Generation of Transgene-Free iPSC Lines from Human Normal and Neoplastic Blood Cells Using Episomal Vectors

Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 04/2013; 997:163-76. DOI: 10.1007/978-1-62703-348-0_13
Source: PubMed


Human induced pluripotent stem cells (iPSCs) have become an important tool for modeling human diseases and are considered a potential source of therapeutic cells. Original methods for iPSC generation use fibroblasts as a cell source for reprogramming and retroviral vectors as a delivery method of the reprogramming factors. However, fibroblasts require extended time for expansion and viral delivery of transgenes results in the integration of vector sequences into the genome which is a source of potential insertion mutagenesis, residual expressions, and reactivation of transgenes during differentiation. Here, we provide a detailed protocol for the efficient generation of transgene-free iPSC lines from human bone marrow and cord blood cells with a single transfection of non-integrating episomal plasmids. This method uses mononuclear bone marrow and cord blood cells, and makes it possible to generate transgene-free iPSCs 1-3 weeks faster than previous methods of reprogramming with fibroblasts. Additionally, we show that this approach can be used for efficient reprogramming of chronic myeloid leukemia cells.

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Available from: Kejin Hu, Jul 19, 2014
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    • "The integration site of retrovirus in the iPSCs may affect the gene expression and change the disease phenotype after redifferentiating them into the original lineages. So, efficient induction of transgene-free iPSCs such as using Sendai virus and episomal systems has been reported [19] [48] [57]. But, we can have in mind, as mentioned by Ramos-Mejia and collaborators (2012), that the difficulties in reprogramming cancer cells do not seem exclusively due to technical barriers or the need for improved reprogramming technologies. "
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