I'd like to think about how epigenetic reprogramming is disrupted in two other technologies, and this is somatic cell nuclear transfer, and reprogramming, or somatic reprogramming. But let me just remind you for the assistive reproductive technologies, while we think the risk of the imprinting disorders occurring following assistive reproductive technologies is extremely low, the reason people are working on it is because they want to optimise this procedure. It's being used more and more every day, and so we want to make sure that it has the best possible outcomes for the patients. So now let's think about cloning. So, you might remember that in the late 90s there was the birth of Dolly the sheep, and Dolly was a clone. That was, she was genetically identical to the cell from which she came. So, rather than us being from our parents. We, of course are not genetically identical to our parents. We have half of the genetic contribution from our mom, and half from our dad. Dolly was identical to the cell that she was made from, which was from a mammary cell. So the way that she was made was that they took an oocyte, so an egg, and removed the nucleus. This is called enucleation. So this oocyte, then, has all of the maternal specific factors that are found being expressed in that cytoplasm, but none of the DNA. And instead, from this mammary cell, from a different sheep, this is what contributed the nucleus. And so you had this newly formed oocyte, which was the oocyte from one animal, but the somatic cell nucleus from another animal. This, produced what would be effectively a fertilised egg, and this was then cultured in vitro to produce a blastocyst which was then implanted into a carrier mother, who then gave birth to Dolly the sheep. So what's happened during this process? And why would we care about what happens? Why would we want to do any cloning at all? We clearly don't want to clone humans. However, cloning is performed in the livestock industry for husbandry reasons, similar to, as I said before, for cattle, that we would want to be able to perform IVF so that you can transport them around the world. Well, if you've got a prize bull, actually you might quite like another bull that's identical to him, that has exactly the same genetic components as that male who produces so well. And so for cattle and deer, they are indeed, investing in cloning to see if they can get this work. But also if we want to resurrect a species for example if we can get the nucleus or the DNA from that species, the idea might be if we can get cloning to work, we can use a oocyte that's from a related species to now be able to resurrect that particular species that's left the planet. So with Dolly the sheep, and in fact with any cloning that's been performed, it's extremely low efficiency, with perhaps less than 1% of clones that actually go on to survive. So, in those clones that either don't make it, or even the ones that do survive, what we see is large offspring syndrome. So, there's placental overgrowth and fetal overgrowth. Now, this is reminiscent of what I said happens in Beckwith-Wiedemann syndrome, is actually true for other imprinted disorders, as well. And so, this suggested that there might be some imprinting defect within these clones, and perhaps this was due to disrupted epigenetic reprogramming. So we actually know that there are many imprinted genes that contribute to growth, as I mentioned when I was talking about the disruption to imprinting in cancer, and this is particularly true in the placenta. One of the places where there are lots of imprinted genes in the placenta we have the genes being expressed from the paternal chromosome, which drive large placental growth and, therefore, large embryo growth because the idea is that the father wants to have a very big and strong offspring. Whereas actually the mother would like to have a small offspring in order that she survive childbirth. So we know we can think about why it might be that you would have disruption to epigenetic reprogramming in this case. We've taken a Somatic nucleus. This somatic nucleus hasn't been through primordial germ cell development and therefore hasn't gone through the appropriate resetting of epigentic marks at this time. Not only that, but it hasn't been packaged into the chromatin that is found particularly in the egg or the sperm. So you remember that I mentioned right at the beginning of this week, that the chromatin that is found within the oocyte or the spermatocyte is actually highly specialised, and it's different to what is found in the Somatic cells. But here if we take a somatic nucleus and just place it into an egg that hasn't got a nucleus of it's own, we've entirely skipped this process. So then we are allowing the embryo to develop first of all in culture just like for IVF, but we seems that maybe these imprint control regions may have, may not be appropriately reset back here, but secondarily are occurring in culture for that first period and perhaps the eroded some of the ones that are appropriate. This could be because of the lack of maternal effect proteins. So while the maternal effect proteins might be in the cytoplasm of the oocyte, they may not be bound to the chromatin appropriately because that chromatin wasn't packaged into an egg or a sperm. So, let's just look