 This is a place I serve, it is the International Center for Genetic Engineering and Biotechnology which was funded upon the same idea as ICTP, 30 years ago, ICTP is 50 years. And basically this is an international organization, different from ICTP it is autonomous and so we are supported by 64 countries which are shown here. And basically we run advanced research in our laboratories in Trieste, New Delhi and Cape Town but at the same time we give fellowships to PhD students and postdocs from these member states and we organize meetings, courses, schools like this one, everything in the field of biotechnology. Physically we are located in this area here, so basically now we are basically here. So it's about a 15 minutes drive from here to the ICGB. So I started with an apology, so I'm a medical doctor by training, I worked always on very gross subjects like gene therapy and more recently cardiovascular disorders so my talk will not be dealing with small objects in quantitative terms as this school would imply but I will deal more with big objects like organs or cells and you will see the approach will be very qualitative. But I think that I will touch a subject that might be of interest for you both as a scientist but also human beings because this subject I think it is very critical in understanding our human body and the problems that we are facing and the modern medicine is essentially facing. Excuse me for that. And the subject is basically led by one important observation that the human lifespan has a fixed term as it has a fixed term basically the lifespan of all living beings on the planet. This is a picture of last year and last year the longest lived person was reported to be this lady Misao Kawa, she was born in 1998 and after she celebrated, she is 117, a couple of months ago later basically she died. So the record now of a long lived person is this other lady here, she is Italian, actually she lives in the province of Vercelli in the northern, northwestern part of Italy and her name is Emma Morano and she is reported to be at 116. So these are testimonies of a long lived person and this fits very well with the concept that we have that the life expectancy has grown a lot over the last century. If you take life expectancy, so life expectancy is a statistical measure to say how many people would believe out of 100 in a certain number of time and basically life expectancy at the beginning of the century, last century was 49 years and after only one century, so in the early 2000, it rose to 76 years, so this is in industrial countries. If you look at the statistics now, these are the latest statistics, the World Health Organization a couple of years ago in Italy, for example a man has a life expectancy of more than 80 years and a women life expectancy of 85 years, the top living countries are Iceland for men, 81 and Japan for women, 86. So this, you want to ask something? Yes? Exactly, exactly. So how do you do the big shift? Sure, sure. All the people. Sure. Let me come to that. Yes, yes. So these are the official statistics, we are witnessing this huge increase, but the interesting point here is a study that was published a couple of weeks ago in nature that is a very simple study. It was performed by a group in the United States and Cornell University and they took 40 countries and 155 years of observation and they simply asked how do this life expectancy data stratify according to the period of observation in terms of age of the person. So the question was if you have an increase from 49 to 76 and now to 85, which is the category of people, the age of people who have benefited more. And as your colleague anticipated it's very obvious that a big part of the increase in life expectancy was a decrease in perinatal and infant mortality, which has occurred thanks to social economical reasons, thanks to medicine and so on. Then there has been increase also from 10 years to 20 years of age from 20 to 30, 30 to 40 and so on, but what happened is that if you analyze data from 90 to 100, to 100 and 110, 110 to 120, the increase has been minimal and unexistent from 110, 120. So basically what we have gained in this last century is not an increase of the maximum lifespan of the species of Homo sapiens, but just an increase in a reduction in the cause of mortalities from zero years of age up to 90 or 100 years. So we are not expanding lifespan, we are just expanding life in this definite period of time. In other terms, we have a fixed lifespan as human species and as Homo sapiens species and this is set to be around 120, 125. In fact, the person who has been reported so far officially to have been with the record for having long lived is this lady here, Jean Calment, she was a French lady who died in 1997 at 122.5 years and nobody has surpassed it. So even from 1995 up to now, so in the last 20 years, nobody has been reported to have lived longer than this lady seemed that basically there is a wall that we cannot surpass by any means. And this wall, so this wall of the fixed lifetime is the same that exists for all species on the planet. As I said, for example, a mouse lives two years, a rat lives three years, a sheep 12 years, turtles are very long-lived animals, it's 150 years, dogs, that's a common experience, it's from 15 to 30 years, the Drosophila melanogasa, the fruit fly, three months and so on. So basically there is a sort of biological program inside each species that sets the maximum lifespan of that species. If you ask me what is this biological program, nobody knows. To me it is one of the most fascinating mysteries of biology, so simply understanding why a mouse lives two years and a man lives 120 years even if they share 97% of the genetic information and basically they have the same anatomy and the same physiology grossly speaking, but they go from two years to 120 years. That really would be a marvelous invention or a marvelous understanding. What we know that's very interesting is that if you take 100 persons and you ask, when will these persons die, one could expect that if we have 120 years, after one year there are 99, then 98, 97, so there is a line starting from 100 and reaches zero in 120 years. Instead the situation is very different. So basically if you take humans or mice, see elegance, even the body needs so the most simple unicellular organism, you see that most people are alive for most of their life and then they start dying just at the end of their life very, very rapidly, so it's not a straight line from here to here, but there is a period by which more than 90% of the population is alive and then it drops down suddenly, which is suggestive of a program, a program by which everything works for a certain time and then suddenly it starts not working anymore. Again, what is this program and why it differs so much among the species is absolutely not known. By the way, this has tremendous implications for medicine because if you take the need for sanitary assistance, for health assistance for people and expenses that society have to make to ensure survival and assistance of people, these are concentrated in the last five years of life. So basically one person spends, every person spends 95% of their whole life expenditures in medicine in the last five years of life and this is what also the society does. So this has a tremendous implications for health policies in the western countries. Please. Yes, these are the curves in terms, this is a lifespan of sea elegance, which is a maximum lifespan is 25 days, and you see that 90% of the population reaches 20 days and then they suddenly start dying, more or less this is the same for humans and mice. Sorry? You're not changing scale, the fact is that you have a fixed program that sets your maximum lifespan and then for 90% of