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.
Highlights
ObjectivesThe main goal of our research was to find suitable conditions for generation of mouse induced Pluripotent Stem Cells (iPSCs) by mRNA transfection of reprogramming factors into mouse somatic cells through minimum exposure time of transfection, to develop a model for further research, in spite of few reports concerning the utilization of mRNAs of reprogramming factors to induce pluripotency in murine species [51]
Great advances have been rapidly achieved in reprogramming of somatic cells to generate cells with greater pluripotency that aid in understanding the mechanisms of both differentiation and dedifferentiation
The mammalian expression plasmid was used as a platform to prepare the mRNA of each transcription factor by in vitro transcription (IVT) using T7 RNA polymerase enzyme
Summary
The main goal of our research was to find suitable conditions for generation of mouse iPSCs by mRNA transfection of reprogramming factors into mouse somatic cells through minimum exposure time of transfection, to develop a model for further research, in spite of few reports concerning the utilization of mRNAs of reprogramming factors to induce pluripotency in murine species [51]
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