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Generation of Induced Pluripotent Stem Cell Lines from Friedreich Ataxia Patients

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Abstract

Friedreich ataxia (FRDA) is an autosomal recessive disorder characterised by neurodegeneration and cardiomyopathy. It is caused by a trinucleotide (GAA) repeat expansion in the first intron of the FXN gene that results in reduced synthesis of FXN mRNA and its protein product, frataxin. We report the generation of induced pluripotent stem (iPS) cell lines derived from skin fibroblasts from two FRDA patients. Each of the patient-derived iPS (FA-iPS) cell lines maintain the GAA repeat expansion and the reduced FXN mRNA expression that are characteristic of the patient. The FA-iPS cells are pluripotent and form teratomas when injected into nude mice. We demonstrate that following in vitro differentiation the FA-iPS cells give rise to the two cell types primarily affected in FRDA, peripheral neurons and cardiomyocytes. The FA-iPS cell lines have the potential to provide valuable models to study the cellular pathology of FRDA and to develop high-throughput drug screening assays. We have previously demonstrated that stable insertion of a functional human BAC containing the intact FXN gene into stem cells results in the expression of frataxin protein in differentiated neurons. As such, iPS cell lines derived from FRDA patients, following correction of the mutated gene, could provide a useful source of immunocompatible cells for transplantation therapy.

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Acknowledgments

The authors are grateful to Ms L Corben for her help in the preparation of the ethics documentation, M. Pera for provision of TG-30 and to L. Li, M. Denham and K. Upton for technical assistance and helpful discussions. This work was supported by the Friedreich Ataxia Research Association (Australasia) and Friedreich’s Ataxia Research Alliance.

Author Contributions

All authors contributed in: conception and design, collection of data, data analysis and interpretation, manuscript writing, final approval. Financial support provided by MD and AP. The authors declare no competing financial interests relevant to this research.

Author information

Authors and Affiliations

  1. Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Melbourne, Australia

    Jun Liu & Paul J. Verma

  2. Bruce Lefroy Centre for Genetic Health Research, Murdoch Childrens Research Institute, Parkville, Australia

    Marguerite V. Evans-Galea, Martin B. Delatycki & Joseph P. Sarsero

  3. Department of Clinical Genetics, Austin Health, Heidelberg, Australia

    Martin B. Delatycki

  4. Monash Immunology and Stem Cell Laboratories, Monash University, Melbourne, Australia

    Anna Michalska

  5. Centre for Neuroscience, The University of Melbourne, Melbourne, Australia

    Jessie Leung, Mirella Dottori & Alice Pébay

  6. O’ Brien Institute, Fitzroy, Australia

    Duncan Crombie & Alice Pébay

  7. Department of Paediatrics, The University of Melbourne, Melbourne, Australia

    Robert Williamson

  8. Department of Pharmacology, The University of Melbourne, Melbourne, Australia

    Mirella Dottori & Alice Pébay

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  1. Jun Liu
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Corresponding author

Correspondence to Alice Pébay.

Additional information

Jun Liu, Paul J. Verma, Mirella Dottori and Alice Pébay contributed equally to this work.

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Supplementary Figure 1

Endogenous and exogenous expression of transgenes in iPS cell lines and fibroblasts. a RT-PCR showed that generated FA3-iPS cells and FA4-iPS cells expressed endogenous human pluripotent markers: OCT-4, SOX2, NANOG, and REX1. Transgene OCT-4 and SOX2 were effectively silenced, while the KLF4 and cMYC transgenes were still expressed. (Endo = endogenous genes, Tg = transgenes). b Integration of four transcription factors OCT4, SOX2, KLF4 and cMYC transgenes in the genome of generated iPS cells was confirmed by genomic PCR using transgene-specific primers, with parental fibroblasts as negative controls, and respective encoding constructs as positive controls. (GIF 114 kb)

High resolution image (TIFF 1734 kb)

Supplementary Figure 2

FRDA-iPS cells form teratomas. Histology of teratoma with gut-like epithelium (a, d), cartilage (b, e) and neural rosettes (c, f) after injection of FA3-iPS cells (a–c) and FA4-iPS cells (d–e) into severe combined immunodeficiency mice. (GIF 296 kb)

High resolution image (TIFF 592 kb)

Supplementary Figure 3

FRDA-iPS cells form EB with cells representative of the three germ layers. Immunostaining of EBs generated from FA3-iPS and FA4-iPS cells immunostained with markers for endoderm (AFP, a), mesoderm (c-kit or CD31, b) and ectoderm (nestin, c). (d): Isotype controls. (GIF 244 kb)

High resolution image (TIFF 2667 kb)

Supplementary Figure 4

GAA expansion PCR for FA3- and FA4-iPS cells. The size of the GAA expansion was determined in control (FAC4) and FRDA (FA3 and FA4) cells by comparison to standard DNA markers (M1 and M2). Genomic DNA isolated from fibroblasts (lanes 1,3 and 6) and iPS cells (lanes 2, 4, 5 and 7–9) were used in long range PCR of the first intron of FXN. Non-expanded alleles yield a product of 810 bp. Positive (BACRP11-265B8 DNA containing the FXN gene; lane C) and negative controls (no DNA; lane N) were included. Multiple bands are observed in each sample since multiple sub-clones were pooled. (GIF 194 kb)

High resolution image (TIFF 576 kb)

FRDA-iPS cells generate beating cardiomyocytes. Generation of functional cardiomyocytes obtained following FA3-iPS derived EB plated onto gelatine and spontaneous differentiation. (MPG 8895 kb)

FRDA-iPS cells generate beating cardiomyocytes. Generation of functional cardiomyocytes obtained following FA4-iPS derived EB plated onto gelatine and spontaneous differentiation. (MPG 6509 kb)

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Liu, J., Verma, P.J., Evans-Galea, M.V. et al. Generation of Induced Pluripotent Stem Cell Lines from Friedreich Ataxia Patients. Stem Cell Rev and Rep 7, 703–713 (2011). https://doi.org/10.1007/s12015-010-9210-x

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