 Okay, I think we can start. Good morning. I have a friend who is a professor at Harvard University who starts his seminar looking at the audience. And then he works in cardiac regeneration, as I do. And then he asks everybody to see who is sitting on the left side, who is sitting on the right side, and then asking the three of them to decide who or the three will die because of cardiovascular disorders. And cardiovascular disorders means more than 50% of the times myocardial infarction and the incapacity of the heart to deal with the repair of myocardial infarction. And this is a reality because 30% of the people in the world die because of these diseases, much more than all cancers put together. And if you think that this is a problem of the industrialized world, you would be wrong because according to the World Health Organization, 80% of deaths due to cardiovascular disorders are occurring in low and middle income countries. So the big cities in Africa and Asia, people die more because of these conditions than because of HIV, malaria, dengue, and all the other infectious diseases. So if a person has a heart attack, so a sudden closure of a coronary artery, he suffers of a myocardial infarction, and he has a chance to die immediately. He doesn't die, nobody dies because the heart doesn't function in terms of contracting power, but they die because after there is a sudden ischemia, so lack of oxygen or a portion of the heart, there is a high probability that other portions of the ventricles start behaving as pacemakers, and so basically there is a subversion of the normal electrical conduction of the electrical pulse, which means that there is a condition called ventricular fibrillation, so basically all the portion of the heart starts contracting in a very asynchronous manner, there is no pump function. If these arrhythmias don't occur, or if the person is resuscitated immediately by heart massage or by defibrillation, so the heart starts pumping again, the person is brought to the hospital, and if it has a chance of being close to an interventional cardiologist unit, so a catheter laboratory in emergency within the two, three hours, most patients here are rivascularized, so an interventional cardiologist inserts a catheter into the femoral artery, reaches the coronary artery, which is occluded by a thrombus, opens a balloon, so the thrombus is dissolved, and the heart is rivascularized. Obviously this determines how much of the heart has been lost during this process, and the sooner the angioplasty, so the opening of the coronary artery occurs, the better is obviously the outcome, the lower is the occlusion in the branches of the coronary artery, the better is the outcome, the higher the bigger is the portion of the heart that dies. When a portion of the heart dies, it is never regenerated. The only way of repairing it is through formation of a scar, but a scar obviously makes the heart pumping difficult. There is an event over the subsequent month that is called ventricular remodeling, so there is a change in the geometry of the heart, and this is a very bad prognostic signal because it means that most likely the patient over the next month will undergo a condition by which the heart will dilate, the ventricular walls will become more thin, and the capacity of contraction will be smaller, so the capacity of pumping blood out of the heart will become smaller, and this can be measured very easily by a parameter which is called ejection fraction, so the percentage of blood which is pumped out of the heart, every heartbeat, and when this starts lowering, then the patient undergoes a condition known as heart failure, this is a condition that I was referring to yesterday, saying that once there is a diagnosed heart failure, 50% of patients are not alive anymore only after four years. So there is a tremendous demand for heart regeneration, so to stimulate formation of new contractile mass, which is a problem that's not trivial because usually when coronary artery is blocked, the portion downstream of the thrombus is relatively big, and it includes from two to four billion cardiomyocytes, so basically the therapeutic target is to induce regeneration, so formation of a billion cardiomyocytes, and people have tried over the last several years to starting from 2001, as we said yesterday, to try to replace this lost portion by injection of stem cells of different derivation. First attempts were there with bone marrow cells and these stroma cells, again from the bone marrow, the adipose tissues, then from a series of putative stem cells that would be occurs residing in the other heart, you see that each of these attempts have a question mark and because they have failed basically, there is no positive results with any of these attempts. More recently, and also we said this yesterday, there is an attempt which has not reached the clinical experimentation yet, which is the monkey level by which cardiomyocytes are implanted, which are derived from ES cells, human embryonic stem cells, or from IPS cells, so the cells which are equivalent to these obtained by the Iamanaka factors. We don't know whether this will be successful for, by now, the problem with arrhythmia seems to be very difficult to overcome. In any case, even if it will be successful, there will be certainly a problem of extending this kind of treatment to the vast mass of people who have this clinical problem. Another possibility is to try to convince the fibroblasts in the scar to become cardiomyocytes. And this can be done, as I also said yesterday, by transferring to fibroblast transcription factors that change the identity of the cell. The idea would be to have a certain number of fibroblasts in the scar, which then are convinced to become cardiomyocytes. Technically, this is very challenging because