Generation of Transgene-Free iPSC Lines from Human Normal and Neoplastic Blood Cells Using Episomal Vectors
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.
Key wordsEpstein–Barr virus Episomal plasmids Reprogramming Induced pluripotent stem cells Human bone marrow Cord blood Chronic myeloid leukemia
We thank Professor James Thomson at Morgridge Institute for Research, Madison, WI, for providing reprogramming plasmids and FGF2, and Patricia Liu for editorial assistance. This work was supported by funds from the National Institute of Health (P01 GM081629 and P51RR000167).
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