Induced pluripotent stem cells: without residual foreign matter

The use of stem cells evokes something that medicine has always dreamed of: replacing damaged tissue with healthy tissue and understanding the principles of pathogenesis step by step. This works best with embryonic stem cells, but there are ethical concerns involved with that approach.

An unobjectionable alternative is the use of “induced pluripotent stem cells” (iPS cells). They are created by returning cells to their original state, even if they are actually already specialized for a certain role. Scientists call this kind of reset a “reprogramming of differentiated cells.” Although therapies with iPS cells are still a long way off, these cells already play an established role in biomedical research. They are used to help understand how cells differentiate, or to test the effect of drugs, for example.    

The genes needed for reprogramming cells are normally active during embryonic development. They control the fate of cells by steering them in a particular direction. When iPS cells are produced, the existing differentiation program is turned off. 

Although only four genes are needed for this reprogramming, the production of iPS cells is not exactly easy. “There are now several generations of reprogramming techniques,” says Vi Chu, in charge of Simplicon^TM at Merck KGaA, Darmstadt, Germany. “These differ in their safety, their efficiency, and how they are carried out.”

In the first generation, the genetic information for the four reprogramming factors was inserted into the differentiated cells by means of viruses and integrated into the genetic material. But when the genes for the reprogramming factors are stably integrated into the genome, they represent a latent risk because they alter the original DNA. Alterations in DNA may give rise to mutations that can cause cancer or affect the ability of cells to function normally. “It is therefore better not to leave the genes for the four factors behind in the iPS cells,” says Chu.

In the first generation, the viral vectors were also left behind in the cells. Because they integrate randomly into the genome, there is a potential for insertional mutagenesis which may trigger diseases. “Therefore the protocol for reprogramming was modified a number of times,” says Chu. In the second generation, the factors were no longer integrated into the genome, nor were viral vectors used. Instead, scientists work with plasmids and minicircles. These autonomously self-replicating DNA molecules are usually ring-shaped and are found outside of the genome with a remote risk that they may integrate into the DNA. 

The third generation, in turn, either uses the proteins exclusively from the very beginning, or else a viral vector that can be removed again later. This is laborious, however, and a small fingerprint of the virus always remains in the genome of the stem cells later on.    

The new Simplicon^TM process, on the other hand, belongs to the fourth generation. It uses only a single, synthetically produced strand of RNA that is completely removed again as soon as the cell nucleus has been reprogrammed. With the help of this RNA, the protein factories (“ribosomes”) of the differentiated cells produce nine proteins. Four of these nine proteins are the desired reprogramming factors with which the existing differentiation is canceled. Four other proteins ensure that new copies of this reprogramming RNA are generated again and again so that it only has to be introduced into the cells once and then reproduces all by itself. The ninth protein makes the cells resistant to the antibiotic puromycin. Only cells that have taken up the reprogramming RNA survive a confrontation with this antibiotic. Their resistance can be used to distinguish and separate cells with reprogramming from those without reprogramming.    

To prevent the reprogramming RNA from being immediately broken down, a protein is added to the cells to make the RNA invisible to the cellular immune system. It has the cryptic name B18R. If it is no longer added, the cells recognize the Simplicon^TM RNA as foreign and break it down. With this mechanism, the RNA can be completely removed after reprogramming. “Simplicon^TM is thus a synthetic, virus-free strand of RNA that replicates itself and only has to be added to the cells once. It is not integrated into the genome. Therefore, none of it is left behind in the iPS cells,” says Chu, summarizing the advantages. “The whole process — from the incorporation of the RNA into the differentiated cells to the time that colonies of induced pluripotent stem cells are generated — takes less than one month. There is currently no faster or more efficient way to reprogram differentiated cells.”