you would need to transfer the transcription factor genes that determine the fate of a cell as a cardiomyocytes into fibroblasts in the heart. But cardiac gene transfer into fibroblast is very, very difficult, very, very low efficiency. And then this would be a stoichiometric process. So you have a certain number of fibroblasts which can become the same number of cardiomyocytes as best. But obviously this would imply that you do gene transfer of these factors into billions of fibroblasts, which is really beyond the technical possibilities at this moment. I thought there is intense research in this field. I showed you this picture before. And to highlight what around the early 2000s was a sort of dogma that is that you can take this beautiful cardiomyocytes from the heart of an adult animal or a man, put them in culture, and that they don't replicate. And this is supported by the evidence I showed you yesterday. You remember the atomic bomb explosions and C-14 measurements and so on. However, if you look at the same data with a more optimistic view, the half full glass view, then you can say, okay, this is true that in a person who is 72 years old, more than 50% of the heart is the same with which he was born. But it's also true that a bit less than 50% has been renewed during the adult life. And there is evidence that this rate of renewal is in the proximity of 1% per year. So 1% of cardiomyocytes is renewed every year, which is at the clinical level, which doesn't make any sense. Heart is post-mythotic. Heart says don't divide. But in principle, biologically, there is a possibility for replication or renewal. And also, it is known that if you look carefully in patients in the border zone of an infant, so in these surviving tissues around the infant, there are some cardiomyocytes that can be seen to divide. So while this is again clinically ineffective, then this biological possibility exists. And so one of the exciting ideas that are emerging in the last two, three years is to see if we can achieve regeneration of the heart from within. That is, instead of implanting cells from the outside, stimulating the endogenous reparative capacity that for some biological reasons is normally ineffective, but still exists so it can be boosted somehow. The idea that the heart is a post-mythotic organ that is cardiomyocytes that don't replicate comes from evidence that is very evident, very, very clear in the post-natal life. But obviously in the pre-natal life, the cardiomyocytes keep dividing because they have to reach a mass which is relevant. And so an obvious question is, when does this replicative capacity stops? And this can be addressed very easily, also experimentally. You can take a mouse, for example, and immediately at birth, inject a nucleotide analogue, which is a Bromades ocuridine. This is an analogue of thymidine that gets incorporated into DNA when the cells replicate so during the ES phases. And then there is an antibody that recognizes BrDU so basically the nuclei of cells that have replicated can be detected specifically with this antibody. This is the kind of appearance that you see you have in the heart. So this is a cardiomyocytes that have replicated in the last 48 hours if you keep the BrDU in fusion, the animal for 48 hours. And basically if you do this experiment immediately, at birth you see that almost more than 35% of cardiomyocytes are replicating in these conditions. So a vast proportion of cardiomyocytes in the heart. If you repeat exactly the same experiment in an adult animal, for example in a mouse at two months, then this number drops virtually to zero. So there is no replication. This is something that occurs immediately at birth. And at the same time you can reproduce this also in culture. So you can take out a heart in adult animals and you recover those beautiful cardiomyocytes that will never divide. But if you recover the heart immediately in a puppet birth and you put in culture cardiomyocytes, you can see that also in culture these cardiomyocytes retain a replicative capacity. For example, the express markers that distinguish S-phase cells. This is a staining of the nucleus of a cardiomyocytes with cyclin A, which is a cyclin, a protein that regulates the cell cycle that is expressed in the late G1 and this phase of the cell cycle. These are other cardiomyocytes. These are cardiomyocytes stained with bromodysosuridine. So they have just incorporated BRDU and they have divided. And you see also mitotic figures. These are chromosomes, the divides before the two cardiomyocytes will divide. So there is this capacity of division immediately at birth and you certainly have heard from KEMPOS that this division also leads to regeneration. So there are experiments performed in the Shamsadek and Rikosso laboratory a few years ago, 2011, by which if you take the neonate heart and you cut a portion of the heart the easiest way of cutting is the apex then this gets completely regenerated in approximately two, three weeks. If you do this immediately at birth during the first week of life if you do that after seven days then you have repaired through a scar. What happens at birth that blocks cardiomyocytes replication? So this is a big question for which we don't have a clear answer but there are at least four dramatic events that occur in the heart immediately at birth. The first one is that before birth the heart of the baby is inside the womb of the mother. So it's very, very far from the oxygen that is in the lung of the mother. So the heart is a venous organ. It's a blood with very low oxygen tension. Immediately after birth the heart finds itself very close to the lungs. So it is inundated by high concentration of oxygen in the blood. So there is an oxidative stress an oxidative shock immediately after birth. And this could be a signal