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
    • "Concerns about unpredictable effects of random genomic integration of retro-or lentivirus as well as tumorigenic risks of the permanent presence of the oncogene c-MYC in the genome led to the development of non-integrating virus-based protocols (e.g. Sendai virus), plasmid-, RNA-or protein-based reprogramming methods111112113114115116117. These approaches induce a transient burst of the three or four reprogramming factors, which appears to suffice to permanently activate an endogenous pluripotency program. "
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    ABSTRACT: Regenerating an injured heart holds great promise for millions of patients suffering from heart diseases. Since the human heart has very limited regenerative capacity, this is a challenging task. Numerous strategies aiming to improve heart function have been developed. In this review we focus on approaches intending to replace damaged heart muscle by new cardiomyocytes. Different strategies for the production of cardiomyocytes from human embryonic stem cells or human induced pluripotent stem cells, by direct reprogramming and induction of cardiomyocyte proliferation are discussed regarding their therapeutic potential and respective advantages and disadvantages. Furthermore, different methods for the transplantation of pluripotent stem cell-derived cardiomyocytes are described and their clinical perspectives are discussed.
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    • "To date, iPSCs have been generated with episomal plasmids, lentivirus, adenovirus, or Sendai virus mediated gene transfer as well as by direct delivery of transcription factor mRNA or protein. Furthermore, attempts have been made to exclude the potential oncogenic factor c-Myc (Yu et al., 2007; Okita et al., 2008; Stadtfeld et al., 2008). Another approach has been developed to replace ectopic transcription factors by small molecule cocktails (Hou et al., 2013). "
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    ABSTRACT: Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a comprehensive epigenetic process involving genome-wide modifications of histones and DNA methylation. This process is often incomplete, which subsequently affects iPSC reprograming, pluripotency, and differentiation capacity. Here, we review the epigenetic changes with a focus on histone modification (methylation and acetylation) and DNA modification (methylation) during iPSC induction. We look at changes in specific epigenetic signatures, aberrations and epigenetic memory during reprogramming and small molecules influencing the epigenetic reprogramming of somatic cells. Finally, we discuss how to improve iPSC generation and pluripotency through epigenetic manipulations.
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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.
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