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.

Induced pluripotent stem cell generation using mRNAs: the effect of valproic acid, 5-azacytidine and ascorbic acid

“…Generation and characterization of iPSC colonies. According to our lab protocol (EL-SAYED et al 2014), five consecutive transfections were done to obtain pluripotent clones. The morphological appearance of the MEF cells showed remarkable changes from spindle shape to compact round epithelial shape, which increased by time.…”

“…1A). The iPS colonies were immune stained for pluripotency markers (Sox2, Nanog, Oct4, Klf4, SSEA-1, and c-Myc), and the obtained colonies were positive for all of them, while the control cells were negatively stained for these markers (EL-SAYED et al, 2014).…”

“…Generation of murine iPSCs using mRNAs of Yamanaka Factors. The technique of murine iPSC generation using mRNAs of the four Yamanaka factors (OSCK) was performed according to our lab protocol (EL-SAYED et al, 2014). The four genes were ligated to the expression vector (pCDNA3) using (T4 DNA) ligase enzyme (Takara, China).…”

“…Several safer strategies have been developed to overcome the potentially harmful effect of using viral integrating methods in reprogramming. These non-integrating safe strategies, such as episomal plasmid (OKITA et al, 2011), protein (ZHOU et al, 2009KIM et al, 2009), andmRNA (WARREN et al, 2010;EL-SAYED et al, 2014) transfection, still have significant genetic instability. Besides, several reports have demonstrated that the reprogramming may be inadequate at both transcriptional and epigenetic levels (XU et al, 2012;LEE et al, 2012;JI et al, 2016).…”

iPSC Preparation and Epigenetic Memory: Does the Tissue Origin Matter?

“…To overcome these and other potential pitfalls that could limit the future use of iPSCs for tissue regeneration, additional methods were developed based on viral and non-viral, non-integrating delivery of the transgenes containing OSKM [5][6][7][8][9][10]. Further modification of the design of the reprogramming expression vectors and new methods of delivery were designed to minimize or eliminate vector sequences that could be integrated into the iP-SCs genome [11,12].…”