that blocks proliferation. This is what Shamsadek believes this is a paper to which we collaborated a couple of years ago basically to show that if you culture cardiomyocytes in very low oxygen then these cardiomyocytes can keep replicating much longer than if you expose them to atmospheric oxygen. The idea would be that high oxygen provokes mitochondria to produce high levels of reactive oxygen species. This would damage DNA, trigger DNA damage response and this would stop the cell cycle. However, this is probably part of the story. Certainly it is true, but it's not the whole story. Another part of the story is that the heart has much more load in terms of pressure immediately after birth. Again, before birth the heart of the baby still keeps beating. It is the first organ that forms so the heart keeps starting beating very early after fertilization, after formation of the embryo. However, before birth it is the heart of the mother that keeps most of the circulation running. It is only after birth that the heart has to keep against a strong gradient of pressure and the heart cells could sense the strong gradient of pressure. There could be a signal that tells the cells stop dividing and now hypertrophies your cytoplasm and build up contractile apparatus to cope with this increased after load. What is the nature of this signal? It is not known at this moment but this is another important component. Then there are two other things that occur. Immediately after birth and this is known for 40 years there is a sudden switch from the use of glucose as a major source for formation of ATP to the use of fatty acid. So basically 80% of metabolism before birth is based on glycolysis, 80% of metabolism after birth is based on fatty acid oxidation. Whether this is a consequence of the stop of replication and increase in hypertrophy or it is the cause, it is completely unknown. There is nobody who has yet studied the possible relationship between metabolism and replication. And then there is something even more provocative, something that the newborn baby is missing after birth is the mother. So it could well be that the mother has some factors in the circulation that are a constant proliferation of the heart which are completely withdrawn at the moment of birth. This has never been considered before. This is a very interesting new way of approaching the problem. In mechanistic terms there is a change in the immediate after birth in the gene expression program of the cells. So neonatal cardiomyocytes still divide, express a set of genes that are different from those that are expressed by the cardiomyocytes in an adult organism. And something that changes very drastically is also the set of non-coding RNA in the cells. Now you know that in our genome we have the capacity to code for approximately 20,000 proteins. These are what we normally call protein-coding genes, those that we have been used to work with for 50 years. But since 10 years or so, 15 years, we know that in addition to these protein-coding genes there is a large number of regions of our DNA that code for RNA which are never translated. And grossly speaking, these RNAs can be divided in small RNAs which are called microRNAs which will be the one we speak of in a moment. And long non-coding RNAs means RNAs longer than 500 nucleotides. Just to give you a numeric value it is expected that the human genome in addition to 20,000 protein-coding genes contain the information for approximately 2,000 microRNAs and something in the order of 80,000, 100,000, perhaps 120,000 long non-coding RNAs which is a world that is very, very rapidly expanding and also much more difficult to tackle in a comprehensive manner. The microRNAs, I mean, are more limited in number. And in fact, if you look at microRNAs in the neonate and you compare those in the adult you see that there are a number of microRNAs which are highly expressed in the neonate and then their expression drops down. And at the same time there are microRNAs whose expression is very low in the neonate and then they become high in the adult. These microRNAs are the product of transcription of genes by RNA polymerase 2 which is the same RNA polymerase that transcribes protein-coding genes. However, these transcripts are highly structured and are immediately recognized. They form these herping loop structures with bulges coming out in this large loop and they are immediately recognized in the nucleus by a complex of proteins, the most important of which are drosha and DGCR8 in humans. This is called the microprocessor. So this microprocessor recognizes these primary transcripts, process them and convert them in pre-microRNAs which are stem and loop structures within perfect pairing. They are approximately 75 base per lung. And these are recognized in turn by a protein which is called the Sporting V which takes them out of the nuclear pore. And once these pre-microRNAs are in the cytoplasm there is another complex containing an endonuclease called Dicer that recognizes them and process them to form the final microRNAs which are 21-22 nucleotides long, double-stranded RNA with the imperfect pairing of the two strands which basically come from these stem and loop structures here. And these microRNAs eventually get assembled to another complex of proteins that's called risk. Here the main dominant protein is argonaut and at this point one of the two strands is displaced from the duplex. Only the other strand remains the displaced strand is called passenger RNA. The stranded remains are called guide RNA. And then this becomes the template for pairing to messenger RNA of the cells. So basically this is a template for best pairing. So messenger RNA are recognized and their function is blocked at the level of translation so they cannot be translated or at the level of