at this again, according with our epigenetic reprogramming picture. So in cloning, we completely skip the primordial germ cell clearing and resetting This epigenetic reprogramming in germ cell development. We know that the Oocyte Cytoplasm that's used and when we take out this particular pronucleus, the female pronucleus, this oocyte cytoplasm certainly has many reprogramming factors to allow this early development reprogramming to happen. However, remember it's now occurring in culture, and what I said from the IVF experiments was that it seemed that even this occurring in culture could contribute to epigenetic abnormalities. So in cloning you have two problems one is the germ cell development problem that you've skipped, and the second is allowing this culture of the embryo at those early stages, and so you've disrupted two phases of epigenetic reprogramming. So what we think about is this epigenetic disruption and leading to imprint disorders. However, if you even look at the clones that survive, so what we would actually term the successful clones, and you then look at expression of the genes genome-wide, so all 25,000 or 30,000 of them at once, we see that there really are large changes in the transcription of genes genome-wide in the surviving clones. And this suggests that other than just the imprint of genes, you have large disruptions, but interestingly you can still get survival. So presumably, there is still, there is quite a lot of leniency if you like in the in the viability of these animals. While this is true in cattle, we know from other studies that humans are far less tolerant to these transcriptional differences or these genome wide differences because we don't tend to have so many babies that have born even, naturally without any screening. We don't tend to have any babies that have born with the same sort of disorders that are seen in livestock. So, the livestock, the cattle and the deer perhaps have different sorts of mechanisms to prevent these sorts of occurrences. But what's also interesting is that they can increase the efficiency of cloning. Remember I said it's extremely inefficient. Perhaps one, less than one percent of clones will go on to survive, and even they may be abnormal. But we can increase the efficiency of cloning by having an incubation during this period of culture during pre-implantation development with particular compounds that will alter epigenetic state. So this, if we can increase the efficiency by these compounds that suggests that it is really all about how the epigenetic machinery is working. And so it's all about really the efficiency of cloning is determined by how well epigenetic reprogramming occurs. So, now let's move on to think about the the last type of disruption that can come from epigenetic reprogramming, and this is for somatic cell reprogramming. So, in about 2006 it was discovered that what you could do is take a somatic cell from an adult and you could reprogram it back into something called an induced pluripotent stem cell or an iPS cell. So, in other words, you were taking a cell that was terminally differentiated and had all these somatic marks on it. So, all of the cells that it was all of the marks of a cell that was just going to perform one function, for example a skin cell, But, then you could reprogram it back to behave like an embryonic stem cell. Now, the feature of embryonic stem cells is that they can be differentiated into any of the tissues of the adult. So, clearly this has fascinating therapeutic potential. Because, now you can take the skin cell or perhaps a buccal cell from the mouth any easily, easily accessible cell type. The cell of the blood, and reprogram it back to something it was like an embryonic stem cell to be able to then differentiate it back out again to produce particular organs to tissue types for that patient. The tantalising idea with this is that it would be patient specific, so it would have all the same DNA as that patient so you would get over any of that graft-versus-host rejection, which often happens with transplants. So here it's shown that you may have a patient that needs to have this lung removed. The lung could be removed and you could make iPS cells for that patient, patient-specific iPS cells with exactly their DNA. Then in vitro, you could regenerate a lung and transplant back a new lung for this patient that is derived completely from their own cells. So clearly this is a fascinating process that has really excited the whole scientific community. But again the problem here is that, as you might expect, production of these iPS cells is incredibly inefficient. So again, only occurs properly in a few percent of cases, but to think about just one more advantage rather than the scientific advantage let's think about the ethical advantage. So now we know that these iPS cells as opposed to embryonic stem cells these iPS cells could not form a whole embryo because they can't make a placenta. So, embryonic stem cells and iPS cells neither of them can make a placenta. So, they can't form an embryo and this ethnically has less issues, but they also were never derived from an embryo in the first place, unlike embryonic stem cells. So, we haven't had to harvest human embryos to make them, but rather they can be made just from a cell that's found in an