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Reprogramming Cells With Protein Power

New Method for Creating Pluripotent Cells May Make Them Safer

reprogrammed mouse cells SOURCE: Hongyan Zhou, et al., Cell Stem Cell Using specially engineered proteins instead of DNA to coax mice cells back into an embryonic state is promising, but doesn’t resolve many potential problems. For regenerative medicine research in humans, embryonic stem cells remain the gold standard. Above: reprogrammed mouse cells.

Previous methods for creating induced pluripotent stem cells have relied upon introducing DNA material into adult cells. A significant problem with much of this work has been that while the cells revert back to an embryonic state, the foreign DNA may cause mutations that lead to tumors. But a new method just described in a paper by my colleagues at The Scripps Research Institute clears this hurdle by using proteins instead of snippets of genetic material.

This method is a landmark in the development of reprogramming technology. Here’s how it works in the mouse cells that they used.

We know that the DNA sequences for the reprogramming factors used in previous research code for proteins, and we know that these particular proteins are transcription factors that bind to other regions of the DNA and turn on other genes. We think that this event, through an as-yet-unknown cascade of other further molecular events, leads to a “resetting” of the state of the cell so that it becomes pluripotent.

We have assumed that the DNA we put into cells to reprogram them works by directing the synthesis of proteins that are produced inside the cells. These proteins, not the DNA, would be the real catalysts for reprogramming, turning on other genes that ultimately result in pluripotency. But we didn’t know this for certain.

Dr. Sheng Ding, lead scientist on the team that published the new research, worked with a company called Fate Therapeutics to obtain purified proteins corresponding to the reprogramming factors. The proteins were engineered in bacteria and had a modified set of amino acids on one end that allowed them to get into cells and not be immediately degraded—cells have robust mechanisms to identify and destroy foreign proteins that enter their cytoplasm.

By adding these proteins to mouse fibroblasts, which are connective tissue cells, in various amounts and combinations, Dr. Ding was able to get a few cells to respond and become pluripotent. Voila! Proteins do seem to be responsible as we thought. Ding also added a drug called valproic acid that wipes DNA clean of its endogenous, or original, binding proteins, which makes it more susceptible to bind to new sets of proteins.

The major benefit of this method is that it does not involve the introduction of new DNA, so there is no chance that integration of foreign DNA might increase tumor risk. Since the proteins do eventually degrade, there should be no trace of their existence in the cells. Demonstrating that the proteins themselves cause reprogramming is also a valuable piece of information for scientists who want to design the next generation of reprogramming methods.

But there are drawbacks to this new method. Producing proteins is enormously expensive compared to making DNA sequences, and while Fate Therapeutics decided to invest a considerable amount in this technology, very few academic labs can afford to make and purify proteins on this scale. Also, the efficiency of protein reprogramming is much lower than the DNA-based methods, meaning that to obtain just a few pluripotent cells, a very large number of fibroblast cultures must be treated.

And the question of safety is still not completely resolved, as we still don’t know whether this method is ultimately going to be less risky than other methods. Using the drug valproic acid may alter DNA, and although we don’t think that proteins will have long-term negative effects on the cells, follow-up studies will be necessary. In fact, we have no idea what any of the existing reprogramming methods do to cells long-term. It’s worth remembering that even mouse cloning—a reprogramming method that transplants the nucleus of a somatic cell into an egg, and which involves neither genetic engineering nor the addition of any proteins—still often results in abnormal embryonic development.

The bottom line is that no matter what the method, we need to focus on understanding the potential dark sides of reprogramming. For the moment, we simply don’t know what those are. That is why it is critical for regenerative medicine research in humans that scientists continue to study embryonic stems cells as the gold standard for understanding pluripotency.

Jeanne F. Loring, PhD, is director of the Center for Regenerative Medicine at The Scripps Research Institute in La Jolla, California.

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