iPSCs derivation methods
1. Lentivirus and retrovirus vectors
2. Plasmid-based episomal vectors
3. Transient transfection
4. PiggyBac transposon
5. Protein transduction
6. Small molecules
7. Synthetic mRNA - September 15, 2011
In 2010, Warren and colleagues developed a technology for reprogramming somatic cells to RNA-induced pluripotent stem cells (RiPSCs) and for redirecting pluripotent cells toward differentiated cells (RNA-mediated directed differentiation) based on administration of synthetic and modified mRNA.
Firstly, they manufactured a synthetic mRNA encoding GFP. The GFP open reading frame (GFP-ORF) is flanked by a 5ʼ untranslated region (GFP-UTR) with a Kosak sequence and a 3ʼ GFP-UTR with an oligo(dt) sequence. In preliminary experiments, GFP-mRNA was transfected (electroporation or cationic vehicle) into murine embryonic fibroblast and human epidermal keratinocytes. During these experiments, it was observed a dose-dependent cytotoxicity that was exacerbated on repeated transfection. The protein cytotoxicity was caused by activated antiviral defenses in mammalian cells. These innate immune responses were reduced by addition a uncapped products bearing 5ʼ triphosphates and by changing some mRNA bases.
Then, using synthetic and modified GFP-mRNA, they were analyzed: cellular penetration capacity, cell localisations for the coexpression of multiple proteins, cell growth and viability, repetitive transfections for high levels of ectopic protein expression and so on. All results showed that mRNA could be utilised to induced RiPSCs to diverse cell types.
Finally, they have developed a technology for reprogramming cells using synthetic and modified mRNAs encoding the four Yamanaka factors Oct4, Sox2, Klf4 and Myc (OSKM) or adding Lin28 (OSKML). mRNAs were transfected into four human cell types: Detroit 551, MRC-5 fetal fibroblasts, BJ postnatal fibroblasts, and fibroblast-like cells from a skin biopsy. The results demonstrated a good efficiency in quantity of iPSCs and reprogramming time. Validation of RiPSCs was done by several methods: immunostaining, DNA fingerprinting, test for developmental potential and so on. Given the efficiency of the RNA-induced pluripotent stem cells (RiPSCs), then, they used the same method to redirect pluripotent cells toward differentiated cells.
In conclusion, authors of mRNA-method have demonstrated that the administration of synthetic and modified mRNA generate iPSCs with high efficiency and that the technology can be applied to efficiently redirect pluripotent cell fate to terminally differentiated fates without compromising genomic integrity. In addition, they mention that this mRNA-technology can be used to express cancer or pathogen antigens for immunotherapy.
Taken together these data, we could hypothesize that this technology might also be utilised to direct conversion of somatic cell types, without the stage of iPSCs. Remain to be determined the molecules (proteins), encoded by mRNAs, for each somatic cells conversion.
Warren et al., Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA, Cell Stem Cell (2010), doi: 10.1016/j.stem.2010.08.012