Article

Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy.

Morgridge Institute for Research, Madison, WI 53715, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 04/2011; 108(16):6537-42. DOI: 10.1073/pnas.1103388108
Source: PubMed

ABSTRACT Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.

Download full-text

Full-text

Available from: Brian Edward McIntosh, Jun 18, 2015
0 Followers
 · 
403 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In gene expression, gene mutations often lead to negative effect of protein translation in prokaryotic organisms. With consideration of the influences produced by gene mutation, a novel method based on error-correction coding theory is proposed for modeling and detection of translation initiation in this paper. In the proposed method, combined with a one-dimensional codebook from block coding, a decoding method based on the minimum hamming distance is designed for analysis of translation efficiency. The results show that the proposed method can recognize the biologically significant regions such as Shine-Dalgarno region within the mRNA leader sequences effectively. Also, a global analysis of single base and multiple bases mutations of the Shine-Dalgarno sequences are established. Compared with other published experimental methods for mutation analysis, the translation initiation can not be disturbed by multiple bases mutations using the proposed method, which shows the effectiveness of this method in improving the translation efficiency and its biological relevance for genetic regulatory system.
    Journal of Theoretical Biology 12/2011; 296:33-40. DOI:10.1016/j.jtbi.2011.11.031 · 2.30 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Here we describe a strategy to model blood vessel development using a well-defined induced pluripotent stem cell-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats.
    Stem Cell Reviews and Reports 09/2014; DOI:10.1007/s12015-014-9549-5 · 3.21 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Age-related macular degeneration (AMD) is a leading cause of severe vision loss in the Western world and is increasing exponentially as the population ages. Despite enormous worldwide efforts, the earliest pathogenic pathways involved in AMD are still not fully understood. It is essential to develop research tools for effective modeling of AMD pathogenesis and for subsequent drug discovery and cell or molecular therapies. This review will focus on the current progress in human pluripotent stem cells for understanding and treating AMD.
    Optometry and vision science: official publication of the American Academy of Optometry 05/2014; DOI:10.1097/OPX.0000000000000282 · 2.04 Impact Factor