this program you are alive and then the organisms start dying. The question is why this happens, so why do we age and how do we age? The molecular mechanisms are really not well understood, but there are more than 300 different theories of aging and when there are many theories and we will see them, some of them in a minute, it means that we don't have a real explanation for this. We can conceive why we age because we are here on the planet, not because we have been creative for our own sake, for the sake of our body, but because simply we fulfill a mechanical purpose of transferring our genes. So evolution on the planet is driven by DNA, it is driven by genes. So basically what is immortal on the planet are genes and our bodies are simple vehicles to transfer these genes. This has been first conceived by an evolutionary biologist and a fantastic science communicator who is Richard Dawkins and published in this book The Selfish Gene in 1976. So this book here he has signed a landmark moment for understanding how life is on this planet. I think that he published this book The Selfish Gene, so genes driving evolution and not the bodies driving evolution, before publishing any paper in any big journal. So basically he came out for this book for the late educated public and this has really shifted our understanding of life. So to my students this is a must, they don't pass my exam if they don't know this book. So I recommend to all of you have not read it. How many people have read this book? Fantastic. I recommend all the others to do so. It's still very, very modern. And so basically once we have transferred our genes to our progeny then we become irrelevant for them on the planet or even worse we become competitor to access to the resources. So evolutionary it makes a very good sense that we are wiped out. And for the human species and for several mama species the period after transferring the genes so the period when fertilization occurs and the period of death is very long afterwards because our babies are born in a condition by which they are not self-sufficient and not sufficiently mature to survive on the planet. So basically the differentiation and development in humans finish by the age of 18 years of age. And so basically there is a need that the parents and the grandparents will survive for sufficiently long period of time. So this is why our maximum lifespan is so extended after the period when our sexual organs are mature. So we can conceive the reason we don't understand the mechanisms and certainly out of these 300 theories there are 12 theories that makes a lot of sense. Certainly there is a deterioration of our organs which is due to accumulation of damage into DNA, into macromolecules which is not repaired. There is an incapacity of the cells progressively to divide because they accumulate this kind of damage. There is a shortening of the extremities of our telomeres of chromosomes so incapacity of driving DNA full DNA replication in the cells. And there are accumulation of errors due to the incapacity of the DNA repair machinery to repair these errors or repairing these errors by introduction of mutations. But all of this still doesn't explain why a mouse lives two years and a human lives 120 years. What we certainly understand is that there is a strong correlation between the aging and death and the exhaustion of the regenerative capacity of organs after birth. So basically there is really a correlation by which critical organs in our body like the heart, the brain and the bone marrow are capable of dealing with the need for renewal of cells in this organ. And while this fades there is also a progression towards aging and death. Somebody believes that this is not just a correlation but this is the cause of aging. So we age because we are incapable of renewing our cells and renewing the tissues and the organs where our cells are located. For example, you know you have seen I'm sure in this course this picture in several different flavors. I will show this also a few times during my presentation. You know that all of us are the product of an egg from our mother fertilized by a sperm cell from our father. Then this cell is the first cell that defines the organism. It divides two times, four times, three times, four times. At this stage of 150 cells it forms a sphere with a mass on one side of the sphere. This is called the inertial mass. The wall of the sphere will give rise to the placenta, the other fetal nexus, the inertial mass to the new organism. This is called the blastocyst. And then immediately after the blastocyst, which is formed about four or five days after fertilization, there is a tremendous reorganization of cells in the embryo, which is called gastrulation, by which three layers are formed, mesoderm, endoderm and ectoderm, and from each of these three layers there is a further specification of specific cell types. So for example, from the ectoderm, the skin and the brain will develop from the mesoderm, the blood, the skeletal muscle, the heart, muscle, the kidney, from the endoderm, all the glands and the internal glands and the lungs. So this specification is a function, a specification that forms about 240 different cell types. So my body and your body is formed by about 10 to the 14 cells, each of which has a specific cell type, has a cell function, and to form 240 different cell types. So a neuron is a cell type, an epithelial cell, red blood cells, or a cardiomyocery is a cell type. During this process, most of the cells at a certain point, when they get specified, they will stop in dividing. So they get terminally differentiated. For example, you can take from the heart of an adult individual the contractile cells, cardiomyocytes, and you can put them in culture. They are these beautifully brick-shaped cells, very big, very large cytoplasm. Sometimes they are benuclated. These striations are the sarcomeres or the contractile apparatus. These cells can be kept in culture. All molecular biology you want, you can do all electrophysiology you want, your immunofluorescence, and all the studies you want in culture. They can survive several days. There is no way of having these cells replicating. So after birth, these cells don't replicate. So we immediately lose the capacity to regenerate in the heart immediately at birth. We came to this conclusion in an equivocal manner through a study which was conducted by the Karolinskaya Institute a few years ago. This is a very fancy study because it took advantage of the fact that in the 15, in the 60, the big nuclear powers like the United States, Russia, but also France, started detonating atomic bombs in the atmosphere. So basically from the atmosphere, in the atmosphere there was a creation of a lot of unstable isotopes for several ions, including carbon. Since carbon is the basis of all biochemistry in living being, there was a certain percentage of carbon incorporated into living beings which was not C12 as in this normal stable state, but it was C14. And by a accelerator of Mach spectrometry, one can recover DNA from a living being and ask how much C14 is there. So if you do this, for example, in the concentric rings in trees, you know that each ring marks one year, you ask what is the concentration of C14 starting in the rings of a long-lived tree. You see that the rings that were formed before the 1960s have very low C14 content. And then starting from the late 50s and the 60s, the content rose. Then this period, the moment there was a treaty signed in St. Petersburg between the USSR at the moment in the United States to stop this nuclear detonation. And so this curve started to go back to normality. It is expected that right in these days it should reach, again, background levels. But this means that if you take, for