“We can rebuild him. We have the technology. We can make him better than he was. Better, stronger, faster”. They did it for Steve Austin in The Six Million Dollar Man, and now researchers from the laboratory of Joseph C. Wu (Stanford) have applied the same thinking to the technology behind human induced pluripotent stem cell (iPSC) generation.
iPSC technology holds great promise for the future of regenerative medicine, but there are currently several concerns regarding the methodologies utilized to “kick-start” the pluripotency network in somatic cells. Integrating expression vectors (e.g. retrovirus) carry a tumorigenic risk, and while non-integrating methods (Sendai virus, mRNAs, and protein/DNA-based methods) are better from this point of view, they tend to be much more expensive, time-consuming, and overall, much less efficient. This promoted the re-evaluation of several aspects of the vectors design, and the development of a new single-plasmid reprogramming system.
The new technology combines:
- Codon optimized (Co) reprogramming factors: genetically modified to replace rare amino-acid coding codons with more common ones to ensure high levels of the reprogramming factors with the right stoichiometry for optimal reprogramming.
- Mini-intronic plasmid (MIP) vector: robust transgene expression DNA vector in vitro and in vivo devoid of bacterial backbone sequences, which provides protection against transgene silencing, an antibiotic-free selectable marker, and higher levels of transgene expression compared to similar mini-circle vectors.
This combination allowed the production of a “CoMIP” vector which expressed reprogramming factors [OCT4, KLF4, SOX2, and c-MYC (OKSM)] controlled by one promoter separated by different self-cleaving 2a peptide sequences, and short hairpin RNA targeting p53.
Application of this system demonstrated:
- Higher transfection efficiency, cell survival, and expression efficiencies.
- Induction of pluripotency after a single transfection of various human/mouse cell types.
- More efficient than other mini-circle or EBNA/OriP episomal plasmid based methods.
- Reduced times for production of iPSC colonies.
- Induction of pluripotency in blood-derived peripheral blood mononuclear cells when used alongside an additional plasmid expressing LIN28 and NANOG.
Overall, this represents a new highly efficient, integration-free, easy to use, and cost-effective methodology for reprogramming somatic cells into therapeutically relevant pluripotent stem cells. This has the potential to bring reprogramming technology to laboratories where the high costs and expertise required previously prohibited such research, and could lead to an explosion in the number of studies in this field.
So do we stand on the verge of another iPSC-revolution? Read more about this potentially significant technological advance in Scientific Reports, January 2015.