At first, then, Yamanaka set about to determine how mouse embryonic cells maintain their pluripotency, the ability to differentiate into any body cell type. He hypothesized that certain proteins would be evident in mouse embryonic cells but not in differentiated cells. He also thought that introducing the genes for these proteins—specifically, transcription factors, which control the activity of other genes—into a normal skin cell’s chromosomes would transform it into an embryonic cell.
After four years of experimentation, he uncovered 24 factors that, when added to ordinary mouse fibroblast cells and subjected to the correct culturing procedures, could create pluripotent cells virtually identical to stem cells. Yamanaka kept examining each factor and found that none could do the job alone; instead a combination of four particular genes did the trick. In 2006 he published a landmark article in Cell identifying them: Oct3/4, Sox2, c-Myc and Klf4.
News of the stunning feat prompted scientists around the world to try to reproduce it using human, rather than mouse, cells. In 2007 Yamanaka reported that triumph with the four transcription factors at the same time as Thomson’s team. “It is actually fairly straightforward to repeat what we have done,” Thomson told the press at the time—still, researchers have likened the breakthrough to turning lead into gold.
The achievement sparked many investigators to switch their efforts from embryonic stem cells to the induced versions. Yamanaka and others have now derived iPS cells from a variety of tissue types, including liver, stomach and brain, and turned the iPS cells into skin, muscle, gut and cartilage, as well as neural cells that can secrete the neurotransmitter dopamine and heart cells that can beat in sync.
Two big safety issues, though, will keep iPS cells out of the clinic for a while. One is that the transcription factor c-Myc happens to be a powerful cancer gene, and the cells produced by Yamanaka’s team tended to become cancerous. “Making iPS cells is very similar to making cancer,” he explains. In principle, c-Myc may not be necessary: in mice, Yamanaka and a group led by Rudolf Jaenisch at the Massachusetts Institute of Technology found a way to avoid using c-Myc, in part, by optimizing culture conditions. Out of 100 mice implanted with iPS cells created without c-Myc in Yamanaka’s lab, none died after 100 days, compared with six out of 100 that died of tumors when c-Myc was used.*
The other risk is the vector used to deliver the genes into target cells—namely, retroviruses. The process results in stem cells full of viruses. Moreover, retroviruses can induce mutations in the cells that lead to cancer. Researchers may soon overcome this hurdle, too. In September a team at the Harvard Stem Cell Institute announced the creation of mouse iPS cells using as a vector the adenovirus, which is safer than retroviruses. In October, Yamanaka’s lab reported success using plasmids, or circular pieces of DNA. Other retrovirus alternatives include proteins and lipid molecules.
Although the surge in interest has led to rapid developments and much competition among labs, Yamanaka and others do not think that iPS cells can replace their embryonic counterparts yet. “We don’t yet know if embryonic stem cells and iPS cells are truly equivalent,” says Konrad Hochedlinger of Massachusetts General Hospital’s Center for Regenerative Medicine. He adds that “at this point, iPS cells are a powerful additional source of pluripotent cells. Time will tell if iPS cells will at some point replace embryonic stem cells. It would be premature to make such a decision now.”
But while insisting iPS cell work remains far from being clinic-ready, Yamanaka trumpets its vast potential for conditions such as diabetes, spinal cord injury, Parkinson’s and even, he chuckles, baldness. “This enormous and striking finding provides a clear framework for regenerative medicine and cell therapy,” says Shinichi Nishikawa, director of the Laboratory for Stem Cell Biology at Japan’s RIKEN Center for Developmental Biology.