title: Activation of pluripotency-associated genes in mouse embryonic fibroblasts by non-viral transfection with in vitro-derived mRNAs encoding Oct4, Sox2, Klf4 and cMyc.
abstract: The first successful reprogramming of differentiated cells to a pluripotent state was done by retroviral introduction of four transcription factors (Oct4, Sox2, Klf4, cMyc) by the group of Yamanaka in 2006. Since then, scientists all over the world have attempted various methods to avoid insertional mutagenesis, a major limitation of the retrovirus-based method, however no technique was found to completely avoid DNA integration. Recently, a non-viral mRNA-based approach, inherent to avoid genomic integration, was implemented to generate stem cell-like cells, yet, seventeen daily transfections were required, inducing substantial stress on the cells. In this work, we demonstrate successful activation of pluripotency-associated genes in mouse embryonic fibroblasts by means of cationic lipid-mediated introduction of mRNAs encoding the four factors. Moreover, our transfection protocol required maximally three transfections. Up-regulation of the transfected factors as well as Nanog and SSEA-1, typical mouse pluripotency markers, was detected already after the first transfection. Nuclear localization of the introduced factors was confirmed. Positive alkaline phosphatase staining of cell clusters further confirmed the onset of the reprogramming process. In conclusion, the transfection method presented here holds great promise for safe generation of induced pluripotent stem cells of mouse origin.
authors: Research Group on Nanomedicines, Ghent University, Harelbekestraat 72, Ghent, Belgium.
authors: Geertrui
authors: G
authors: Tavernier
authors: Katharina
authors: K
authors: Wolfrum
authors: Joseph
authors: J
authors: Demeester
authors: Stefaan C
authors: SC
authors: De Smedt
authors: James
authors: J
authors: Adjaye
authors: Joanna
authors: J
authors: Rejman
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005786:Gene Expression Regulation; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D016256:Lewis X Antigen / Q000235:genetics / Q000378:metabolism; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D015854:Up-Regulation
chemical_list: C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D016256:Lewis X Antigen; C489428:Myc protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D000469:Alkaline Phosphatase