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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell Stem Cell. Author manuscript; available in PMC 2011 November 5.
Published in final edited form as:
PMCID: PMC2987635

Friedreich’s Ataxia Induced Pluripotent Stem Cells Model Intergenerational GAA•TTC Triplet Repeat Instability


The inherited neurodegenerative disease Friedreich’s ataxia (FRDA) is caused by GAA•TTC triplet repeat hyper-expansions within the first intron of the FXN gene, encoding the mitochondrial protein frataxin. Long GAA•TTC repeats causes heterochromatin-mediated gene silencing and loss of frataxin in affected individuals. We report the derivation of induced pluripotent stem cells (iPSCs) from FRDA patient fibroblasts by transcription factor reprogramming. FXN gene repression is maintained in the iPSCs, as are the global gene expression signatures reflecting the human disease. GAA•TTC repeats uniquely in FXN in the iPSCs exhibit repeat instability similar to patient families, where they expand and/or contract with discrete changes in length between generations. The mismatch repair enzyme MSH2, implicated in repeat instability in other triplet repeat diseases, is highly expressed in pluripotent cells, occupies FXN intron 1, and shRNA silencing of MSH2 impedes repeat expansion, providing a possible molecular explanation for repeat expansion in FRDA.


Friedreich’s ataxia (FRDA), the most common inherited ataxia, is caused by heterochromatin-mediated silencing of the nuclear FXN gene, encoding the essential mitochondrial protein frataxin (Herman et al., 2006). The genetic mutation in FRDA is a GAA•TTC triplet-repeat expansion in the first intron of FXN (Campuzano et al., 1996), with unaffected alleles having 6–34 repeats in contrast to 66–1700 repeats in patient alleles. Longer repeats are associated with more severe gene repression, lower frataxin protein levels and earlier onset and increased disease severity (Bidichandani et al., 1998; Campuzano et al., 1996). Frataxin insufficiency leads to progressive spino-cerebellar neurodegeneration and associated movement disorders along with an increased risk for diabetes and cardiomyopathy, the latter being the most common cause of death in FRDA.

Unlike many triplet-repeat diseases (e.g., the polyglutamine expansion and the RNA toxicity diseases (Orr and Zoghbi, 2007)), GAA•TTC expansions in FXN are intronic and do not alter the frataxin protein sequence; thus, gene activation would be of therapeutic benefit (Gottesfeld, 2007; Herman et al., 2006). However, studies in FRDA pathogenesis and therapeutics are limited by poor cellular models, and available mouse models do not fully recapitulate gene silencing and frataxin protein levels (Al-Mahdawi et al., 2004; Miranda et al., 2002). Recent studies have shown that human fibroblasts can be reprogrammed to a pluripotent state by transduction of transcription factors (Takahashi et al., 2007), and importantly, the same has been demonstrated with fibroblasts from repeat-associated neurodegenerative disease patients such as Huntington’s disease (HD) and Fragile X syndrome (Park et al., 2008a; Urbach et al., 2010). We now report the derivation of FRDA iPSCs. We find that the FXN GAA•TTC repeats in FRDA iPSCs exhibit a repeat instability pattern similar to the human disease, where repeats expand and/or contract with discrete changes in length between generations (Campuzano et al., 1996; Pianese et al., 1997). We also provide evidence for the role of the mismatch repair (MMR) enzyme MSH2 in repeat instability. Our observations provide a cellular model system for mechanistic studies of repeat instability in FRDA and potentially in other triplet repeat diseases.


Derivation of iPSCs from FRDA Patient Fibroblasts

Primary fibroblasts from two FRDA patients (GM03816 and GM04078 from the NIGMS Coriell Cell Repository) were reprogrammed by transcription factor overexpression (Takahashi et al., 2007), and colonies with ES/iPS morphology were selected and expanded (Figure 1A). Analysis by qRT-PCR shows that our FRDA iPSC lines are indeed pluripotent (Figure 1B) and retain marked repression of FXN mRNA (Figure 1C). Further, expression of the integrated transgenic reprogramming factors is silenced in the iPSCs (Figure S1A, available online), a hallmark of full reprogramming (Lowry et al., 2008).

