Molecular Medicine Israel

Oct4, Considered Vital for Creating iPSCs, Actually Isn’t Needed

Dropping the transcription factor from the four so-called Yamanaka factors reduces the efficiency of inducing the production of stem cells, but the resulting cells are of greater quality

Since 2006, when Shinya Yamanaka, now the director of the Center for iPS Cell Research and Application at Kyoto University, discovered a method that could guide fully differentiated cells back to their pluripotent state, scientists have been using his recipe to produce induced pluripotent stem cells. The protocol relies on overexpressing the so-called Yamanaka factors, which are four transcription factors: Oct4, Sox2, Klf4, and cMyc (OSKM). While the technique reliably creates iPS cells, it can cause unintended effects, some of which can lead to cells to become cancerous. So researchers have worked to adjust the cocktail and understand the function of each factor.
No one had succeeded in creating iPS cells without forcing the overexpression of Oct4. It was thought that this was the most crucial factor of the four. At least until now.
Four years ago, Sergiy Velychko, a graduate student at the Max Planck Institute for Molecular Biomedicine in Hans Schöler’s lab, and his team were studying the role of Oct4 in creating iPS cells from mouse embryonic fibroblasts. He used vectors to introduce various mutations of the gene coding for Oct4 to the cells he was studying, along with a negative control—one that didn’t deliver any Oct4. He was shocked to discover that even using his negative control, he was able to generate iPS cells.
Velychko’s experiment was suggesting that it is possible to develop iPS cells with only SKM.
“We just wanted to publish this observation,” Velychko tells The Scientist, but he knew he’d need to replicate it first because “reviewers wouldn’t believe it.”
He and his colleagues, including Guangming Wu, a senior scientist in the lab, repeated the experiment several times, engineering vectors with different combinations of the four factors. SKM—the combination that didn’t include Oct4—was able to induce pluripotency in the cells with about 30 percent of the efficiency of OSKM, but the cells were of higher quality, meaning that the researchers didn’t see evidence of common off-target epigenetic effects. They reported their results yesterday (November 7) in Cell Stem Cell.
“Efficiency is not important. Efficiency means how many colonies do you get,” explains Yossi Buganim, a stem cell researcher at the Hebrew University of Jerusalem, who was not involved in the study. “If the colony is of low quality, the chances that eventually the differentiated cells will become cancerous is very high.”
Finally, the team employed the ultimate test, the tetraploid complementation assay, in which iPS cells are aggregated with early embryos that otherwise would not have been able to form a fully functional embryo on their own. These embryos grew into mouse pups, meaning that the iPS cells the team created were capable of maturing into every type of cell in the animal.
What’s more is they found that the SKM iPS cells could develop into normal mouse pups 20 times more often than the OSKM iPS cells, suggesting that the pluripotency of iPS cells can be greatly improved by omitting Oct4 from the reprogramming factor cocktail.
The results will need to be verified in human cells, Buganim cautions. His team has developed methods for creating iPSCs that worked well in mouse cells only to be completely ineffective in humans.
Yamanaka himself was enthusiastic about the results, telling The Scientist in an email that his team would definitely try the method in other cell types, especially “adult human blood cells and skin fibroblasts. If this works in adult human cells, it will be a huge advantage for the clinical applications of iPS cells.”

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