example, the brain, this was the first study that was performed, and let's start first with the gut. The gut, contrary to the heart or the brain, renews every two weeks. So we renew the epithelial cells in the gut or the epithelial cells in our skin every two weeks. So if I take epithelial cells in my gut or in my skin and extract DNA and measure the C14 content, I can ask the question, is the C14 content compatible with the today conditions or the content is higher suggesting that the cells were produced before? And the answer is no, the C14 content is the same as today. But if you take the DNA from the brain, which was performed in this study here, then the DNA from the cortex of the brain has the C14 content, which was identical to that of the date of birth of the person. So indicating that, basically, we are born with a certain number of neurons in our cortex and these are never renewed. The numbers say that we are born with, perhaps, a bit more than this number here, so 50 billion of neurons. And we lose 80 neurons every day. So while we're here speaking, you are losing neurons, I'm losing neurons. And the capacity of regenerating these neurons during the adult life is zero. So we are programmed to be born with a certain number of neurons and no capacity to regenerate them. The same study was performed after a few years. We are here 2009 in cardiomyocytes and the conclusion is the same. A person at 72 years has more than 50% of his heart formed exactly by the same cells with which this person was born. So this means that in my heart, in your heart, there are cardiomyocytes that will go along with us until the time of our death, which are exactly the same that has been produced at the moment of our birth. And this is remarkable because if you think these cells have a mechanical property of contracting, so these cells contract billions of time during a lifespan and they are exactly the same. They have been formed exactly at that moment. Now, having this notion that we are increasing our lifespan, but we cannot regenerate the organs and that our terminal maximum lifespan is fixed, would you be surprised to see that starting from the 1900 when this expansion of life expectancy has occurred, would you be surprised to see that there is such a huge increase of degenerative conditions? So we have an enormous increase in cases of heart failure. We have degeneration of the cartilage with arthrosis. We have loss of beta cells in the pancreatic diabetes. We have a tremendous increase of degenerative disorders and so on and so forth. So medicine at the beginning of the 1900s was a medicine of infectious diseases. Now, medicine is a medicine of degenerative diseases. We have to cope with the effects of prolonging life expectancy without having any tool to regenerate the organs that get lost. Even in organs that are known to regenerate themselves, like the bone marrow, you know that all cells in our body, so red blood cells, white blood cells and platelets, come from an individual cell which is, during our life, has a continuous turnover in which part of the progeny remains an hematopoietic stem cell and part of the progeny differentiates to become a red blood cell, white blood cells and a platelet. This is the hematopoietic stem cell, stem cell which stays in spongy bones. So it is estimated that if I take me on you, all cells in the body come from approximately 30,000 hematopoietic stem cells. So hematopoiesis is sustained by 30,000 stem cells that continue replicating and differentiating, considered that some types of white blood cells, granulocytes have half life or four, five hours, so they need to be continuously produced by the bone marrow. Well, there have been scientists, this is a very remarkable study that there are scientists in the Netherlands who were interested to ask the question whether aging in an individual is due to the progressive accumulation in mutations in the genome. So basically what they did was to take a certain number of individuals and measure the amount of mutations over years, and one of these individuals, they were so lucky that she was a lady and she survived up to 115 years old before dying because of a cancer. And basically, they had these samples for 40 years and they measure the accumulation of mutations in the blood of this lady for 40 years. And basically they came to the conclusion that mutations are not important for aging. So this lady, over 40 years, accumulated 45 mutations, but our genome is made by three billions, best pair. And 98% doesn't code for proteins, so it is either between genes or inside introns in genes. So finding that you accumulate 45 mutations is probably telling us that there is no relationship between aging and mutations, so basically it is not because we accumulate mutations that we age. But the interesting thing is that by finding mutations, these scientists had the opportunity of using these genetic variations as a marker of the cells. Mutations always occur not in the differentiated cells, but in the replicating cells. So basically finding a mutation means also using this mutation as a marker of all the red blood cells, all the white cells and all the de-platelets that are formed from this cell. So it is a marker of clonality. And so they asked the question, how many clones when this person died were sustaining her hematopoiesis? So how many stem cells this lady had out of the 30,000? And the answer was two. So all her hematopoiesis was sustained only by two hematopoiesis stem cells. So during her life, she has exhausted completely the capacity for regenerating her hematopoietic system. So suppose that she didn't get the cancer and she survived an additional couple of years after six months, she would have had one. And then after that, zero. And at that point, she would have died by a plastic anemia, so incapacity of producing blood cells. So to me, this is a fantastic example of this concept that aging is accompanied or perhaps caused by exhaustion of the regenerative capacities, even in organs that are known to be regenerative like the bone marrow. The numbers are really impressive. So every year, there are 15 million people diagnosed with heart failure, so incapacity of the heart to contract properly in the world. And once you have a diagnosis of heart failure, heart failure is the condition where with the heart dilates, the walls become very thin. And there is the incapacity of the pump to supply the sufficient amount of oxygen and nutrients that the body needs. Once a person is diagnosed with heart failure, this person has a probability of 50% on not being alive only after four years, which is a prognosis that is much worse than most cancers. If you take persons after eight years of age, one person out of three of them has a dementia, which in most cases is Alzheimer's disease. One person out of three after eight years, we have just told you that life expectancy in industrial countries is more than 80 for men and more than 85 for women. A real epidemic here in Europe and the United States. There are more than 170 million people with diabetes who needs insulin because simply they have exhausted their beta cells in the pancreas and there is no possibility for beta cells to be regenerated. After 75 years of age, there are 30% of people don't see well because they have had degeneration of the retina and there is no possibility for retinal regeneration. And again, after 75 years, one person out of two doesn't hear well because they have degeneration of the neuroepithelia cells in the inner ear and no possibility for degeneration. We even tend to think that not hearing well is a sort of hallmark of aging because it is so common that we associate this with aging. But this is a pathology. This is due to the fact that you have degenerated the