title: Generation of transgene-free iPSC lines from human normal and neoplastic blood cells using episomal vectors.
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.
authors.affiliation: Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Kejin
authors.initials: K
authors.lastname: Hu
authors.forename: Igor
authors.initials: I
authors.lastname: Slukvin
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D048430:Cell Shape; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics; D018920:Coculture Techniques; D015925:Cryopreservation; D017077:Culture Media, Conditioned; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry; D010957:Plasmids / Q000235:genetics*; D014162:Transfection; D019076:Transgenes
chemical_list: D017077:Culture Media, Conditioned; D020778:Matrix Metalloproteinase 2