George Daley, Harvard University

In January 2007, George Daley of Harvard University published proof of principle that pluripotent stem cells could be created so that they would not cause an immune response when differentiated for cell transplantation1. His forthcoming publication in January 2008 shows much the same thing, but through an entirely different technique. The first paper used unfertilized mouse eggs; the more recent one uses a skin biopsy from a human volunteer2. It is the first to demonstrate such complete reprogramming without starting from embryos or cell cultures available from commercial vendors.

These bookends highlight the major stem cell advance of this past year: multiple laboratories have now shown that adult human skin cells can be reprogrammed to an embryonic stem (ES) cell–like state.

Daley's lab began work shortly after Kyoto University's Shinya Yamanaka announced the four genes that could transform skin cells, or fibroblasts, cultured from newly born mice, into an embryonic-like state3. This summer, Yamanaka and two other groups proved that the mouse fibroblasts could be made truly pluripotent by showing that the cells could become sperm and eggs4,5,6 (see Skin cell to stem cell).

Any group that knows how to keep human or mouse embryonic stem cells alive will probably be able to make and maintain induced pluripotent stem cells. George Daley, Harvard University

Meanwhile, labs across the world were racing to reprogram human cells. In November, Yamanaka and James Thomson, of the University of Wisconsin–Madison, became the first labs to announce that they had done so7,8. “Our paper was already submitted when the others were published,” Daley says. “It was frustrating, but the point is that this is a robust technology that lots of people can reduce to practice.” Indeed, he says, any group that knows how to keep human or mouse ES cells alive will probably be able to make and maintain induced pluripotent stem (iPS) cells.

Daley's initial strategy was to try to use fewer genes to reprogram adult stem cells. His group began working with spermatagonial stem cells, as they may be epigenetically poised for reprogramming.

It didn't work. Eventually, Daley's postdoctoral fellow In-Hyun Park figured out that the genes introduced to induce pluripotency were silenced extremely quickly in the spermatagonial stem cells, so he decided to try human fibroblasts instead. Daley says he was initially reluctant. “I didn't think it was a good idea to do experiments that everyone else was doing.”

The group first created fibroblasts from human ES cells and, in the spring, began testing whether the so-called 'Yamanaka factors' would reprogram these cells. “We found out pretty quickly that they did,” Daley recalls.

Genes for the Yamanaka factors are inserted into cells through a retrovirus and are silenced as the original pluripotency machinery of the cells becomes active. Because the viruses insert randomly, every reprogrammed cell might reprogram in a different way, and so Park wanted to study descendents of single reprogrammed cells rather than mixed populations. That's tricky because embryonic (and embryonic-like) stem cells tend to die when separated. But with the help of a technique published in June9, Park learned to create cultures derived from individual cells.

Next, the researchers made induced pluripotent cells using established cultures of human fibroblasts originally derived from fetuses, newborns and adults. The Yamanaka factors readily reprogrammed ES-derived and fetal fibroblasts, but seemed to cause adult-derived cells to grow slowly, so the Daley team supplemented the Yamanaka factors with two genes known to prevent cell death and were able to isolate iPS lines. (The extra genes were not found in the final iPS lines.)

But for the technique to have clinical applications, scientists will want cells obtained from particular patients rather than established cultures, so Daley decided to try reprogramming cells obtained from a fresh skin biopsy.

Even though skin biopsies are routine procedures, Daley's institutional review board performed a full scientific review and took about two months deciding that the experiments could go forward. All prospective volunteers had to be made aware of all the “sensitive issues,” says Daley. They had to realize, for example, that reprogrammed cells could generate many cell types (including, potentially, sperm and eggs), that the cells would be cultured indefinitely and that the cells could be genetically matched to the donor, even if the volunteer wished to remain anonymous. Eventually, a skin biopsy was taken from a man's forearm and grown into fibroblasts, which were then used to make iPS cell lines.

While many, many scientific and ethical issues remain, Daley believes such skin biopsies could be used to create a bank of cell lines matched to much of the population. When mismatched, 'transplant antigens' on cell surfaces cause a recipient's immune system to attack transplanted cells. This peril is averted if a patient uses his or her own cells for transplantation, but few believe that stem cell therapies will routinely create cell lines for individual patients. In the past, Daley and others have proposed that a stem cell bank could be populated with cell lines derived from unfertilized oocytes. These can be manipulated to be homozygous for transplant antigens and thus made more likely to match more people. However, some individuals are naturally homozygous. Compared to asking women to donate their eggs, it should be easy to ask such people for a piece of their skin.