cells and our body has not the capacity to regenerate that. Now, these conditions have in common that they are due to the loss of cells, have in common that this loss of cells is irreversible, but also that they have in common that we have no therapy. There is not a single drug that might induce regeneration of the heart. There is not only that, but the drugs that you have of heart failure are only drugs that are aimed at having the surviving cells contracting a bit better. No way of regenerating the cells. And the last of these drugs was developed 20 years ago. So it means that a person now is treated with the same therapies as 20 years ago. We have zero drugs for Alzheimer's disease. There was a monoclonal antibody from Haley Lilly that underwent phase three clinical trial with a lot of promises for the previous experimentation, and last week it failed in the phase three clinical trial. The Haley Lilly quotations in the market dropped 15% suddenly in a few minutes when the result of this trial was communicated. At this moment we have no promise for anything for Alzheimer's disease. We have no way of regenerating beta cells. We have nothing to regenerate the retina. As you know, we have nothing to regenerate the inner ear. The only way is to try with devices to amplify the hearing. And this is the reason why, please, zebrafish, yes, it can pause. Yes, so, yeah, yeah. Zebrafish has a fixed lifespan in the day-long period despite the fact that it regenerates its organs. But the fact is that it regenerates some specific organs but doesn't regenerate them completely. So if you take an aging zebrafish, the capacity of regeneration is much lower than young zebrafish. So one might well argue that also in zebrafish, over time, you lose the regenerative capacities. Most of the experiments that we know in zebrafish and the salamander or other amphibians are performed with young animals. If you look at aging animals, then the capacity of regeneration is much lower. So the pyramid would fit also there. The reason why there is no drug for these conditions is because the pharmaceutical industry basically is very well suited to develop small chemical drugs, so small molecules. But it's very difficult also to conceive that a small molecule could trigger regeneration of an organ. It's much more conceivable that if we want regeneration, we should gear up a regenerative program, so a biological program. And now we have different tools that are coming from biotechnology. So we can regenerate an organ, for example, implanting a stem cell or using a stem cell in culture to produce new cell types, new cells of that specific cell type to be implanted. Or we can use, for example, growth factor to stimulate this endogenous regeneration. Or we can use genes to stimulate regeneration. And genes doesn't mean only protein-coding genes, but it means all regulatory genes in the domain of small regulatory RNAs, long regulatory RNAs, DNA that sequester other RNAs in the cell, and so on. So all nucleic acid therapeutics. So basically what I'm going to tell you today now is what is the state of the situation with the stem cell applications for organ regeneration. And tomorrow instead, I will deal with the possibility of stimulating endogenous regeneration of the heart using microRNA. So tomorrow it will be more a cardiovascular presentation showing our work and seeing how we would like to transform a mouse or a pig, possibly a human heart, into a zebrafish heart for achieving endogenous regeneration. Okay, so basically this is the same scheme as before. If you take this blastocyst, this blastocyst is basically formed at four or five days. And this is a time point where the blastocyst implants into the uterus wall and gives rise to a new organism. In 1981, Marcus Evans made a very simple experiment in which he took a blastocyst from a mouse, disrupted the inner cell mass, put this cell in culture, and he basically discovered two very, very surprising things at the time. First, these cells were immortal. So they could grow these cells in certain conditions for indefinite periods of times, without them changing their carotide and without them becoming tumor cells. So a very specific cell type. And then the other thing that he knew is that if he takes these cells and implants these cells into another blastocyst, and suppose that these cells are marked with a gene that makes these cells blue, so a black Z gene, then the embryo that is formed is a chimeria. So these cells can take part in the formation of any cell type and any organ in the body. So he called, in the scientific community, he called these cells embryonic stem cells because these cells are virtually capable of becoming any cells of the body. So if you like nomenclature, you could say that these cells are pluripotent. So they can come any part, any cells in the body. If you implant an embryonic stem cell in a uterus, then nothing happens. So there is a difference between the embryonic stem cells from the blastocyst and the zygote because if you implant a zygote in the uterus, then there is a new organism. So basically, if you take a zygote and you weight one division, you have two cells, you desegregate two cells, two uterine, you have two organisms, you weight four cells, four uterine, four organisms, eight cells, eight organisms, then depending on the species from the stage of eight cells and the stage of 16 cells, this totipotency is lost but still the cells in the blastocyst retain a multipotent capacity. This concept will become important also later. It took 17 years to show that this is exactly the same for humans. So basically, this is an experiment made by Thomson in 1998. Basically, he fertilized an egg in the laboratory, grew the small embryo up to the blastocyst cell in the laboratory, desegregated the inner cell mass, put the stem in culture. In this case, he couldn't make a transgenic human but basically what he did was to show that these cells, human embryonic stem cells, can form any kind of cell in the body. So basically, it took 17 years because the culture condition of embryonic stem cells from the mouse and from humans are significantly different. We know a lot now on embryonic stem cells. There has been really an immense work from several top laboratories in the world. We know they come from the blastocysts. They can renew indefinitely. They maintain a stable karyotype. We know that they are clonogenic so from one cell we can have a progeny and that under specific conditions we can differentiate them in all cell types in the body. Obviously, there has been a tremendous work on understanding which are the molecular mechanisms that maintain pluripotency of these cells and then drive their capacity to differentiate. There are a number of factors which have been identified to characterize these cells, the molecular level. The most important of these are three specific transcription factors which are called oct4, nanograms, and sox2. These are absolutely essential to maintain the proliferative and indifferentiated state of embryonic stem cells. Again, this was the first clue to show that the characteristic of a cell type is due to the characteristic of transcription factor that this cell expresses. For those of you who are not biologists transcription factor is a master factor that drives an expression of a set of specific genes. When we say that there are 250 different cell types in our body, it means by cell type a cell that has a specific function because it expresses specific proteins and it expresses these specific proteins because it has a specific set of transcription factors. The identity of the cell is due to the transcription factor this cell expresses. I don't want to enter into the Baleo-Biogeodes, which is very interesting, but out of the main scope