title: Pluripotent state induction in mouse embryonic fibroblast using mRNAs of reprogramming factors.
abstract: Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs) generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF) cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1) and Rex-1 (ZFP-42, zinc finger protein 42). Using embryonic stem cells (ESCs) conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs) using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF) cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.
authors.affiliation: Provincial Key Laboratory of Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China. ahmedkamelvet@gmail.com.
authors.forename: Ahmed Kamel
authors.initials: AK
authors.lastname: El-Sayed
authors.affiliation: Provincial Key Laboratory of Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China. yzuztzhang@gmail.com.
authors.forename: Zhentao
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Provincial Key Laboratory of Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China. leizhang5807@gmail.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Provincial Key Laboratory of Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China. liuyunfei198@gmail.com.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Veterinary Integrative Biosciences (VIBS), College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4458, USA. labbott@cvm.tamu.edu.
authors.forename: Louise C
authors.initials: LC
authors.lastname: Abbott
authors.affiliation: Provincial Key Laboratory of Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China. ynzhang@yzu.edu.cn.
authors.forename: Yani
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Provincial Key Laboratory of Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China. yubcli@yzu.edu.cn.
authors.forename: Bichun
authors.initials: B
authors.lastname: Li
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D065150:Cellular Reprogramming / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D044127:Epigenesis, Genetic; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D016513:Mice, SCID; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection
chemical_list: D015415:Biomarkers; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D012333:RNA, Messenger; D014157:Transcription Factors; D049452:Green Fluorescent Proteins