Figure 1
Characterization of FRDA iPSCs

Immunostaining of FRDA iPSCs for pluripotent markers (SSEA3 and SSEA4; Oct4; and Tra1–60 and Tra1–81) was also found to be comparable to that of H1 ESCs (Figure 1D). Genotyping of the FXN gene GAA•TTC repeats and cytogenetic analysis demonstrated that the iPSCs indeed originated from FRDA fibroblasts and are karyotypically normal (Figures 2A and S1B), and ChIP experiments confirm heterochromatin histone marks near the FXN GAA•TTC repeats (Al-Mahdawi et al., 2008; Herman et al., 2006; Rai et al., 2008) (Figure S1C to E). Finally, teratoma analysis shows full in vivo differentiation capacity (Figure S1F), providing additional evidence of the pluripotent nature of the FRDA iPSCs.

Figure 2
Analysis of GAA•TTC repeat lengths

Global mRNA Expression Profiles of FRDA iPSCs

Hierarchical clustering of global gene expression profiles of four FRDA iPSC lines (two from GM03816, two from GM04078) with a set of unaffected iPSCs, hESCs, and various human tissues and cell lines show that our iPSC lines group to the same cluster as other iPS/ESCs, though in a separate, distinct subset (Figure 1E). We attribute this distinction to a 5–7% global expression difference between our FRDA iPSCs and other iPS/ESCs, whereas an internal variation of only 2–3% was observed among other iPS/ESCs, a difference that likely reflects the diseased nature of the FRDA genetic background. Moreover, functional annotation clustering using the Database for Annotation, Visualization and Integrated Discovery (DAVID) of the top differentially expressed genes in FRDA iPSCs identified gene groups related to mitochondrial function, DNA repair, and DNA damage response (Table S1). DNA repair also appeared as a top GO category significantly enriched in our dataset (Table S2), consistent with recent studies on FRDA patients (Haugen et al., 2010). Additional significant GO categories were related to cell cycle, protein modification/ubiquitination, lipid metabolism and carbohydrate biosynthesis, all of which have been previously associated with altered function in FRDA patients (Coppola et al., 2006; Haugen et al., 2010). Global microRNA profiling also shows that the FRDA iPSCs express many miRNAs associated with pluripotency, but distinct differences, again presumably due to FRDA pathogenesis, were also noted (Figure S1G–H and Table S3).

GAA•TTC Repeat Expansion in FRDA iPSCs

PCR analysis of the FXN GAA•TTC repeats showed repeat instability in the iPSC lines (Figure 2A), a phenomenon not seen in donor fibroblasts (data not shown). PCR products from unaffected (GM15851) and FRDA (GM15850) lymphoblasts and an unrelated patient DNA are also shown for comparison. In all cases, an apparent expansion of both alleles of FRDA iPSCs was observed (with certain caveats addressed below). PCR analysis of iPSCs from a second patient (GM04078) similarly showed repeat expansion (Figure 2A, middle panel), confirming the general nature of this observation. In contrast, wild-type FXN alleles do not change in size in a non-FRDA iPSC control (Figure 2A; right panel, GM03813 spinal muscular atrophy iPSCs) and in non-disease unaffected iPSCs (data not shown). Due to the allele ambiguity of our PCR assay, shifts in PCR bands could represent expansion of both alleles or contraction of one and expansion of the other. Therefore, iPSCs were generated from carrier parents of a third FRDA patient and subjected to PCR analysis, showing repeat expansion in both parental pathogenic alleles (Figure 2B). The wild-type FXN allele, as expected, did not expand, suggesting a gender-neutral instability in pathogenic FXN alleles. As in somatic cells, GAA•TTC repeat expansions at two unrelated genetic loci (2q36, 16 repeats; 4q31.1, 30 repeats; (Rindler et al., 2006)) remain unchanged between our iPSCs and their three corresponding donor fibroblasts (Figure 2C), even though these loci are at Alu elements similar to FXN intron 1. Altogether, the data suggest that changes at the FXN gene are a consequence of its particular repeat expansion (and perhaps its length) and are not a general phenomenon throughout the human genome. Further, we find that GAA•TTC repeat lengths in FRDA iPSCs change over time in culture (Figure 2D).