of this which is more applicative of this presentation which is more applicative, you might well argue, well, then I need the regeneration in the heart or I need the regeneration in the brain, the problem is solved. I take an embryonic stem cell, I put this into the heart, I will go this cell, regenerating the tissue. Unfortunately, it's not so easy because if you do like that, basically you don't obtain regeneration so it is true that if you take an embryonic stem cell you put this in the heart that this cell will start expanding, so replicating and then differentiating, but it will do that in a very chaotic manner forming big masses, more or less aggressive, they are less aggressive, they are called teratomas, if they are more aggressive, they are called teratocarsinomas and these masses, if you cut them, they are formed by basically all kind of tissues. You have these chaotic masses in which you see some of the epidermis, the skin, some connected tissues, some cartilage, some bone, some red blood cells, some epithelia, so these are very monstrous masses in which differentiation has occurred by completely and specifically. Well, what you can do with the embryonic stem cells is instead of implanting embryonic stem cells in vivo, you can expand these cells in culture and obtain billions of embryonic stem cells and then convince these cells to become differentiated cells. For example, you want to repair the heart, you simply convince these billion cells to become a billionaire cardiomyocytes. This can be obtained now very easily because we know all the differentiation steps that leads embryonic stem cells to become a cardiomyocytes and we can modulate this simply by adding or removing factors in the supernatant and changing the cultural conditions. So you have one billion cells, embryonic stem cells, you have one billion cardiomyocytes, efficiency is now more than 90%. And you, as you can see here, for example, this is in the mouse, these are embryonic stem cells, the right cardiomyocytes, the only stem cells that we convince to become cardiomyocytes, they are cardiomyocytes with the expressed cardiomyocytes-specific marker, which is alpha-actin, staying red here. Most important, you see that this culture starts beating synchronously and for a culture of cardiomyocytes, beating synchrony is a marker of differentiation because a cardiac cell in our heart beats synchronously because all cells are joined by a specific type of gape junction which contains proteins like Conexin-43 that permits a rapid passage of mechanical information and electrical information. So just one single cell in the culture starts contracting and this electrical signal is passed very rapidly to all the other cells that start beating synchronously. This is the reason why our heart beats synchronously because of these formation of these gape junctions. So you take these cells, you implant them in a mouse with a myocardial infarction or heart failure, you completely repair and regenerate the heart. So fantastic achievement and this is exactly what he's trying to do, a laboratory in Seattle. Chuck Murray is the PI involved. He's working in monkeys as a very close model for myocardial infarction. So he's basically ligating the coronary artery in monkeys inducing damage to the heart, the myocardial infarction. So he takes human embryonic stem cells converted into cardiomyocytes. He takes one billion of these cardiomyocytes, puts them in the monkey, and he regenerates the wall. With a lot of problems still because the implanting of these cells suddenly means also disturbing the electrical connectivity of the existing tissues. So these cells take time to make electrical connections with the rest of the myocardium. And so basically these monkeys have very severe arrhythmias and it is unclear whether these arrhythmias will prevent clinical use. Murray is believed that this will not be a big prejudice. Others in the field are a bit more cautious and skeptical, but this is the most advanced way of really regenerating the heart using stem cells. For those of you who have a religious belief or in any case scientists have to take into account what this society believes, then this would imply destroying an embryo because to achieve this kind of stem cell population and then transform this into cardiomyocytes or any other cell type, then you would need to obtain an embryo by in vitro fertilization and destroy this embryo at the stage of blastocyst. So after four or five days. So this means creating an embryo in the laboratory and destroying this embryo. And for example for the Catholic Church this is not allowed. In Italy you have a terrible law that doesn't even permit the creation of embryonic stem cells line for research purposes which is very crazy because scientists here in Italy who want to work on human embryonic stem cells simply cross the border to Switzerland and do the embryonic stem cell derivation. They come back and they work with cells because our law permits working but does not permit regenerating. There are a number of topics that might be interesting for discussion in this case. For example in the fertility clinics usually women who go to fertility clinics to have babies, there are several blastoses that are generated during vitro fertilization. Most of these are thrown away in the United States or here in Europe in the Catholic country they are saved in the refrigeration but they cannot be used. So it would be simply this as could be used. This blastosage could be used for research and it is also known that in normal conditions almost 20% of the diagodes do not implant after conception. So our deficiency that we humans have in terms of generating babies is as low as 80% of the blastosage that are formed they don't implant. In the United States there has been a strong reaction against the creation of the U.M. embryonic stem cells under the Bush administration so the use of federal money for this application were forbidden. When Obama came into power eight years ago he released the use of public money and in 2009 a first trial was started with a very difficult application. So you heard yesterday you heard from Campos a few days ago that if you take a zebrafish you cut, I'm sure he told you that you cut the spinal cord then after a while there is a 40 days there is complete regeneration by the spinal cord and there are some factors involved in this activity. Well, if you take a human person who has a car accident he has a damage to the spinal cord instead there is no regeneration and this person remains paralyzed for the rest of his life. So this trial was aimed at injecting human embryonic stem cells derived neurons into the spinal cord or persons with a damage at the thoracic level. A few patients were injected but then the trial was stopped and the company moved to cancer therapy they invested lost a lot of money on that and the reason is that it is really difficult to think that regeneration of the neurons could occur so efficiently in terms of forming connections. So if you think of the spinal cord motor neurons can be formed by injecting neurons inside the spinal cord but you also need that these motor neurons emit an axon that travels for many many centimeters to exactly reach the muscle that it should innervate and this is really too demanding as my opinion is too demanding also to think that we can have brain regeneration in Alzheimer's disease because we can have neuron regeneration and implantation and then thinking that these neurons make the same synapses as in normal conditions is very difficult to conceive. There is an organ however in which the possibility of spontaneous regeneration is much easier in which this is the eye this is work from the SASI laboratory a few years ago in which you show that you can