adult human in fact the patient's in this case So I'd just like to spend a little bit more time explaining what's happening in this Epigenetic reprogramming for Somatic cell reprogramming to get induced pluripotent stem cells. So if you think of the landscape, the epigenetic landscape, and this was first proposed by Conrad Waddington back in the middle of the last century. We think that actually we'd have these pluripotent cells sitting at the top of this epigenetic landscape, these cells that are able to contribute to many different lineages, all the lineages of the adult, and at the bottom of that landscape we have the differentiated cells for example, here shown is the neuron. So what, needs to happen when we reprogram is that we need to go back up this mountain. So, we need to remove all of those particular lineage-specific epigenetic marks. We need to restore the pluripotent epigenetic marks. And actually, restore the specialised chromatin structures that are found in pluripotent cells. I haven't mentioned this so far, but pluripotent chromatin seems to be much more open and flexible than that found in, the Somatic nuclei. At the same time as these things are reprogrammed, and the Epigenetic marks are removed You need to make sure to retain the imprints that are found at imprinting control regions and as we know, this is a very difficult task, as we've mentioned already. And you need to make sure that you remove the X inactivation marks in females. Because one of the hallmarks of the Pluripotent cell, as you remember from last week, was that they have two active X chromosomes. So, we know that, actually, some lineages are more readily reprogrammed than others. They're all extremely inefficient however, some are better than others and this has probably got to do with what the state of their chromatin is. So, how well you can actually reprogram. So let's think again with in terms of Epigenetic reprogramming what we required to happen. We are taking a cell from an adult and we are asking it to go back in time and restore the marks that were found in the inner cell mass of the blastocyst. So as I said, removing many lineage specific marks, we are removing the X inactivation mark if it was a female donor and we're retaining the imprint control region methylation, which is very difficult. So it's a very big ask, and not really like anything else that happens in normal biology. So the way that they can get this to happen is you can take your differentiated cell, and you can imagine that it would be incredibly inefficient to mount this barrier and climb the mountain if you'd like to make it back to a pluripotent cell. The way that they make this more efficient, and by more efficient I mean it still happens at less than 1% efficiency, is by adding particular transcription factors. And this pool of a few transcription factors, decrease the barrier. So, once this barrier is decreased and it becomes slightly easier, there is still a mountain to climb, but that mountain is smaller. So this will mean that the efficiency can be as high as perhaps 1%, of cells that are able to do this. But interestingly again, what we know is that you can change the reprogramming efficiency by inhibiting or adding back epigenetic factors. So again we know that this is an epigenetic event. So if we add in not only these transcription factors that are, the transcription factors here, sorry, that are acquired into the cells, but also add drugs to inhibit some of the epigenetic machinery, or activate the epigenetic machinery, you can increase the efficiency from perhaps less than 1% to 5%. So clearly we haven't overcome all of the barriers, by any means and this will be for research over the next several years to work out, or maybe a decade. But we can begin to get this to happen more efficiently by modifying the epigenetic reprogramming that's happening here. And for therapy, this would be an amazing win for health. So, if we summarise then how epigenetic reprogramming happens in these three different scenarios. We know that there's two reprogramming windows, the primordial germ cell development, and early embryonic development. are really sensitive periods for epigenetic reprogramming and if you disrupt them by altering the environment in which they happen, or you speed the up or avoid them entirely with any of these processes. Then obviously this has consequences for how efficiently the reprogramming occurs. So in IVF or ICSI, we know that the germ cell harvest seems to disrupt germ cell development, then the culture, ICSI itself, IVF itself and embryo handling, seem to disrupt it again for early development. We know in cloning, you've entirely skipped primordial germ cell development, and then you go on to still culture the early embryo. And in reprogramming, as I said, it's really not like any other scenario that's occurred in normal development. And so, you are skipping all of these stages and trying to get it to occur in reverse. We do know that modifications to these procedures are resulting in higher efficiency for reprogramming for the case of cloning and Somatic cell nuclear transfer. And we know that if we can learn more about how IVF and ICSI can be disrupted, then we'll be able to optimise this technique, which will be the best for all of the patients undergoing these procedures.
