Why not?
We need a little cell biology here. The nucleus contains the vast majority of the DNA/genes that encode us – our blueprint. There’s a miniscule fraction of DNA that isn’t in the nucleus, it’s in tiny structures called mitochondria, but we don’t need to worry about that here. When we’re first taught about cells in school it’s almost as if the nucleus is all powerful and the rest of the cell – the cytoplasm – is a bag of liquid that doesn’t really do much. Nothing could be further from the truth, and this is especially the case for the egg, because the toads and Dolly have taught us that the cytoplasm of the egg is absolutely key. Something, or some things, in that egg cytoplasm actively reprogrammed the adult nucleus that the experimenters injected into it. These unknown factors moved a nucleus from the bottom of one of Waddington’s troughs right back to the top of the landscape.
Nobody really understood how the cytoplasm of eggs could convert adult nuclei into ones like zygotes. There was pretty much an assumption that whatever it was must be incredibly complicated and difficult to unravel. Often in science really big questions have smaller, more manageable questions inside them. So a number of labs tackled a conceptually simpler, but technically still hugely challenging issue.
Remember that ball at the top of Waddington’s landscape. In cellular terms it’s the zygote and it’s referred to as totipotent, that is, it has the potential to form every cell in the body, including the placenta. Of course, zygotes by definition are rather limited in number and most scientists working in very early development use cells from a bit later, the famous embryonic stem (ES) cells. These are created as a result of normal developmental pathways. The zygote divides a few times to create a bundle of cells called the blastocyst. Although the blastocyst typically has less than 150 cells it’s already an early embryo with two distinct compartments. There’s an outer layer called the trophectoderm, which will eventually form the placenta and other extra-embryonic tissues, and an inner cell mass (ICM).
Figure 2.1 shows what the blastocyst looks like. The drawing is in two dimensions but in reality the blastocyst is a three-dimensional structure, so the actual shape is that of a tennis ball that’s had a golf ball glued inside it.
Figure 2.1 A diagram of the mammalian blastocyst. The cells of the trophectoderm will give rise to the placenta. During normal development, the cells of the Inner Cell Mass (ICM) will give rise to the tissues of the embryo. Under laboratory conditions, the cells of the ICM can be grown in culture as pluripotent embryonic stem (ES) cells.
The cells of the ICM can be grown in the lab in culture dishes. They’re fiddly to maintain and require specialised culture conditions and careful handling, but do it right and they reward us by dividing a limitless number of times and staying the same as the parent cell. These are the ES cells and as their full name suggests, they can form every cell of the embryo and ultimately of the mature animal. They aren’t totipotent – they can’t make placenta – so they are called pluripotent because they make pretty much anything else.
These ES cells have been invaluable for understanding what’s important for keeping cells in a pluripotent state. Over the years a number of leading scientists including Azim Surani in Cambridge, Austin Smith in Edinburgh, Rudolf Jaenisch in Boston and Shinya Yamanaka in Kyoto have devoted huge amounts of time to identifying the genes and proteins expressed (switched on) in ES cells. They particularly tried to identify genes that keep the ES cells in a pluripotent state. These genes are extraordinarily important because ES cells seem to be very prone to turn into other cell types in culture if you don’t keep the conditions just right. Just a small change in culture conditions, for example, and a culture dish full of one-time ES cells can differentiate into cardiomyocytes and do what heart cells do best: they beat along in time with one another. A slightly different change in conditions – altering the delicate balance of chemicals in the culture fluid, for example, can divert the ES cells away from the cardiac route and start the development of cells that give rise to the neurons in our brains.
Scientists working on ES cells identified a whole slew of genes that were important for keeping the cells pluripotent. The functions of the various genes they identified weren’t necessarily identical. Some were important for self-renewal, i.e. one ES dividing to form two ES cells, whereas others were required to stop the cells from differentiating[9].
So, by the early years of the 21st century scientists had found a way of maintaining pluripotent ES cells in culture dishes and they knew quite a lot about their biology. They had also worked out how to change the culture conditions so that the ES cells would differentiate into various cell types including liver cells, heart cells, neurons etc. But how does this help with the dream we laid out earlier? Could the labs use this information to create new ways of driving cells backwards, to the top of Waddington’s landscape? Would it be possible to take a fully differentiated cell and treat it in a lab so that it would become just like an ES cell, with all the potential that implies? Whilst scientists had good reason to believe this would be theoretically possible, that’s a long way from actually being able to do it. But it was a wonderfully tantalising prospect for scientists interested in using stem cells to treat human diseases.
By the middle of the first decade of this century, over twenty genes had been identified that seemed to be critical to ES cells. It wasn’t necessarily clear how they worked together and there was every reason to think that there was still plenty we didn’t understand about the biology of ES cells. It was assumed that it would be almost inconceivably difficult to take a mature cell and essentially recreate the vastly complex intracellular conditions that are found in an ES cell.
Sometimes the greatest scientific breakthroughs happen because someone ignores the prevailing pessimism. In this case, the optimist who decided to test what everyone else had assumed was impossible was the aforementioned Shinya Yamanaka, with his postdoctoral research associate Kazutoshi Takahashi.
Professor Yamanaka is one of the youngest luminaries in the stem cell and pluripotency field. He was born in Osaka in the early 1960s and rather unusually he has held successful academic positions in high profile institutions in both Japan and the USA. He originally trained as a clinician and became an orthopaedic surgeon. Specialists in this discipline are sometimes dismissed by other surgeons as ‘the hammer and chisel brigade’. This is unfair, but it is true that orthopaedic surgical practice is about as far away from elegant molecular biology and stem cell science as it’s possible to get.
Perhaps more than any of the other researchers working in the stem cell field, Professor Yamanaka had been driven by a desire to find a way of creating pluripotent cells from differentiated cells in a lab. He started this stage of his work with a list of 24 genes which were vitally important in ES cells. These were all genes called ‘pluripotency genes’ – they have to be switched on if ES cells are to remain pluripotent. If you use various experimental techniques to switch these genes off, the ES cells start to differentiate, just like those beating heart cells in the culture dish, and they never revert to being ES cells again. Indeed, that is partly what happens quite naturally during mammalian development, when cells differentiate and become specialised – they switch off these pluripotency genes.
Shinya Yamanaka decided to test if combinations of these genes would drive differentiated cells backwards to a more primitive developmental stage. It seemed a long shot and there was always the worry that if the results were negative – i.e. if none of the cells went ‘backwards’ – he wouldn’t know if it was because it just wasn’t possible or if he just hadn’t got the experimental conditions right. This was a risk for an established scientist like Yamanaka, but it was an even bigger gamble for a relatively junior associate like Takahashi, because of the way that the scientific career ladder works.
9
For a useful review of the state of knowledge at the time see Rao, M. (2004)