take embryonic stem cells committed to become eye cells and they form an optical cap in the laboratory so basically this cells even in cell culture have the capacity to spontaneously differentiate and form a three-dimensional structure that resembles the eye this SASI by the way has a very tragic story and it has given a fantastic contribution to eye regeneration two hours and ten years of eye biology then two years ago he remained trapped in a fraud story that involved one of the major centers of the rick in Japan and a laboratory in Harvard by which there was the proof published in the false proof published in nature that you can obtain embryonic stem cells simply taking a fibroblast in the pH of the medium this turned out to be false he found himself as one of the co-authors of this paper he was one of the middle authors so they gave him not much contribution but he is a pride and honor was so that he killed himself so he committed suicide however this idea of regenerating the eye using embryonic stem cells has moved to clinical trials in many groups in the United States we are trying to regenerate the rachina using human embryonic stem cells most advanced one is in the Boston area and apparently there are patients now that have recovered part of their vision at least the night vision the black and white visions are seeing shadows of embryonic stem cells for regeneration of the rachina this is the state of the art for embryonic stem cells the one question is can we avoid going through this process of fertilizing the egg with the sperm cell to obtain embryonic stem cells exactly exactly so at the abutment or in the slow manner that could be a way I suppose that for example if you take a heart and you would find a way of implanting 10 million cells every second day for a long period then these cells will coordinate better with the existing cells obviously this is clinically impracticable so all what you can do is to do that in a single bolus in a single lamp and this is for the heart for the heart there are other problems the cells, the cardiomyocytes that you can obtain in cell culture from embryonic stem cells are embryonic cardiomyocytes they are not the mature cells that you find in the adult cells and there are some steps that still need to be to be fulfilled by these cells and how to stimulate this part of the maturation is not known at the moment several laboratories are trying to find ways of stimulating maturation of these cells I believe it is more a technical problem than a conceptual problem and I'm strongly convinced that you could achieve a real regeneration of the plant in stem cells from the outside for a mechanical organ like the heart I'm a bit more skeptical that this can be a solution for a disease that affects 15 new million persons every year in the world so I see that a medicine based of having embryonic stem cells one billion culture in the laboratory differentiated into cardiomyocytes implanted in the heart very destined to a very tiny fraction of very rich people in a very specialized clinic in the United States or in Europe but it's not a medicine for to tackle a problem that is so spread and so important also socially as heart disease for the brain for the nervous system in particular I'm much more skeptical because if you take a child when he's born his neurons have stopped dividing so basically he has a certain number of neurons in his brain and in his spinal cord and then this person however takes 18 years to reach full maturation of his intellectual functions and this full maturation is not cell production but it just contacts sign ups can we really think that implanting an adult said from the outside these neurons will make the same connections even projecting axons very distant part I'm very skeptical about that they can try to fit without needing to review I find this very difficult to conceive also very difficult to conceive that you can have projection going from one hemisphere to the other one in a normal aging in a normal person you lose 10-15% of your normal neurons in Alzheimer you lose up to 30% of core-energic neurons all throughout the brain how can you replace these from a disorders stem cells I find it very difficult I'm 57 so I'm approaching more than you this fatidic limit the first to be pleased to be wrong but I find it very difficult this moment that's a big question that's a big question and one should hope that once implanting a cell in an adult individual there are still there some signals that direct these neurons as they did during development but I find it very difficult to conceive so how can we obtain embryonic stem cells without fertilization so it is reached this stage here and the first experiment trying to prove that this could be done was performed in frogs this is a very old experiment performed by Gordon who won the Nobel Prize in 2012 for this experiment he basically took a frog and took a skin cell of the frog a nucleus of one of the skin cells and injected this nucleus into an egg and then this egg started to develop this nucleus as it would the product of fertilization and basically he obtained several frogs which were identical genetically to the donor of the skin cells this is called somatic cloning so cloning from somatic cells this happened in the seventies and people try to repeat this for decades in mammals and they all failed so the dogma was that this process of differentiation from embryonic stem cells towards differentiated cells was irreversible until in the scientific community was a bit shocked by the publication by a group that was working at the Edinburgh University in an institute that now belongs to the Edinburgh University it's called the Rosling Institute the name of the scientist is a veterinarian Jan Wilmut he showed in 1997 that basically you can apply the same principle to generate also a mammalian organism in that case the ship so basically what he did was to take a cell from the mammary gland of a ship so this cell is a specialized cell producing milk proteins and then he took this cell and took another ship from which he recovered an egg cell he sucked out the nucleus of this egg cell with the pipette so this egg cell remained just a small bag and then he put the nucleus of this cell the empty bag together gave an electrical pulse so more than 2,000 volts for a few milliseconds so this depolarized the membrane so the nucleus can enter the cell and at that point this nucleus started thinking of himself not as a specialized nucleus of a specialized cell to produce milk proteins but as the nucleus which was the product of fertilization so he started to behave as a zygote and then after a few days a blastocyst was formed he took the blastocyst and implanted the blastocyst on a third ship and after a certain number of days Dolly's ship was born this is Dolly with her surrogate mother so the mother that donated the uterus for creation was seen as a landmark discovery not for the reason that the newspaper reported the process is still very inefficient so Wilmut Klondoli out of 243 attempts and efficiency in all mammalian species is in the order 1-2% of the attempt so it's impossible to think of using this for reproductive cloning so you cannot have armies of soldiers clonally identical because this is really stupid but it was a landmark moment in biology because it was the first demonstration that in mammals you can basically revert genetic programs so think the way again I present this to the lay public it is a very nice metaphor that you are taking a brand new computer home you plug the computer into the wall outlet then you open switch on the computer it starts loading the routines for the operating systems and then you are on your operating system and you say well now I want to do a graph I want to analyze