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


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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    • "Indeed, such iPSC-based disease models are rapidly developing into a key platform for drug discovery and preclinical testing of candidate compounds (Lee et al., 2009, 2012a; Merkle and Eggan, 2013; Wainger et al., 2014). In the case of PD, recent studies have demonstrated that neurons derived from patient-specific iPSCs could successfully reproduce several disease-related phenotypes such as increased α-synuclein levels, mitochondrial dysfunction and hypersensitivity to toxins such as compounds that trigger mitochondrial stress or ROS (Byers et al., 2011; Devine et al., 2011; Seibler et al., 2011; Soldner et al., 2011; Cooper et al., 2012; Imaizumi et al., 2012; Liu et al., 2012; Sánchez-Danés et al., 2012; Miller et al., 2013; Reinhardt et al., 2013; Su and Qi, 2013; Sanders et al., 2014; Woodard et al., 2014). Most of these models showed evidence of biochemical changes that are directly dependent on the disease-specific genetic defects (Fig. 1). "
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    ABSTRACT: In contrast to the successful modeling of early-onset disorders using patient-specific cells, modeling of late-onset neurodegenerative diseases such as Parkinson's disease remains a challenge. This might be related to the often ignored fact that current induced pluripotent stem cell (iPSC) differentiation protocols yield cells that typically show the behavior of fetal stage cells. Acknowledging aging as a contributing factor in late-onset neurodegenerative disorders represents an important step on the road towards faithfully recreating these diseases in vitro. Here, we summarize progress in the field and review the strategies and challenges for triggering late-onset disease phenotypes.
    Development 09/2015; 142(18):3085-3089. DOI:10.1242/dev.120667 · 6.46 Impact Factor
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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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