Read The Epigenetics Revolution Online
Authors: Nessa Carey
Tags: #Science/Life Sciences/Genetics and Genomics
The Epigenetics Revolution (5 page)
Endless potential
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.
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.
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.
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.
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.
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
1
.
1
.
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.
The triumph of optimism
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.
When faced with the exposure of damaging personal love letters, the Duke of Wellington famously responded, ‘Publish and be damned!’ The mantra for scientists is almost the same but differs in one critical respect. For us, it’s ‘publish
or
be damned’ – if you don’t publish papers, you can’t get research funding and you can’t get jobs in universities. And it is rare indeed to get a paper into a good journal if the message of your years of effort boils down to, ‘I tried and I tried but it didn’t work.’ So to take on a project with relatively little likelihood of positive results is a huge leap of faith and we have to admire Takahashi’s courage, in particular.
or
be damned’ – if you don’t publish papers, you can’t get research funding and you can’t get jobs in universities. And it is rare indeed to get a paper into a good journal if the message of your years of effort boils down to, ‘I tried and I tried but it didn’t work.’ So to take on a project with relatively little likelihood of positive results is a huge leap of faith and we have to admire Takahashi’s courage, in particular.
Yamanaka and Takahashi chose their 24 genes and decided
to test them in a cell type known as MEFs – mouse embryonic fibroblasts. Fibroblasts are the main cells in connective tissue and are found in all sorts of organs including skin. They’re really easy to extract and they grow very easily in culture, so are a great source of cells for experiments. Because the ones known as MEFs are from embryos the hope was that they would still retain a bit of capacity to revert to very early cell types under the right conditions.
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 (
neo
R
) 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
neo
R
gene, they will survive. When Yamanaka created the mice he needed for his experiments he inserted the
neo
R
gene in a particular way. This meant that the
neo
R
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.
neo
R
) 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
neo
R
gene, they will survive. When Yamanaka created the mice he needed for his experiments he inserted the
neo
R
gene in a particular way. This meant that the
neo
R
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
neo
R
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.
neo
R
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
neo
R
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.
Oct4
,
Sox2
,
Klf4
and
c-Myc
something quite extraordinary happened. The cells survived in neomycin, showing they had switched on the
neo
R
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
2
. 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.
Cell
, the world’s most prestigious biological journal
2
. 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.
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