Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming FactorsReport as inadecuate




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1

Provincial Key Laboratory of Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China

2

Department of Anatomy and Embryology, College of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt

3

Department of Veterinary Integrative Biosciences VIBS, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4458, USA





*

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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. View Full-Text

Keywords: pluripotency; reprogramming; epigenetics; induced pluripotent stem cells; in vitro transcription pluripotency; reprogramming; epigenetics; induced pluripotent stem cells; in vitro transcription





Author: Ahmed Kamel El-Sayed 1,2, Zhentao Zhang 1, Lei Zhang 1, Zhiyong Liu 1, Louise C. Abbott 3, Yani Zhang 1,* and Bichun Li 1,*

Source: http://mdpi.com/



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