some data in excel so you start the excel application your data say now I want to make a graph and then you open the excel routine to prepare graphs you work on the graph you change the lines change the color change from a bar to a pie chart then you are happy you print this graph and you say well now I am happy with my work I want to go and check my mail and you realize that you have not an exit or a quit command so basically you have a very brand new computer the hard disk which is immense harmless of application in the hard disk but you have started working in excel and the only way you can use your computer for the rest of the life of the computer is to make that single graph in excel this is differentiation so you start from the zygote you end up with the cardiomyocytes this cardiomyocytes will remain a cardiomyocytes for 85 years when the person will die they will never become a neuron a fibroblast and keratinocytes in genetic terms it is like thinking that we have 20,000 genes at the time of fertilization we are out of these 20,000 we have a switched on so we express proteins for these green genes and then immediately after you have blastocystis so this remains home there are other four red genes then these are switched off cardiomyocytes it becomes a mesoderm so it has these blue genes and so on and then it becomes a cardiomyocytes expressing actin, myosin actinine, troponin and so on it will remain a cardiomyocytes and will never go back what did Vilmo do? He unplugged the computer from the wall and replugged it or as in non-computer there was a reset button he pressed the reset button so he unplugged and started the program again this is the first demonstration that any cell in our body has exactly the same information as the zygote that formed us years ago and has the potential to become another god zygote and to form a new organism so it's all a matter of programs it's not a matter of information, it's a matter of applications not a matter of hardware or content of information yes the shortening why there should be shortening of DNA no, this slide is probably what you are meaning it's not DNA that is shortened it is that one of the theories of aging one of the observations of aging is that cells are born during development with long repetitions of DNA and the extremity of telomers and then when cells stop dividing after birth the enzymes that makes the repetition are switched off, it's not expressed anymore so this long repetition at every division becomes shorter every time the cell divides becomes shorter and at a certain point it's so short that the cell cannot replicate anymore this was a theory that holds true 20 years ago so this idea of telomers shortening because of aging but it's not true at all, for example, cardiomyces stop dividing with very long telomers neurons stop dividing with very long telomers certainly with short telomers you cannot divide but this doesn't mean that you cannot age if you have long telomers at the beginning when the cloning of Dolly was performed a laboratory in the UK made a study, a very rapid study published in Nature saying that the telomers of Dolly were shorter than the telomers of the donor of the egg the cell that, the ship that donated the mammary gland and this, and the old journals in the world reported this as saying Dolly is born old this was completely false in fact the length of the telomers was re-analyzed later and was shown to be as that of a normal ship which is normally obtained by fertilization normal fertilization so basically cloning resets the length of the telomers simply it re-activates the enzyme which is called telomerase that makes telomers long as tumor cells do, for example tumor cells grow indefinitely because they re-express telomerase in fact a normal ship lives about 12 years so the life expectancy of a ship is 12 years 10 years and a half, 11 years because she was suffering and so she was euthanized but she basically would have made all her life through and in fact again also if you look at other species this is in cows you cloning resets the telomer length you see that these are other people say, extension of lifespan and telomer length and you can even do cloning for senescent cells and they re-completely reset the time information so basically after dolly immediately mice were cloned goats were cloned pigs were cloned, rabbits were cloned and also pets were cloned, first cats then a scientist in Korea became very famous his name is Vossuk Wang and because he did the first cloning of a dog as a pet the dog was called Snoopy and so this seems to work all throughout the species so there is no reason why should not work also in humans and in humans it is a very important application because basically you can't use this for human infertility but you can do a very simple thing and suppose you have a patient with a heart disease or a liver disease he needs regeneration of his cells so you can take a nucleus for example from a cell of a skin or for simply a white cells in the blood you take the nucleus, you have an egg from a female donor you transfer the nucleus into this egg and you grow these cells in the laboratory until they form a blastocyst then you destroy the blastocyst you put them the cells in culture, you take one billion cells you transform these cells into cardiomyces or liver cells you implant them in vivo, you have regenerated the difference is enormous because here we are working on the same cells as the patient so it is his cells it's not donating cells from a donor in fact the monkeys my friend Jack Murray injected in the heart with human embryonic stem cells had to be immunocompromised because they were receiving cells from another individual another species in that case here instead it's like having this patient when 70 years ago he was a blastocyst somebody would have taken one cell of the blastocyst put that in the freezer and then thawed these cells and grow this again after 70 years when he needed these cells really revolutionary, this is called therapeutic therapeutic cloning it remained for many years unknown whether this could happen also in humans until this Fosu Wang published a paper in size in 2004 that he obtained human embryonic stem cell but then something happened because this Fosu Wang for which the Korean government built a big institute for stem cell research in Seoul became a sort of national hero then a female person of his laboratory sued him because he apparently wanted their post-docs and female post-op students to spontaneously donate eggs for this kind of research but donating an egg is a procedure used in fertility clinics it's an invasive procedure so you need hormonal treatment and then with the needle you have to recover the eggs and so on he was apparently forcing so a committee an investigation committee was set up he was investigated and the investigation committee discovered that this not only was true but also that all the experiment on human cloning were fabricated so there was a big scandal he became a sort of rejected person in Korea to California and set up a company quirky clones pets if you go to the website of this company they even show you how to save cells from your died dog putting the dog in the refrigerator you can keep it for 2-3 days and then take a sample of cells you send them to this company and for 66 pounds you can obtain a clone of your favorite dog instead you want to spend you have to spend $100,000 you can have even a copy an exact copy of a dog called trucker which was a German shepherd who became famous in the 9-11 disaster of the Twin Towers in New York for having saved so many lives so with $100,000 you can buy an exact genetic copy of these dogs there is apparently a very very florid market for these among very wealthy people in the United