Induced Pluripotent Stem Cells Generated Without Viral Integration

Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, 185 Cambridge Street, Boston, MA 02114, USA.
Science (Impact Factor: 33.61). 10/2008; 322(5903):945-9. DOI: 10.1126/science.1162494
Source: PubMed


Pluripotent stem cells have been generated from mouse and human somatic cells by viral expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc. A major limitation of this technology is the use of potentially harmful genome-integrating viruses. We generated mouse induced pluripotent stem (iPS) cells from fibroblasts and liver cells by using nonintegrating adenoviruses transiently expressing Oct4, Sox2, Klf4, and c-Myc. These adenoviral iPS (adeno-iPS) cells show DNA demethylation characteristic of reprogrammed cells, express endogenous pluripotency genes, form teratomas, and contribute to multiple tissues, including the germ line, in chimeric mice. Our results provide strong evidence that insertional mutagenesis is not required for in vitro reprogramming. Adenoviral reprogramming may provide an improved method for generating and studying patient-specific stem cells and for comparing embryonic stem cells and iPS cells.

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Available from: Jochen Utikal, Aug 12, 2014
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    • "However, using iPS cells carries a risk of tumourigenesis (Miura et al., 2009). Therefore, researchers have attempted to reduce the risk of tumourigenesis by establishing integration-free methods for generating iPS cells (Fusaki et al., 2009; Okita et al., 2008; Stadtfeld et al., 2008) and using selected cell lines (Koyanagi-Aoi et al., 2013). One solution may be to eliminate undifferentiated cells from transplants, and the use of terminally differentiated cells is desirable (Doi et al., 2012). "
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    ABSTRACT: The present study examined the efficacy of a neural induction method for human induced pluripotent stem (iPS) cells to eliminate undifferentiated cells and to determine the feasibility of transplanting neurally induced cells into guinea-pig cochleae for replacement of spiral ganglion neurons (SGNs). A stepwise method for differentiation of human iPS cells into neurons was used. First, a neural induction method was established on Matrigel-coated plates; characteristics of cell populations at each differentiation step were assessed. Second, neural stem cells were differentiated into neurons on a three-dimensional (3D) collagen matrix, using the same protocol of culture on Matrigel-coated plates; neuron subtypes in differentiated cells on a 3D collagen matrix were examined. Then, human iPS cell-derived neurons cultured on a 3D collagen matrix were transplanted into intact guinea-pig cochleae, followed by histological analysis. In vitro analyses revealed successful induction of neural stem cells from human iPS cells, with no retention of undifferentiated cells expressing OCT3/4. After the neural differentiation of neural stem cells, approximately 70% of cells expressed a neuronal marker, 90% of which were positive for vesicular glutamate transporter 1 (VGLUT1). The expression pattern of neuron subtypes in differentiated cells on a 3D collagen matrix was identical to that of the differentiated cells on Matrigel-coated plates. In addition, the survival of transplant-derived neurons was achieved when inflammatory responses were appropriately controlled. Our preparation method for human iPS cell-derived neurons efficiently eliminated undifferentiated cells and contributed to the settlement of transplant-derived neurons expressing VGLUT1 in guinea-pig cochleae. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.
    Journal of Tissue Engineering and Regenerative Medicine 08/2015; DOI:10.1002/term.2072 · 5.20 Impact Factor
    • "To address the safety issues that arise from target cell genomes harbouring integrated exogenous sequences, a number of modified genetic protocols have been further developed: These protocols produce hIPS cells with potentially reduced risks, and include non-integrating adenoviruses to deliver reprogramming genes [25], transient transfection of reprogramming plasmids [26], piggy-Bac tansposition systems [27], Cre-excisable viruses [28] and or iP/ENNA1- based episomal expression system [29]. "
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    ABSTRACT: Human pluripotent stem cells in their characteristics of immortal self-renewal and pluripotency have gained high expectations during last fifteen years regarding their ability to cure near any human disease. Regenerative Medicine was the new term for the novel cell-based treatments, which had the possibility of curing genetic diseases as well by previous correction of endogenous mutations in patient-specific induced pluripotent stem (iPS) cells. The possibility of developing combined genetic/ stem cell therapies for at least inherited monogenic diseases, meanwhile surpassing ethical rightness to destroy otherwise non-usable human embryos, still raised even more interest in the field of translational pluripotency. Aternative sources of stem cells from adult human tissues, gave rise to the research competition between the pluripotent stem and adult stem cells. The latter surpassed ethical debates but imposed slowness on the development of the field to translational medicine: these cells were multipotent in their vast majority, not immortal, and obtaining relevant amounts for research was more difficult compared to pluripotent stem cells. Despite their advantages in terms of availability, induced pluripotent cells have been mainly obtained by means of genetic modification, which impairs safeness of the downstream therapeutic product. The present review outlines the most recent inventions related to the preparation of safe clinical-grade hIPS cells. Its main objective is to contribute a concise compilation of the advances in the reprogramming of adult cells to pluripotency, the choice of original tissues and cell types, the methods of reprogramming to pluripotency, and above all the alternatives for elimination of genetic material from the reprogramming protocol. Registered patents and related bibliography reported mostly from the past two years have been reviewed, although basic literature has been included where needed to explain recent developments.
    06/2015; epub ahead of print(1). DOI:10.2174/2210296505666150318231456
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    • "However, the commonly employed method of viral gene delivery of the reprogramming factors is associated with considerable risk of insertional mutagenesis and genotoxicity (Wu and Dunbar, 2011). To overcome these risks, alternative methods, such as non-integrating adenoviral vectors (Stadtfeld et al., 2008), plasmids (Yu et al., 2009), recombinant proteins (Zhou et al., 2009), modified mRNAs (Warren et al., 2010) and small molecules (Shi et al., 2008) were successfully used for iPS cell derivation. However, the efficiency of reprogramming using these methods is significantly lower than that of retro-or lentiviral vectors, and the alternative methods may require repetitive treatments to maintain pluripotency (Kumar et al., 2014). "
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    ABSTRACT: Induced pluripotent stem cells (iPSCs) are a seminal breakthrough in stem cell research and are promising tools for advanced regenerative therapies in humans and reproductive biotechnology in farm animals. iPSCs are particularly valuable in species in which authentic embryonic stem cell (ESC) lines are yet not available. Here, we describe a nonviral method for the derivation of bovine iPSCs employing Sleeping Beauty (SB) and piggyBac (PB) transposon systems encoding different combinations of reprogramming factors, each separated by self-cleaving peptide sequences and driven by the chimeric CAGGS promoter. One bovine iPSC line (biPS-1) generated by a PB vector containing six reprogramming genes was analyzed in detail, including morphology, alkaline phosphatase expression, and typical hallmarks of pluripotency, such as expression of pluripotency markers and formation of mature teratomas in immunodeficient mice. Moreover, the biPS-1 line allowed a second round of SB transposon-mediated gene transfer. These results are promising for derivation of germ line-competent bovine iPSCs and will facilitate genetic modification of the bovine genome.
    Cellular Reprogramming 04/2015; DOI:10.1089/cell.2014.0080. · 1.79 Impact Factor
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