Soon after the exciting discovery of a method to transform human skin cells into stem cells in 2007 came the frustration of actually trying to make a sufficient amount of these induced pluripotent stem (iPS) cells.* The process is so inefficient that scientists typically only get 0.01 percent of a sample of human skin, or fibroblast, cells to form iPS cell colonies after they infect fibroblasts with the retroviruses used to induce pluripotency. "We almost gave up three years ago," says Dr. Duanqing Pei, director general and professor at the Guangzhou Institutes of Biomedicine and Health in Guangzhou, China.

But Pei's group at the Chinese Academy of Sciences plugged away and now has found that a simple chemical can give iPS cell transformation a big boost. By adding vitamin C to the medium in which human skin cells are grown, the researchers managed to turn 1 to 2 percent, compared to 0.01 percent, of cells into iPS cell colonies. "Once you reach the single digit frequency, that is no longer an infrequent event," Pei says. This yield, he believes, could allow scientists to finally study what is happening inside the cells as they undergo transformation. The results were published December 24 in Cell Stem Cell.

The ultimate goal is to make iPS cells a valid therapeutic option by replacing the potentially dangerous retroviruses with a safer cocktail of proteins and chemicals, including vitamin C.

The path that Pei's group took to identifying the beneficial role of vitamin C started with the realization that the factors that induce cells to become pluripotent were causing the cells to make the free radicals known as reactive oxygen species (ROS). "A high level of ROS is definitely very bad for the fibroblasts," Pei says, because it induces cell death at a faster rate.

To enhance the survival of cells and their likelihood of becoming iPS cells, Pei's team tested a variety of chemicals with anti-oxidant properties. The researchers started with fibroblast cells from mouse embryos, which have been shown to revert to iPS cells at a higher frequency than human fibroblasts. First, they infected the cells with retroviruses that trigger the expression of four embryonic cell-specific genes in the cells. Then they let the cells grow, either with or without vitamin C.

Ten days later, when cells typically begin to be reprogrammed into iPS cells, Pei's group found that 30 percent more mouse cells were alive in the Petri dish that contained vitamin C than in the one that did not, suggesting that the vitamin helps keep cells alive. One possible mechanism to explain this improved survival is that the cells exposed to ascorbic acid had lower levels of a tumor suppressor protein called p53, which can elicit cell death.

Surprisingly, the researchers found that vitamin C not only helps cells survive, but it enhances their progression to pluripotency by a factor of 100 . After 14 days, when cells start to become fully pluripotent, 10 to 20 percent of the mouse cells that were grown with vitamin C expressed genes associated with pluripotency compared with only 0.1 to 0.2 percent of the colonies grown without vitamin C. The group saw a similar improvement in human fibroblast reprogramming, from 0.01 percent in the absence of vitamin C to 1 percent in the presence. When other anti-oxidants were tested, none boosted the development of pluripotency.

Pei believes that ascorbic acid is spurring the induction of pluripotency in mouse and human cells through both its anti-oxidant properties as well as an as-yet unknown mechanism. Too much ROS and the reprogramming "machine" won't start, he says. But once ROS is reduced, the machine starts, and vitamin C makes it run more smoothly. "If you have too much ROS, the whole [reprogramming] machine will not move, but then once you [reduce] ROS, I think vitamin C does something [else] to make the machine move more smoothly," he says.

"Overall I think this is quite impressive progress," says Kwang-Soo Kim, director of the Molecular Neurobiology Lab at Harvard Medical School. Kim has developed a system to improve the safety of iPS cells by delivering the four proteins that induce pluripotency directly into cells. Because it avoids retroviruses, which integrate their genomes into cell chromosomes, his system carries less risk of potentially cancer causing mutations. But the efficiency is low—one to 10 percent of the viral method—and it's much slower. "We need a different kind of approach," he says.
This approach could be adding the four pluripotency-inducing proteins to cells to jumpstart the reprogramming "machine" and then improving the efficiency with a combination of chemicals, including vitamin C. Pei is currently trying to optimize the medium in which cells are grown. Although he doubts that any other anti-oxidant will have the same effect as ascorbic acid, Pei thinks that certain growth factors could further improve the culture conditions. In fact, his group found that combining vitamin C with valproic acid, which helps induce pluripotency, can improve transformation efficiency of human fibroblasts from 1 to 6 percent.

This level of efficiency could be enough to advance studies with iPS cells. "We don't need to generate 50 percent of the cells…as long as we can reproducibly generate a sufficient number of iPS lines," Kim says, adding that a 1 percent transformation efficiency could be enough.

Eventually, researchers would have to differentiate the iPS cells into certain cell types, says Kim, who himself has differentiated the more controversial embryonic stem cells into neurons to try to treat Parkinson's disease.

But, first, several studies with these vitamin C-induced pluripotent cells should be done, Kim notes. One possible problem – vitamin C causes cells to express lower levels of p53, which is important for the repair of DNA damage. Although Pei's group did not find any chromosomal abnormalities in cells grown with vitamin C, Kim says that higher resolution analysis is needed to ensure there are no mutations.

Because of the relatively high yield of Pei's method, these analyses and other studies of iPS cells should be possible. Now researchers might be able to generate enough cells to study the mechanism of and improve the safety and efficacy of iPS cells. "It's a worldwide effort to boost efficiency and make this more practical for much wider participation from the scientific community," Pei says.

* Note (12/29/09): This sentence was edited after publication to correct the year of the first human iPS cells.