Molecular Basis For Repeat Instability

Previous reports have implicated the MMR enzymes MSH2, MSH3 and MSH6 in CAG•CTG and CTG•CAG somatic and intergenerational repeat instability in HD and DM1 (myotonic dystrophy type I) transgenic mice, respectively (reviewed in (Dion and Wilson, 2009)). Other studies have implicated the oxoguanine-DNA glycosylase OGG1 in somatic instability (Kovtun et al., 2007). In FRDA iPSCs, mRNA expression analysis shows large increases in MSH2 compared to donor fibroblasts (Figure 3A). No differences in MSH3 mRNA, however, were found, and a small decrease in OGG1 mRNA was noted in the iPS/ESCs. Western blotting further shows corresponding increases in MSH2 protein in FRDA iPSCs and H1 ESCs compared to FRDA fibroblasts (Figure 3B). ChIP assays, at a resolution of ~1 kb, show increased occupancy of MSH2 and MSH3 downstream of the GAA•TTC repeats in FRDA iPSCs compared to an unaffected iPSC line but, in contrast, not 1254 bp upstream of the FXN transcriptional start site nor directly upstream of the GAA•TTC repeats (Figure 3C). No differences in MSH6 occupancy were found at any of the regions probed.

Figure 3
MSH2 expression and localization in FRDA iPSCs

To further investigate the role of MSH2 in repeat instability, lentiviral shRNA constructs were integrated into single colony-expanded FRDA iPSCs (to limit repeat length heterogeneity) and assayed for GAA•TTC repeat length (summarized in Figure 4A). As shown in Figures 4B–D, stable expression of MSH2-targeted shRNA achieved relatively high levels of mRNA and protein knockdown compared to a scrambled shRNA. Additionally, we verify that MSH2 silencing does not affect pluripotency (Figure S2A). After eight passages, repeat length PCR analysis showed that MSH2 knockdown results in a significantly smaller large allele compared to a scrambled shRNA (Figure 4E–G). No statistical significance, however, was observed for the smaller allele based on pooled data, contrary to the single-point data shown in Figure 4E–F. shRNA silencing of MSH2 in FRDA fibroblasts followed by reprogramming also yielded similar results (Figure S2). Collectively, these data implicate the involvement of MSH2 in GAA•TTC repeat instability.

Figure 4
shRNA silencing of MSH2 in FRDA iPSCs


To our knowledge, this is the first report of triplet repeat instability occurring in patient-specific iPSCs. Previous studies have analyzed triplet expansions in disease-specific iPSCs but either did not compare donor fibroblast repeats or the iPSCs did not show any changes after reprogramming (Park et al., 2008a; Urbach et al., 2010). In our case, we observe repeat expansion, perhaps resembling intergenerational instability as in FRDA families with one exception. In patient families, only the maternal pathogenic allele is reported to undergo intergenerational expansion, whereas the paternal allele usually remains the same length or can contract (Campuzano et al., 1996; Pianese et al., 1997). However, we find expansion of both parental pathogenic alleles in iPSCs. One interpretation for this difference is that both alleles undergo the same cellular changes during in vitro reprogramming as opposed to differential gametogenesis.

Our finding of MSH2 and possibly MSH3 as components involved in repeat expansion is supported by extensive prior studies in various triplet repeat disorders (Dion and Wilson, 2009). Early work in Fragile X syndrome models pointed towards MSH2 as a component responsible for repeat instability (Kramer et al., 1996). Other studies have shown that MSH2 has a role in both intergenerational and somatic instability in various HD models (Dragileva et al., 2009), and similar findings have been presented in DM1 studies as well (Savouret et al., 2003). Along similar lines, our present results implicate MSH2 as one of the proteins responsible for GAA•TTC repeat expansion. Although we have no direct evidence for an MSH2–MSH3 complex (MutSβ), we believe that such a complex is responsible in our case, as not only do both MSH2 and MSH3 localize near pathogenic FXN alleles, but other studies also point to the involvement of MutSβ as well (Dragileva et al., 2009; Kim et al., 2008).

