysis of the FMR1 promoter reported as relative methylation level at indicated CpG positions in indicated neural differentiated iPSC lines (FMR1 promoter CpG site schematic not to scale). (C) Western blot analysis of FMRP protein levels in indicated neural differentiated iPSC lines, β-actin is shown as a loading control.
Figure 5
Figure 5. Aberrant Neural Differentiation of FXS iPSC-derived Neuronal Progenitors.
Immunocytochemical analysis of Tuj1 expression (green) in neuronal progenitor cells differentiated upon mitogen removal of (A) FMRP+ control line 8330-8 and (B) FMRP- FXS line 848-3, overlaid with nuclei DNA staining (blue) (see Figure S1 for additional images). (C) Quantification of neurite process length in indicated iPSC-NP lines. Two wells (6-well plate) were imaged, 9 images per well (n = 18 images per sample). P-values as indicated *1.5e-13, **1.1e-10, ***3.1e-13, ****1.4e-4.
Figure 6
Figure 6. Rescue of Aberrant Neural Differentiation of FXS iPSC-derived Neuronal Progenitors by FMRP Expression in Premutation Clones.
Immunocytochemical analysis of expanded neuronal progenitor cells differentiated upon mitogen removal of FMRP+ control line 8330-8 (A) and FMRP- FXS lines 848-3 and 131-1 (B and C) and FMRP+ FXS line 131-3 (D), overlay of Tuj1 staining (green), GFAP (red) and nuclei DNA staining (blue) (see Figure S1 for additional images).
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