destruction. So they are depolydinated and disrupted. The ultimate result is that the protein for which this mRNA were coded is down-regulated. So basically each microRNA can down-regulate the expression of the protein but since pairing is not perfect each microRNA can recognize tens of hundreds of different messenger RNAs. So they down-regulate basically the expression of tens of hundreds of different proteins. So these are sort of real stats that block simultaneous expression of a variety of proteins. Obviously in evolutionary terms they have been selected to block expression of a lot of proteins and change the fate of the function or the destiny of the cells. In biology now there is virtually no process that in which microRNAs are not involved, ranging from every step of development and differentiation to cancerous genesis, cell proliferation and so on and so forth. If you knock out the component of these processor complexes, this is incompatible with life. So the idea that there are microRNAs that somehow are involved in all processes suggested that there might be perhaps some microRNAs also involved in regulating proliferation of cardiomyocytes and we have available a facility that for high-throughput screening so these are robotic stations for automated screenings of chemical compounds or small nucleic acids and we have libraries corresponding to microRNAs of human, mouse, rat origin or even libraries of nucleic acid that block microRNAs by base pairing with them. So a few years ago we asked ourselves whether we can find among the human microRNAs some microRNA that could stimulate cardiomyocytes proliferation. So basically the screening was a high-throughput screening, 96 well plates. We had at that time available about 1000 microRNAs, synthetic microRNAs so in each of these wells we plated cardiomyocytes from neonatal mice and then to each of the well in a robotic manner we added one specific microRNAs and the screening was performed by high-content microscopy. It means that this is an image of a cardiomyocytes that you see at microscopy in red, in green you see cardiomyocytes in blue all nuclear stained with chemical compound called Hext and then we added BRDU for 48 hours and then stained with an antibody so where you see red, violet which means red plus blue, these are nuclei so replicating cells. Some of the nuclei are inside cardiomyocytes, some are outside and these are fibroblasts that contaminate always this kind of preparation, about 5% of preparation from the heart are fibroblasts which is a good internal control. Then this image is taken up by a microscope that converts this to a computer generated image in which the contours of cardiomyocytes are evident and then in which each red nucleus inside the cardiomyocytes here is shown in yellow. So basically the machine tells you what is the percentage of yellow nuclei inside cardiomyocytes and the percentage in a normal condition is around 12%. These are cells treated with two microRNA that increase proliferation and basically the percentage is almost 50%, 50%. So basically at the end of this story we identified almost 40 microRNAs human microRNA that very significantly stimulate cardiomyocytes proliferation. So this was the first demonstration that you can really play with the proliferative potential of these cells by playing with the microRNA network. When I see proliferation I'm not saying only cardiomyocyte incorporation or BRGU, so passage through the S phase. By C I say real proliferation. So this can be seen for example by checking other markers in different phases of the cell cycle. For example antibody against phosphorylative form of histone H3 which accumulates only in G2M. You see that if you take the top microRNAs all of these increase the percentage of cardiomyocytes that go through G2M. This is instead a protein Aurora B which localizes in these elongated structures. I hope you can see them here. These are called mid-bodies and this is the final step of cardiokinase. When a cell divides two daughter cells divide and they are connected for a limited amount of time by a sort of tube where this protein accumulates. These are called mid-bodies. So seeing increasing mid-bodies means that there is really division of cells. And what's most important is you see that for example this is one of the most proliferative microRNA. The plate is filled by cardiomyocytes after six days where they control cardiomyocytes stop dividing. So that was very, very exciting. It was even more exciting that this work in vivo also in neonatal cardiomyocytes and these are neonatal hearts. This is the control. This is treated with a microRNA from C. elegansum so which should not have find no targets in mammals. You see that this is a left ventricle. This is a longitudinal session of the heart. Left ventricle, right ventricle. And here cardiomyocytes are in green and replicating cells are in red. You see that this is the ordinary vein. So the big vessels have a lot of replicating cells. These are two hearts injected with two of the most effective microRNAs, 590 or 199A. You see immediately that the hearts are bigger and that the ventricles here so the size of these ventricles is bigger than here. And these are bigger because simply they have many more cells. So you see here that birth replication is very limited in the ventricles so the mass, the contractile mass is limited is a bit more evident to the endocardium which is the endothelial cells lining the center of the ventricle or the epicardium. However if you inject the microRNA you see many replicating cells inside the ventricles themselves. So this is what is called in pathology hyperplasty. So you increase the number of cells in an organ if you look at my higher resolution you see that there are a lot of cardiomyocytes with a nucleus that has incorporated