Protocol

Pluripotent Stem Cells

Volume 997 of the series Methods in Molecular Biology pp 163-176

Date:

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

  • Kejin HuAffiliated withWisconsin National Primate Research Center, University of Wisconsin-Madison
  • , Igor SlukvinAffiliated withWisconsin National Primate Research Center, University of Wisconsin-Madison

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Abstract

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 words

Epstein–Barr virus Episomal plasmids Reprogramming Induced pluripotent stem cells Human bone marrow Cord blood Chronic myeloid leukemia