Induced pluripotent stem cells made by inserting genes at just one location.
Adult mouse cells can now be reprogrammed to a stem-cell-like state with the help of a single genetic insertion — rather than the multiple gene insertions required in the past. The advance also enables reprogrammable mice to be maintained in the lab generation after generation.
Three years ago, Shinya Yamanaka at Kyoto University and his colleagues made a splash by creating the first induced pluripotent stem (iPS) cells, which can develop into any of the body's cell types. Because they are obtained using adult body cells, iPS cells hold the potential for being used to develop human therapies without the ethical concerns associated with stem cells obtained from embryos. So far, iPS cells have been reprogrammed from a wide variety of somatic cell types, including skin, blood and liver cells, but scientists are still unsure how iPS cells compare to true embryonic stem cells.
One challenge has been the fact that to induce pluripotency, four reprogramming genes must be inserted into the genome — Oct4, Sox2, Klf4 and c-Myc. This requires the use of multiple retroviruses, meaning the genes end up in random locations in the mouse genome, which can interfere with the function of the mouse's own genes. Moreover, offspring of these mice must be screened to ensure that they contain all of the required reprogramming genes.
Now, two teams of researchers — one led by Rudolf Jaenisch at the Massachusetts Institute of Technology in Cambridge and the other led by his former student Konrad Hochedlinger at Harvard University, also in Cambridge, Massachusetts — describe a technique in Nature Methods that avoids these difficulties1,2.
The researchers combined the four mouse reprogramming genes onto a piece of DNA, known as a cassette, which they inserted at a single locus in the mouse genome. The mice were then bred, and their somatic cells were transformed into iPS cells following the addition of the antibiotic doxycycline, which triggers the cassette to express the four reprogramming genes.
“The problem with a virus is that you never really know where it landed in the genome. Konrad Hochedlinger , Harvard University”
"The advantage of this method is that the single gene has been introduced to a defined locus," says Hochedlinger, "The problem with a virus is that you never really know where it landed in the genome or how well it was expressed." By eliminating this variability, Hochedlinger says that the technique will eliminate the need for further screening in the mice and free up the equivalent of one full-time employee in his lab. "I'm very happy," he says.
The technique may also help to answer lingering doubts about the differences between iPS cells and embryonic stem cells. A study earlier this year in Cell Stem Cell showed that hundreds of genes are differentially expressed in the two cell types3, and another revealed that iPS cells are not as efficient as embryonic stem cells at differentiating into all cell types4. Matthias Stadtfeld of Harvard University, who is first author on one of the reprogramming studies2, says that it will now be possible to compare two genetically matched cell types and ask if iPS cells are as useful as embryonic stem cells. "We are fairly confident you can reprogram any cell type, the question is: are we ending up with the same quality of cells in the end?"
Other experts agree that the advance will circumvent limitations with iPS technologies. "I've been hoping these guys would make these strains of mice," says stem-cell biologist George Daley of the Children's Hospital in Boston, Massachusetts, who was not involved in the research.
Although some researchers have developed non-genetic systems to reprogram cells using proteins or small molecules (see 'Stem-cell therapies closer to the clinic'), Daley points out that such methods are currently "incredibly inefficient". To improve efficiency and safety so that these techniques can be used in humans, scientists could potentially create lines of mice with just three of the four reprogramming genes, and screen for chemicals that could be used as an alternative to inserting the fourth reprogramming gene.
"Fundamentally, everyone is looking to improve the efficiency of reprogramming using chemicals, proteins and the like," Daley says. "These two papers give you a substrate on which to work."
Carey, B. W., Markoulaki, S., Beard, C., Hanna, J. & Jaenisch, R. Nature Methods advance online publication doi:10.1038/nmeth.1410 (2009).
Stadtfeld, M., Maherali, N., Borkent, M. & Hochedlinger, K. Nature Methods advance online publication doi:10.1038/nmeth.1409 (2009).
Chin, M. H. et al. Cell Stem Cell 5, 111-123 (2009).
Zhao, X.-Y. et al. Nature 461, 86-90 (2009).