Mechanistically, there is also debate over GAA•TTC repeat instability, and our results are consistent with several models of FRDA pathogenesis (Ditch et al., 2009; Dragileva et al., 2009; Iyer and Wells, 1999; Shishkin et al., 2009). One model proposes the formation of an exposed single-stranded DNA hairpin (resembling mismatched DNA) originating from a triple-stranded structure formed by long GAA•TTC repeats, which recruits MMR machinery (Wells, 2008). This recruitment then stabilizes the slipped-stranded intermediates, leading to repeat expansion (reviewed in (Mirkin, 2007)). Alternatively, sense/antisense transcription at the FXN locus (de Biase et al., 2009) could allow for transcription-coupled DNA repair and GAA•TTC repeat tract expansion (Ditch et al., 2009). Interestingly, transcription-coupled repair has been shown to interact with the MutSβ complex (Zhao et al., 2009), further supporting our observations.

There currently exist few model systems in which one can study GAA•TTC expansions (Al-Mahdawi et al., 2004; Ditch et al., 2009; Iyer and Wells, 1999; Shishkin et al., 2009). Despite the fact that a recent report revealed cellular physiological differences between a human disorder and its iPSC model and urged caution when making associations between the two (Urbach et al., 2010), these key differences are likely to be highly context-dependent. In our case, we expect that FRDA iPSCs will provide a valuable, more accessible resource to study repeat instability mechanisms as well as for differentiation into cell types affected in this human disease (sensory neurons and cardiomyocytes). Such cellular models will be useful to dissect disease mechanisms and to screen potential therapeutic agents.


Cell culture

Fibroblasts were grown at 37°C and 5% CO2 with 10% FBS (Lonza) in MEM, 2 mM glutamine, 1% NEAA, 20 mM HEPES, and 1% antibiotic-antimycotic (all from Invitrogen). ES/iPSCs were grown at 37°C and 5% CO2 on γ-irradiated MEFs (GlobalStem) in D-MEM/F12 with 20% Knockout Serum Replacement, 1 mM glutamine, 1% NEAA, 15 mM HEPES, 0.1 mM β-mercaptoethanol (all from Invitrogen), 20 ng/mL basic FGF (Stemgent) and were passaged manually every five to seven days. Phoenix cells were grown with 10% FBS (Lonza) in DMEM, 2 mM glutamine, 20 mM HEPES, and 1% NEAA (all from Invitrogen).

Isolation of primary fibroblasts

Dermal explant cultures were established from dispase-treated skin biopsies on fibronectin underneath a glass coverslip with fibroblast media after 5–7 days. After establishment, primary dermal fibroblasts were cultured as described above. Biopsies were performed at the University of California, Los Angeles, under an approved Human Subjects Protocol.

Generation of viruses

Retroviruses were packaged using Phoenix cells (a gift from the laboratory of W. Balch) and Fugene6 (Roche). The four reprogramming vectors ((Takahashi et al., 2007); were packaged individually and pseudotyped with VSV-G. Lentiviruses were generated by co-transfecting into 293T cells shRNA constructs with psPAX2 and pMD2.G. Virus-containing ES media supernatant was collected throughout 48 hours after transfection (See Supplemental Experimental Procedures).

Derivation of iPSCs

Previous methods were followed with minor deviations (Park et al., 2008b). Donor fibroblasts were transduced daily for three consecutive days, and four to six days after the last transduction, cells were replated onto MEFs. Beginning one day following, cells were given ES media daily. Colonies were picked between 21 and 28 days after transduction.

Lentiviral shRNA transduction

FRDA iPSCs were subjected to two lentiviral transductions with lentivirus overnight at 37°C with 5 µg/mL polybrene. Cells were then expanded and subjected to 6 days of puromycin selection (0.4 µg/mL) on DR4 drug-resistant MEFs (GlobalStem).


Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (all in PBS). Primary antibodies were incubated at 4°C overnight, and secondary antibodies were incubated at room temperature for one hour followed by nuclear staining with DAPI (See Supplemental Experimental Procedures for antibodies used).