BRDU and they are happily integrated with the muscle fibers. Of these two this is up-regulated in the neonates and the embryo and down-regulated in the adult so basically we are restoring the level that were before. This is never expressed in the heart. So this is a microRNA that does not belong to the heart because the screening obviously didn't take into account what is expressed, what is not expressed. We wanted a therapeutic agent for proliferation and this is not expressed in the heart but probably what it does is to impart a program that is not the same as the one in embryogenesis. And so some of these in general are physiological and some of these are therapeutic. These are neonates and you simply open up the chest, injecting the heart close the chest, wait a few days and sacrifice the animals and do the sections. This was where experiment started in 2011, so five years ago and this was the only library that was available. So this is a library from Dar McConn it's a commercial library of synthetic microRNAs it was the compilation of microRNAs according to the knowledge five years ago. Now we know that we have almost double these and we are we have also screens for all the 2,000 microRNAs of human origin and there are additional microRNAs that do the job. Simply because we put thresholds to decide that we wanted to work on this the threshold was very simple, so we wanted those that increase at least two-fold proliferation in both human mouths and rat. This was a screening of human sequences in the rat but we wanted a universal reagent that worked also in mice and also in human cells and so this is why we shortlisted them to 40. Yeah, you will see a lot of functional data now. Yes, yes, yes. Obviously, if you want to have a perspective, a translation perspective, you want these microRNAs to be expressed for a long period of time and so a single shot injection and then neonate I mean it goes to our favor because this is a condition when still cardiomyocytes have a capacity to proliferate and the assumption at that moment was that if you inject double-stranded RNA this would last very little, so even our cells were surprised to see such a big effect. But obviously if you want to have a prolonged expression you have to I mean you have to use a system that permits endogenous expression of these microRNAs and the way that you can express genes in the cardiovascular system is by using a vector, a viral vector system based on a virus that is called adenossociated virus. So this is the best and in my opinion even the only way of expressing genes for a prolonged period of time and very high efficiency in the heart. So this AAV has a small virus that is broadly diffused in the population so almost 90% of us have antibodies against this virus and nevertheless we don't have any disease associated with this infection so probably this is a virus that circulates in the population during our infancy we all get infected and then it remains as a commensal in the human population. It is a very small virus when I say small it means that it has a diameter of approximately 20 nanometers and it consists of a small genome which is single-stranded with the exception of these two herpes surrounded by 60 proteins that come from the same gene. So basically when I describe this in nanotechnology I describe this as a perfect nanobiotechnology particle so 20 nanometers 60 proteins and one nucleic acid surrounding it. And genetically this is very simple so it has only two genes which are called repeccate completely remove these genes and substitute them with any genes you are interested in provided it is expressed from a promoter it has a polydenylation site. The remains of the virus is only these two herpes which are 146 nucleotide long the two extremities they don't code for any proteins so the virus once the virus is inside the cell it is completely unseen and transparent to the immune system it doesn't cause inflammation and for some very interesting reasons that are pertinent to the biology of this virus to transfer genes inside cells in the laboratory it doesn't work the worst system you can use. However if you inject it directly in vivo you will find that the virus goes to specific cell types for example if you inject it in the muscle it goes to the skeletal muscle fibers if you inject it in the heart it goes to the heart but in the heart or in the skeletal muscle muscle fibers or cardiomyocytes but not for example to endotelial cells not into fibroblasts in these organs if you inject it in the brain it goes to neurons but not to the glial cells if you inject it in the retina it goes basically to all cells in the retina so basically it has a specific tropism for post mitotic cells so cells that have exceeded the cell cycle and never replicate which is fantastic because yesterday the problem of regenerative medicine and the big problem of medicine in general is that we have cells in our body that don't replicate so they don't regenerate and these are the cells in the heart in the heart, our neurons in the brain our beta cells in the pancreas our retina cells and this vector is perfect exactly to transfer genes into this cell so a perfect vehicle if you want we can discuss that we understand why there is this specific tropism and there are some variations of these surface proteins of these 60 proteins that are on the coat of the virus that amino acid variations that auto-permits the virus to circulate and go to these organs for example this defines a serotype that's called number 9 this AV9 can be injected intraperitone intravenously it goes in the circulation in the muscle and it stays there forever because these cells don't divide and so they survive as the animal survives so the virus is there we have some experiments performing dogs 5, 6 years ago in which the virus has survived