States in Europe nobody knew whether this human cloning was possible until 2013 when this group in a primal center in Oregon provided real proof that there is no reason why humans could also not be cloned the church however was not the Catholic church was not happy at all at this moment because cloning means that you don't have to create an embryo because you obtain this through the formation of a blastocyst through nuclear transfer but still you have to destroy the blastocyst and so the Catholic church doesn't want to destroy a blastocyst and then the procedure is a bit cumbersome and the discussion immediately started when this person here this is a picture taken one month ago in a congress in Finland this person here published a paper he was also completely unknown to the field published a paper in cell in 2006 showing that basically any cell in the body can become directly an embryonic stem cell in the cloning procedure like Dolly you don't obtain embryonic stem cell the homolog of a zygote and so it is before the embryonic stem cells here instead he showed that you can take a fibroblast from the skin you simply transfer into this fibroblast four transcription factors which are this one in red here and you transform a fibroblast directly into an embryonic stem cell that he called IPS IPS cells the guy who did this is Shinya Yamanaka Shinya has a very interesting story he is an orthopedic surgeon that at the age of 42 decided that orthopedic surgery was very interesting but very limited to him he wanted to do something for humanity so he left completely surgery he set up a laboratory and started to see if he would find a cocktail of transcription factor to do exactly like this to transform any cell into an embryonic stem cell succeeded, he won the Nobel Prize in 2012 for this discovery only six years after the discovery the year later one famous laboratory in embryonic stem cell research a lot of Yenishi, the MIT proved that you can use these cells injecting them in a blasto-utene mouse and now the situation is very very different because you can simply take any cell type on an individual reprogram these cells with these transcription factors and then you have embryonic stem cell you can expand and implant so basically here the church is also very happy because if you take one of these IPS cells you put them in you choose nothing happens as an ESS and so you don't have to go through the creation and destruction of an embryo so everybody is happy the problem is that in technical terms this is still not very effective and the application would be immense because they are the same application of embryonic stem cells so you could cure Parkinson's disease spinal cord injury, heart failure beta cell production liver failure and so on and so forth but unfortunately the efficiency of this is still very low so you can obtain an embryonic stem cell with this Yamanaka factor only one cell is out of two 10 to the 4 but if our body is made by 10 to the 14 cells and 10% of these are in the liver it means that the liver my liver, your liver is made by the 10 to the 13 cells and obtaining 10 to the 13 cells starting with an efficiency of 1 per 10 minus 4 so one out of 10,000 is very difficult however this could be achieved in organs that are very small and again verityna comes into play verityna is very small and very defined and in fact two years ago the first trial for veritynal regeneration was started again in Japan with the collaboration of Yamanaka and then of Thalmodis there who is called Masao Takahashi now apparently two people have been injected to regenerate the verityna is very good however it appears that there are mutations that are introduced into these cells so for safety reasons this trial is being stopped now and we will see where we resume I think that one of the major conceptual contribution of this work is that to define again that you can change fate of cells simply changing transcription factor of the express and forcing expression of transcription of transcription factor for example you can transform mesoderm cells into heart tissues with these transcription factors or you can transform fibroblast into cardiac cells with specific transcription factors you can also convert fibroblast to neurons that use dopamine as a neurotransmitter with other specific transcription factors or fibroblast to neuro progenitors or fibroblast again to neurons so basically you can change all cell types simply changing the transcription factors they express and I think that this is a very important piece of information so this is where we are now in terms of application of stem cells for regeneration everything is at a very experimental level before ending I'm seeing that I'm running late so I will skip the last part but I want just to leave you a notion that is if you take if you take again this scheme we are here and these cells can really regenerate any kind of cell type there are cells also here that have the capacity of being stem cells but these are much more specific and they can do only a specific cell type and here however there has been a lot of publicity also on journals for the lay public in stem cells that can regenerate any kind of the body you take stem cells from the adipose tissues from the bone marrow and they can regenerate anything this has been supported by very many big papers also in important journals a certain point 15 years ago there was this notion that for example the bone marrow could become brain the brain could become muscle the muscle could become blood the blood would become vessels the bone marrow could become liver and so on somebody even proposed that in our circulation there are stem cells that circulate like in a highway according to the exit they take then they differentiate and specify into specific cell type this has led to thousands of patients injected for regeneration with cells taken from the bone marrow from the adipose tissue and all this has failed this is completely forbidden in Europe and the United States but these are clinics in Thailand in the former Soviet Union republics in the Caribbean islands in Costa Rica which offer this miraculous stem cell treatment for incurable diseases this is totally bullshit and unfortunately these treatments are very very very very expensive one of the laboratory who initially tried to prove that bone marrow cells can regenerate the heart is a laboratory in Harvard who then went under investigation there have been a couple of papers who have been retracted and apparently there is an FBI investigation for misuse of federal funds in the order of 70 millions for this research and so there are huge problems in this because there is a huge demand by the public by patients but the only therapy for adult stem cell that work are those based for regeneration of the blood so in matopoeia stem cells 50 years of bone marrow transplantation or use oipithelial stem cells so cells from the skin essentially that can be used for three application one is to regenerate the skin and the big burns have a kid with a big burn you take a normal portion of the skin put the epidermis in culture you have big sheets of epidermis and you put this into the into the wound and the application is for the cornea you have patients with the cornea which becomes opaque you can regenerate this with the oipithelial cells or you can cover artificial scaffolds this is a trachea made of an artificial material you can cover this with oipithelial cells these are the only applications of adult stem cells this is how to regenerate organs with stem cells as we already mentioned there are other organs like fish and the oorodils that regenerate organs through a completely different mechanism so they have endogenous regeneration and then tomorrow we will speak about that ok