Whitehead Institute researchers report that they have simplified the creation of induced pluripotent stem (iPS) cells by cutting the number of viruses used in the reprogramming process from four to one. The one catch is that while the new single-virus technique integrates all four genes into the same location, it has proven to be about 100 times less efficient than older approaches.
The earliest reprogramming efforts relied on four separate viruses to transfer genes into the cells’ DNA, or one virus for each reprogramming gene (Oct4, Sox2, c-Myc, and Klf4). Once activated, those genes converted the cells from their adult, differentiated status to an embryonic-like state.
This method, however, poses significant risks for potential use in humans, according to the scientists. The viruses used in reprogramming could potentially trigger the expression of oncogenes, because they may insert DNA anywhere in a cell’s genome, they explain.
The new method works by joining in tandem the four reprogramming genes through the use of bits of DNA that code for polymers known as 2A peptides. The researchers manufactured a polycistronic virus capable of expressing all four reprogramming genes once it is inserted into the genomes of mature mouse and human cells.
When the cells’ protein-creating machinery reads the tandem genes’ DNA, it begins making a protein. But when it tries to read the 2A peptide DNA that resides between the genes, the machinery momentarily stops, allowing the first gene’s protein to be released. The machinery then moves on to the second gene, creates that gene’s protein, stalls when reaching another piece of 2A peptide DNA, and releases the second gene’s protein. The process continues until the machinery has made the proteins for all four genes.
Using the tandem genes, the team created iPS cells containing just a single copy of the polycistronic vector instead of multiple integrations of the viruses. The investigators point out that that this approach can be made safer by combining it with technologies such as gene targeting, which allows a single transgene to be inserted at defined locations.
The scientists say that they are working on figuring out why this method is less efficient. “We're not sure why, but we need to look at what's going on with expression levels of the polycistronic virus's proteins compared to separate viruses' proteins,” says Bryce Carey, an MIT graduate student working in the lab of Whitehead member Rudolf Jaenisch, who led this investigation.
The article appears online between December 15 and December 19 in PNAS.