The Epigenetics Revolution

Remember how John Gurdon used donor and acceptor toad strains that had different genetically-encoded markers, so he could tell which nuclei had generated the new animals? Yamanaka did something similar. He used cells from mice which had an extra gene added. This gene is called the neomycin resistance (neoR) gene and it does exactly what it says on the can. Neomycin is an antibiotic-type compound that normally kills mammalian cells. But if the cells have been genetically engineered to express the neoR gene, they will survive. When Yamanaka created the mice he needed for his experiments he inserted the neoR gene in a particular way. This meant that the neoR gene would only get switched on if the cell it was in had become pluripotent. The cell had to be behaving like an ES cell. So if his experiments to push the fibroblasts backwards experimentally into the undifferentiated ES cell state were successful, the cells would keep growing, even when a lethal dose of the antibiotic was added. If the experiments were unsuccessful, all the cells would die.

Professor Yamanaka and Doctor Takahashi inserted the 24 genes they wanted to test into specially designed molecules called vectors. These act like Trojan horses, carrying high concentrations of the ‘extra’ DNA into the fibroblasts. Once in the cell, the genes were switched on and produced their specific proteins. Introducing these vectors can be done relatively easily on a large number of cells at once, using chemical treatments or electrical pulses (no fiddly micro-injections for Yamanaka, no indeed). When Shinya Yamanaka used all 24 genes simultaneously, some of the cells survived the neomycin treatment. It was only a tiny fraction of the cells but it was an encouraging result nonetheless. It meant these cells had switched on the neoR gene. This implied they were behaving like ES cells. But if he used the genes singly, no cells survived. Shinya Yamanaka and Kazutoshi Takahashi then added various sets of 23 genes to the cells. They used the results from these experiments to identify ten genes that were each really critical for creating the neomycin-resistant pluripotent cells. By testing various combinations from these ten genes they finally hit on the smallest number of genes that could act together to turn embryonic fibroblasts into ES-like cells.

The magic number turned out to be four. When the fibroblasts were invaded by vectors carrying genes called Oct4, Sox2, Klf4 and c-Myc something quite extraordinary happened. The cells survived in neomycin, showing they had switched on the neoR gene and were therefore like ES cells. Not only that, but the fibroblasts began to change shape to look like ES cells. Using various experimental systems, the researchers were able to turn these reprogrammed cells into the three major tissue types from which all organs of the mammalian body are formed – ectoderm, mesoderm and endoderm. Normal ES cells can also do this. Fibroblasts never can. Shinya Yamanaka then showed that he could repeat the whole process using fibroblasts from adult mice rather than embryos as his starting material. This showed that his method didn’t rely on some special feature of embryonic cells, but could also be applied to cells from completely differentiated and mature organisms.

Yamanaka called the cells that he created ‘induced pluripotent stem cells’ and the acronym – iPS cells – is now familiar terminology to everyone working in biology. When we consider that this phrase didn’t even exist five years ago, its universal recognition amongst scientists shows just how important a breakthrough this really is.

It’s incredible to think that mammalian cells carry about 20,000 genes, and yet it only takes four to turn a fully differentiated cell into something that is pluripotent. With just four genes Professor Yamanaka was able to push the ball right from the bottom of one of Waddington’s troughs, all the way back up to the top of the landscape.

It wasn’t surprising that Shinya Yamanaka and Kazutoshi Takahashi published their findings in Cell, the world’s most prestigious biological journal[10]. What was a bit surprising was the reaction. Everyone in 2006 knew this was huge, but they knew it was only huge if it was right. An awful lot of scientists couldn’t really believe that it was. They didn’t for one moment think that Professor Yamanaka and Doctor Takahashi were lying, or had done anything fraudulent. They just thought they had probably got something wrong, because really, it couldn’t be that simple. It was analogous to someone searching for the Holy Grail and finding it the second place they looked, under the peas at the back of the freezer.