title: Exploring refined conditions for reprogramming cells by recombinant Oct4 protein.
abstract: The generation of human induced pluripotent stem (iPS) cells would represent an appealing option for the derivation of pluripotent patient-specific cells, as no embryos or oocytes are required. However, crucial safety issues have to be addressed in order to create human iPS cells that are clinically useful, as the classical iPS technique involves permanent genetic manipulation that may result in tumor formation. Various experimental strategies have been suggested to accomplish transgene-free derivation of iPS cells, including the use of non-integrating viruses, site specific recombinases to excise transgenes after reprogramming, or RNA transfection. Protein transduction, i.e. the direct delivery of biologically active proteins into cells, has been employed to generate iPS cells but has been found to have very low efficiency. In fact, success of protein transduction is limited by poor stability and solubility of recombinant factors, as well as their poor endosomal release. We recently reported the generation of cell-permeant versions of Oct4 and Sox2 and showed that both can be delivered intracellularly as biologically active proteins. Here we explore conditions for enhanced protein stabilization and delivery into somatic cells. Employing optimized conditions, we demonstrate that Oct4 protein delivery can substitute for Oct4 virus, yielding iPS derivation efficacy comparable to a four virus transduction protocol. The number of colonies is strictly dependent on the dose and duration of cell-permeant Oct4 exposure. We expect our transduction system to reach a thus far unattained level of control over reprogramming activity, turning it into a valuable tool for both the analysis of the reprogramming mechanism and the derivation of transgene-free iPS cells.
authors.affiliation: Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, University of Bonn - Life and Brain Center and Hertie Foundation, Bonn, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Thier
authors.forename: Bernhard
authors.initials: B
authors.lastname: Münst
authors.forename: Frank
authors.initials: F
authors.lastname: Edenhofer
mesh_terms: D000328:Adult; D000818:Animals; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics*; D005347:Fibroblasts; D015870:Gene Expression; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae; D014161:Transduction, Genetic; D019076:Transgenes; D016662:Virus Integration
chemical_list: D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D011994:Recombinant Proteins