Turn back the cell clock / Tim Horniak

Shinya Yamanaka discovered how to return adult cells to an embryonic state. These induced stem cells may soon replace embryonic stem cells as the next medical promise

When future historians record the stem cell wars, Shinya Yamanaka will probably be remembered as a peacemaker. The Japanese scientist helped bypass, in a surprising way, the moral debate surrounding the need to destroy embryos to create embryonic stem cells. In 2007, Yamanaka headed one of two research groups that showed that the genes of normal human skin cells could be reprogrammed to become stem cells. It seems that these cells, called induced pluripotent stem cells (iPS cells), are practically identical to embryonic stem cells, and they have the ability to differentiate into any other cell type.

The 46-year-old Yamanaka is clean-shaven and clean-cut, almost like a military man. His small office in a shabby wing of the Institute of Advanced Medical Sciences at Kyoto University is spotlessly clean, with nothing to indicate his achievements in creating iPS cells. It is possible that a Nobel Prize will one day grace one of the shelves. Yamanaka looks around and says, "About 10 meters below us is a room I have never entered. I am not allowed to enter there because I do not have permission from the government. It contains the only stem cells in Japan that originate from human embryos."

Although Japan is permissive in law, in fact it imposes strict laws on the production and, unlike the USA, also on the use of stem cells derived from human embryos. Researchers can spend almost a year filling out forms before they get permission to use the cells.

It was Japan's suffocating scientific culture, which made Yamanaka a pioneering pioneer. He was originally an orthopedic surgeon in Osaka, but in the mid-90s he decided to do post-doctoral training at the Gladstone Institute of Cardiovascular Diseases in San Francisco, and investigate the reprogramming of cancer-related genes in mice. There he had easy access to mouse embryonic stem cell lines, as well as an environment that included stable funding and exchange of views between leading researchers from around the world. But when he returned home he sank into depression. "When I returned to Japan, I lost all those incentives," Yamanaka recalled. "I had little funding and few good scientists around me, and I had to treat about 1,000 mice on my own."

Out of desperation, he almost left everything and returned to the operating room. But two things encouraged him to continue: an offer to head a small lab at the Nara Institute of Science and Technology, and the creation of the first generation of human stem cells by James A. Thomson of the University of Wisconsin-Madison (who headed the second group that produced human iPS cells last year).

Following Thomson's success in producing embryonic stem cells, many researchers have attempted to control the differentiation of these cells into different cell types that may replace diseased or damaged tissues. This treatment, if successful, will cause a revolution in the world of medicine. "It was too competitive for our small lab," says Yamanaka, "so I thought I should do the exact opposite. Instead of turning embryonic stem cells into something else, I will produce embryonic stem cells from something else.” Due to Ian Wilmot's success in cloning animals like Dolly the sheep, he says, “We knew that even fully differentiated cells could revert to an embryonic-like state. But we also thought it would be a very long project," that would last 20 or 30 years.

It lasted less than ten years. Yamanaka was eager to solve two main problems in the field of stem cells. One was the embryonic origin of the cells. He tells how he visited a friend's fertility lab and looked through the microscope at very young embryos. The sight of fragile and nascent life touched his heart, even though he emphasizes that he is not opposed to the use of embryonic stem cells "to save patients". The second problem is the risk that the immune system will reject the embryonic cells after they are implanted in the patient. There is no such danger when it comes to cells differentiated from the patient's own iPS cells.

First of all, Yamanaka tried to find out how mouse embryonic cells maintain their pluripotency, that is, their ability to differentiate into all types of cells in the body. He hypothesized that certain proteins would be found in embryonic mouse cells but not in differentiated cells. He also hypothesized that if the genes containing the code for the production of these proteins were inserted into the chromosomes of normal skin cells, and especially the genes for creating transcription factors that control the activity of other genes, the skin cells would turn into embryonic cells.

After four years of research, Yamanaka discovered 24 factors that, under the right conditions, can transform a simple mouse fibroblast cell into a pluripotent cell virtually identical to a stem cell. Yamanaka went on to investigate each of these transcription factors and discovered that none could act on their own. But a combination of four particular genes did the trick. In 2006, he published in the scientific journal Cell an article that became a landmark, in which he described the identification of these genes: Oct3/4, Sox2, c-Myc and Klf4.

The news of the amazing achievement spurred scientists around the world to repeat the experiment in human cells instead of mouse cells. In 2007, Yamanaka reported his achievement in combining the four transcription factors at the same time as Thomson's group. "It's actually quite simple to repeat what we did," Thomson told reporters at the time. And yet, researchers compared this breakthrough to turning lead into gold.

These discoveries led many researchers to shift their efforts from embryonic touch cells to these induced cells. Yamanaka and others have so far created iPS cells from a variety of tissue types, including liver, stomach and brain, and turned iPS cells into skin, muscle, intestine and cartilage cells, as well as nerve cells capable of secreting the neurotransmitter dopamine and heart cells capable of beating together.

But two safety aspects currently prevent the clinical use of iPS cells. One problem is that the transcription factor c-Myc is also a powerful cancer gene, and the cells Yamanaka created tended to become cancerous. "The creation of iPS cells is very similar to the development of cancer," he explains. Basically, c-Myc may not be needed at all. In mice, the research groups of Yamanaka and Rudolf Janisch from the Massachusetts Institute of Technology (MIT) found a way to avoid using c-Myc, in part by adjusting the culture growth conditions. All 100 mice transplanted with iPS cells created without c-Myc in Yamanaka's lab survived after 100 days compared to 6 out of 100 who died of cancerous tumors when transplanted with cells with c-Myc.

The second danger is the means used to introduce the genes into the target cells: a retrovirus. The process caused the creation of stem cells infected with viruses. Researchers may soon overcome this hurdle as well. In September 2008, a research group at the Harvard Stem Cell Institute announced the creation of iPS cells using an adenovirus that is safer to use than a retrovirus. In October, Yamanaka's laboratory reported success in using plasmids, that is circular pieces of DNA. Other substitutes for retroviruses include proteins and fat molecules.

Despite the great interest that has led to accelerated progress and much competition among labs, Yamanaka and others do not believe that iPS cells can replace their embryonic counterparts just yet. "We don't know if embryonic stem cells and iPS cells are really equivalent," says Konrad Hochdlinger of the Center for Tissue Regenerative Medicine at Massachusetts General Hospital. And he says that "currently, iPS cells are used as another powerful source of pluripotent cells. In time we will know if iPS cells will replace embryonic stem cells. Right now it's too early to decide."

But while he insists that research on iPS cells is very far from the clinical stage, Yamanaka also proclaims the tremendous promise of these cells in treating conditions such as diabetes, spinal cord injuries, Parkinson's disease, and even, he chuckles, baldness. "This amazing and important finding provides a clear framework for tissue regenerative medicine and cell-based therapies," says Shinichi Nishikawa, Director of the Stem Cell Biology Laboratory at the Rikan Center for Developmental Biology in Japan.

Over the next five years, Yamanaka's group of about 20 researchers will focus on how iPS cells can help predict the side effects of drugs and explain mechanisms underlying toxicity and disease. In spite of all the excitement, possibilities and rivalries that his findings provoked, the former doctor moderates his expectations very carefully. "A lot of basic research is still needed on the safety of using iPS cells," emphasizes Yamanaka. "This is not an international competition like the Olympic Games. It should be international cooperation. This is the beginning of a long process."

Tim Horniak lives in Tokyo

Three years after the publication of the article, Shinya Yamanaka won the Nobel Prize in Medicine or Physiology together with John Gordon