Nucleic Acid Purification

Total RNA was purified using the RNeasy Plus Mini kit (Qiagen) according to the manufacturer. Genomic DNA was purified by phenol/chloroform extraction followed by isopropanol precipitation of cell lysates prepared in total cell lysis buffer (100 mM Tris, 5 mM EDTA, 0.2% SDS, 0.2 M NaCl, 200 µg/mL proteinase K, pH 8).

PCR and Quantitative RT-PCR

For GAA•TTC repeat length PCRs, Phusion polymerase (New England Biolabs) was used according to the manufacturer. Quantitative RT-PCR analysis was done using the qScript One-Step SYBRGreen qRT-PCR kit (Quanta Biosciences) according to the manufacturer. All primers for pluripotent markers were as described (Park et al., 2008b). FXN, MSH2, MSH3, OGG1, and GAPDH primers are described in Supplemental Experimental Procedures. Analysis of relative qRT data was performed using the ΔΔCT method (Livak and Schmittgen, 2001). Detection of retroviral transgenes was done by absolute qRT-PCR using the same kit as above with previously described retrovirus transcript-specific primers (Takahashi et al., 2007). (See Supplemental Experimental Procedures.)

Western analysis

Whole cell extracts (in 50 mM Tris pH 7.4, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, protease inhibitor; Roche) were electrophoresed in polyacrylamide gels and transferred onto nitrocellulose membranes. Primary antibodies were incubated overnight, and secondary antibodies were incubated one hour at room temperature. Signals were detected using HRP-conjugated secondary antibodies and enhanced chemiluminescence (SuperSignal West Pico, Thermo Scientific). (See Supplemental Experimental Procedures for antibodies used.)

Chromatin immunoprecipitation

Cells were crosslinked first with 1.5 mM dithiobis-succinimidyl propionate (DSP) followed by 1% formaldehyde. Subsequent ChIP procedures were as described (Herman et al., 2006) with MSH2 antibody (Santa Cruz). Analysis by qPCR using primers for the FXN promoter, the region upstream of the GAA•TTC repeats, and the region downstream of the repeats were as described (Herman et al., 2006). Additional primers are listed in Supplemental Experimental Procedures.

Microarray analysis

RNA purification with the MirVana RNA extraction kit (Ambion), labeling with the TotalPrep kit (Ambion), and hybridization to Illumina HT12 arrays were as according to the manufacturers. Data were then filtered, normalized, and hierarchically clustered (Eisen et al., 1998). Differentially expressed genes compared to a set of unaffected iPSCs were detected using a false discovery rate-corrected Student’s t-test at a significance level of αFDR < 0.01. Genes with expression level changes greater than 1.75 were then subjected to functional annotation analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID; (Dennis et al., 2003; Huang et al., 2009)) at a significance level of αFDR < 0.01.


  • FRDA iPSCs retain key molecular features characteristic of the human disease
  • GAA•TTC repeat instability is observed in iPSCs in the disease-relevant FXN gene
  • Repeat instability is dependent on the mismatch repair enzyme MSH2

Supplementary Material



We thank S. Perlman (UCLA) for providing skin biopsies and I. Singec, J. Clark, and C. Desponts for valuable advice and guidance. Additionally, we thank V. Lukiyanchuk for virus expertise, K. Clingerman for veterinary assistance, and C. Lynch for microarray analysis. This work was supported by the National Institutes of Neurological Disorders and Stroke (NIH), The Friedreich’s Ataxia Research Alliance (FARA), GoFAR, Ataxia UK, Friedreich’s Ataxia Society Ireland, and Repligen Corporation (Waltham, MA) to J.M.G. and by a fellowship from Families of Spinal Muscular Atrophy (to S.K.). L.C.L. is supported by NIH WRHR K12 Career Development Award and the Hartwell Foundation, and G.A. and J.F.L. are supported by CIRM (CL1-00502, RT1-01108, TR1-01250), NIH (R21MH087925), the Millipore Foundation, and the Esther O’Keefe Foundation. M.N. is supported by the National Ataxia Foundation and the FARA "Kyle Bryant Translational Research Award."


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Microarray data are deposited in the Gene Expression Omnibus as Accession No. GSE22651.


Supplemental Information includes two figures, three tables, Supplemental Experimental Procedures, and can be found with this article online at


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