Generation of Isogenic Pluripotent Stem Cells Differing Exclusively at Two Early Onset Parkinson Point Mutations

The Whitehead Institute, 9 Cambridge Center, Cambridge, MA 02142, USA.
Cell (Impact Factor: 32.24). 07/2011; 146(2):318-31. DOI: 10.1016/j.cell.2011.06.019
Source: PubMed

ABSTRACT Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc finger nuclease (ZFN)-mediated genome editing and iPSC technology, we provide a generally applicable solution to this problem, generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinson's disease by modifying the underlying point mutations in the α-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents significant progress for basic biomedical research and an advance toward hiPSC-based cell replacement therapies.

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Available from: Dmitry Guschin, Sep 28, 2015
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    • "Thus, if you find something different between the control and the patient-derived iPSCs you have to worry whether the difference is due to the pathology of the disease or to the variability between cell lines. For this reason, years ago, we started generating isogenic cell lines that differed from one another exclusively by a disease-causing mutation in order to generate a molecularly defined system in which we could study genetic diseases (Soldner et al., 2011). However, an important part of medically relevant diseases is that most are sporadic. "
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    ABSTRACT: Rudolf Jaenisch is a Professor of Biology at Massachusetts Institute of Technology, a founding member of the Whitehead Institute for Biomedical Research and the current president of the International Society for Stem Cell Research (ISSCR). His contributions to the stem cell field span from making the first transgenic mouse to seminal advances in the reprogramming field, and much more. In recognition of his pioneering research leading to induced pluripotency, he recently received the 2015 March of Dimes Prize in Developmental Biology. At the recent Keystone Meeting on 'Transcriptional and Epigenetic Influences on Stem Cell States' in Colorado, we had the opportunity to talk to him about his life and work. © 2015. Published by The Company of Biologists Ltd.
    Development 06/2015; 142(12):2085-7. DOI:10.1242/dev.126128 · 6.46 Impact Factor
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    • "In contrast, iPSCs (Takahashi et al., 2007) can be isolated from patients directly and still maintain pluripotency and an unlimited self-renewal capacity . Accordingly, when conjugated with made-to-order genetic correction technologies, human iPSCs derived from a patient with a genetic disorder (Park et al., 2008; Hotta et al., 2009) might be applicable to autologous transplantation as ex vivo gene therapy (Sebastiano et al., 2011; Soldner et al., 2011; Zou et al., 2011a, 2011b). "
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    ABSTRACT: Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Stem Cell Reports 11/2014; 4(1). DOI:10.1016/j.stemcr.2014.10.013 · 5.37 Impact Factor
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    • "Patient-derived pluripotent stem cells, including embryonic stem (ES) and induced pluripotent stem (iPS) cells, are important tools to model pathology1011121314. Although in vitro studies using DS-ES and DS-iPS cells reproduced the haematopoietic abnormalities in DS151617, DS-derived pluripotent stem cells with an acquired GATA1 mutation have not been generated. "
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    ABSTRACT: Infants with Down syndrome (DS) are at a high risk of developing transient abnormal myelopoiesis (TAM). A GATA1 mutation leading to the production of N-terminally truncated GATA1 (GATA1s) in early megakaryocyte/erythroid progenitors is linked to the onset of TAM and cooperated with the effect of trisomy 21 (Ts21). To gain insights into the underlying mechanisms of the progression to TAM in DS patients, we generated human pluripotent stem cells harbouring Ts21 and/or GATA1s by combining microcell-mediated chromosome transfer and genome editing technologies. In vitro haematopoietic differentiation assays showed that the GATA1s mutation blocked erythropoiesis irrespective of an extra chromosome 21, while Ts21 and the GATA1s mutation independently perturbed megakaryopoiesis and the combination of Ts21 and the GATA1s mutation synergistically contributed to an aberrant accumulation of skewed megakaryocytes. Thus, the DS model cells generated by these two technologies are useful in assessing how GATA1s mutation is involved in the onset of TAM in patients with DS.
    Scientific Reports 08/2014; 4:6136. DOI:10.1038/srep06136 · 5.58 Impact Factor
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