Coaxing a humble skin cell to become a jack-of-all-trades pluripotent stem cell is feat so remarkable it was honored earlier this month with the Nobel Prize in Physiology or Medicine. Stem cell pioneer Shinya Yamanaka, MD, PhD, showed that using a virus to add just four genes to the skin cell allowed it to become pluripotent, or able to achieve many different developmental fates. But researchers and clinicians have been cautious about promoting potential therapeutic uses for these cells because the insertion of the genes could render the cells cancerous.
Now researchers at the Stanford University School of Medicine have devised an efficient and safer way to make these induced pluripotent stem cells, or iPS cells, by using just the proteins that the genes encode.
It's not the first time such an approach has been tried. Many researchers have shown that using proteins to make a cell pluripotent, although possible, is far less efficient than the virus-based method. The unprecedented success of the Stanford researchers, however, was due to an unexpected discovery: The virus used in the original method is critical for more than just gene delivery.
"It had been thought that the virus served simply as a Trojan horse to deliver the genes into the cell," said John Cooke, MD, PhD, professor of medicine and associate director of the Stanford Cardiovascular Institute. "Now we know that the virus causes the cell to loosen its chromatin and make the DNA available for the changes necessary for it to revert to the pluripotent state."
Cooke is the senior author of the research, published in the Oct. 26 issue of Cell. Postdoctoral scholars Jieun Lee, PhD, and Nazish Sayed, MD, PhD, are co-first authors of the study.
iPS cells, which don't require human embryos, offer a possible alternative for some of the ethical dilemmas associated with stem cell research. They're created from adult cells that have already assumed a specialized function in the body. Until Yamanaka's discovery, it was thought that these cells could never revert to the pluripotent stem cell from which they originated. But Yamanaka showed that these highly specialized cells are more developmentally flexible, or plastic, than previously thought. In the presence of just four genes (identified because they are highly expressed by embryonic stem cells), they can assume the characteristics of embryonic stem cells and, under the right conditions, can become nearly any cell type.
Now Cooke's research has identified an important component of how this transformation happens.
"We found that when a cell is exposed to a pathogen, it changes to adapt or defend itself against a challenge," said Cooke. "Part of this innate immunity includes increasing access to its DNA, which is normally tightly packaged. This allows the cell to reach into its genetic toolbox and take out what it needs to survive." It also allows the pluripotency-inducing proteins to modify the DNA and transform a skin or other specialized cell into an embryonic-stem-cell-like changeling.
Because the cells activate an immune response similar to inflammation in the presence of viral genetic material, the researchers termed the process "transflammation." They believe their finding could pave the way to the use of iPS cells in humans and shed light on the biological pathways by which pluripotency occurs.
Cooke and his colleagues began by working to optimize the use of cell-permeable proteins to reprogram adult, specialized cells to become pluripotent. They knew that the proteins made it into the cell's nucleus and that, in the laboratory, they were able to bind to the correct DNA sequences. They were also able to maintain pluripotency in cells that had been reprogrammed by other means. So why were the proteins so much more inefficient than the viral-based method?
The breakthrough came when they compared the gene expression patterns of the cells exposed to the cell-permeable proteins with those of cells infected by the gene-bearing virus: They were quite different. Cooke wondered if some property of the virus could be responsible.
The researchers repeated the experiment with the cell-permeable proteins, but also included an unrelated virus. The efficiency of the pluripotency transformation increased dramatically. Further investigation revealed that the effect was due to the activation within the cell of what is called the toll-like receptor-3 pathway; triggering the pathway with a small molecule mimicking the viral genetic material had a similar effect.
"These proteins are non-integrating, and so we don't have to worry about any viral-induced damage to the host genome," said Cooke, who also noted that the use of cell-permeable proteins can confer a greater level of control of the reprogramming process and may lead to the use of iPS cells in human therapies. It also opens up new alternatives.
"Now that we understand that the cell assumes greater plasticity when challenged by a pathogen, we can theoretically use this information to further manipulate the cells to induce direct reprogramming," said Cooke.
Direct reprogramming involves inducing a specialized cell like a skin cell to become a different type of cell, like an endothelial cell, without first going through an intermediate pluripotent state. Stanford researcher Marius Wernig, MD, PhD, used direct reprogramming to successfully transform human skin cells into functional neurons.
Other Stanford co-authors of the study include postdoctoral scholars Arwen Hunter, PhD, Kin Fai Au, PhD, and Wing T.J. Wong, PhD; research associate Eduard Yakubov, PhD; and Renee Reijo Pera, PhD, professor of obstetrics and gynecology.
The research was supported by the National Institutes of Health and the Tobacco Related Disease Research Program of the University of California.
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