all throughout these years without causing any kind of disease and the system is very efficient this is a virus expressing fluorescent protein injecting injected IP and you see the extent and specifically into into cardiomyocytes and obviously we the first thing that we did was to adapt this virus to have as a gene the gene coding for the microRNA so basically this is the same very simple so you put the microRNA gene a promoter which in our case was constitutive promoter a polyethylation site and then these transcripts produced are then processed through the RNA processing machinery for microRNAs and in this case the heart were much bigger even microscopically so we could wait for example 12 days from injection this again neonates and you see that immediately you see these huge hearts in the animals if you measure function of these hearts by standard functional measurements echocardiography then you see no difference from normal hearts I still remember the moment when the postdoctoral disease experiment came to my office with two tubes in one there was a normal heart in another big heart and she said guess which one has microRNA which one is the control but this heart is pretty enormous taking the heart from the chest of a bigger animal and putting this into a smaller animal and what was more interesting was that when we injected these AV vectors immediately after myocardial infarction basically they completely helped cardiac function this is a measurement of this parameter I mentioned before ejection fraction so this is a percentage of blood pumped out every heart beat normal conditions in my city is this dotted line so almost 60% of the heart is pumped out if you do myocardial infarction after 12 days this is significantly reducing white and then it goes progressively down in two months this is heart failure in mice basically if you give an AV vector expressing the microRNAs the parameters remain at the basal level this is another parameter called fractional shortening so how much the heart wall can contract you see almost preservation to normal levels this is left ventricular anterior wall thickness so how big is the ventricular wall when the heart contracts again complete preservation what's even more interesting is that after two months when we sacrificed these animals in control animals there were huge scars here this is a transversal section of the heart so this is the left ventricle the right ventricle and the scar here is in blue the muscle is in red you see that this part of the ventricle has been completely substituted by a huge scar a human would never be able to live with such a big scar rodents do but the animal that received this microRNA 590 or 199A then the scar is much smaller and you can measure this also there is a very significant difference so one question that we pose ourselves after these the results was which are the cells on which this you might say it worked because these experiments were published in 2012 and this was the time when a lot of people in the world it's not a long time ago but the paradigm has really shifted over the last two or three years a lot of people believe that there are stem cells in the heart that regenerate the heart so we did an experiment that in genetics is called the fate mapping experiment so basically we wanted to see which is the cell type that replicates after we give the microRNA is it that already exist that are pushed to proliferate or is it another cell type that comes there and then proliferates because of the microRNA and this experiment was performed in this manner we had a mouse that contains the clear recombinase which is sensitive to tamoxifen which is activated by tamoxifen under the control of a promoter which is expressed only in adult cardiomyocytes in differentiated cardiomyocytes the alpha myosin heavy chain promoter and we crossed these with mice which contain the target sequences of this recombinase so the loxpecite to flank the GFP protein so basically these mice are all dark but if Cree is activated in some organ then the cells of that organ become green and the organ in which is activated Cree is an organ that expresses the alpha myosin heavy chain promoter so basically this is a way to have the cells in the heart, the cardiomyocytes in the heart of these animals becoming green and only those cells and in fact after seven days we give tamoxifen to activate the recombinase 85% of the cardiomyocytes in the heart becomes green and these are normal animals with green cardiomyocytes in the heart then at that point these animals will ligate the coronary artery so we induce an infarction injecting the AV vector expressing the microRNA in the presence of bromozoceuridine or EDU this is another analog that we use sometimes and then the question is are the cells that start incorporating this so that they replicate green it means that the pre-existing cardiomyocytes were pushed to proliferate if they are black it means that the cardiomyocytes the cells that proliferate comes from another source so the blood, the circulation other cells in the heart fibroblasts whoever knows the answer was that all the cells that become green had a red nuclear were also green before you see these are cardiomyocytes with the red nucleus and it's clearly these nuclei belong to pre-existing cardiomyocytes here there are some pictures you see these big cardiomyocytes that have been pushed by this microRNA or this microRNA to proliferate so clearly it seems that this microRNA stimulates pre-existing cardiomyocytes to proliferate so we became more say brave and started to ask the question well let's see if they can push proliferation also of those cells that nobody has seen to proliferate and so basically we took this big cardiomyocytes that you have seen before we added some serum to have some source of growth factors nothing happens and then at that time we transfected the microRNAs and this you see that all these microRNAs with one exception all these microRNAs push proliferation of these big cardiomyocytes you see that the nuclei starts incorporating BRDU and they express marker of S phase this is the control and these are big cardiomyocytes these monster cells start entering the S phase they also go through G2 mitosis this is staining for this phosphorylite E3 you see these big cardiomyocytes in G2M some of these you also see this mitotic condensation of chromosomes start dividing so you have these big cells starting to divide which is very very remarkable because this is the first time that we can really see adult cardiomyocytes that dividing response to these cells and so basically this is surprising for mammals and has never been seen before let me skip this it's not relevant but it is not surprising for biology in general because this is exactly what happens and you've seen that with Campos in zebrafish or in salamander when you have regeneration of the heart in these animals what you have is that existing cardiomyocytes go a little back in their differentiation process so they start disassembling the contralaparatus, the sarcomen dividing until regeneration occurs so what we believe that we are doing here with these microRNAs is simply to go back to a program in which there is the same program as physiologically zebrafish and the salamander keep for the rest of their life and then for some reasons we have lost mammals immediately immediately after birth then one can ask is it a specific effect of these microRNAs for cardiomyocytes or it is a proper liferative effect that is exerted in all cell types and here I'm going to answer indirectly this question showing you another of the screen that we do we love biology made of screenings so we think we are in an era in which we can do screens instead of doing candidate gene studies you know the molecular biology and genetics and started 40 years ago studying candidate genes so the first genes that was studied in detail were the genes coding for globins so hemoglobin and it's component this is a targeting approach and that has been the basis of all studies for 30 years and the people had their own candidate gene they studied all the gene how it function how it interacts with other proteins how individual mutation of amino acids can lead to change in this function and then in most cases you study your favorite genes and at the end of your paper you ask well perhaps it has a function in vivo so you do a few mouse experiments and then at the end of the story I show a paper in which figure 5 and figure 6 has the results and say now I've studied these genes very interesting these are the mutations and it works also I confirm this in vivo now if you ask yourself and this has generated a tremendous amount of biochemical and biological information but if you ask yourself how many of these proteins are relevant for function then very few and how many of these proteins that have been studied in this way for example are therapeutic proteins less so for example genes that are involved proteins that are involved in repairing the myocardium you can have a list of 50 different proteins in the literature of the last 30 years if you ask how many of these have become therapeutics the answer is 0 none because obviously by studying one candidate gene you cannot judge the relevance of these genes in the more broad context is this the best gene you have to study nobody knows you try to prove this in figure 5 or figure 6 of this experiment this was the first wave of studies biochemical study which was followed by the second wave of studies in which people started doing omics approaches omics approaches means that you take a picture you take a picture of what is changing from normal and pathology or normal conditions and treatment you can take a picture of the metabolome a picture of the transcritome a picture of the myronome a picture of the interactome a picture of the lipidome whatever yeah just pictures at the end of the pictures you end up with big excel files that are very difficult to study we know this very well so it's very difficult to understand inside these changes what is the real set of genes or individual genes that could be a trigger how many drugs or how many treatments are you aware of that came from omics studies I don't know anyone I know several studies that gives you a broad picture of what's going on but if you want to find a trigger that can be used therapeutically this is probably not the way to go now however we have a complete information on the genome complete information on the transcript myronome and we have robotic stations to study directly function so why don't doing as we like to do a biology discovery path which is based on screening for function first so for example we are interested in cardiomyocidal proliferation we screen for function of microRNAs and then for those who have a function for example the stimuli proliferation we study how they work so we go we are sure first that these are the best ones and they are effective and then we try to understand how they work I think that this is a much more straightforward pathway for biological discovery now so this is just to say that we do a lot of screenings in the laboratory one of the screenings that we did was a game for proliferation but was for proliferation in another completely different setting was proliferation of senescent fibroblasts so yesterday we spoke a bit about senescence and a part of senescence which is still not clear how does it refer to real aging in vivo but is cellular senescence so if you take fibroblasts from an individual you can culture them and then at a certain point they stop dividing and they stop dividing depending on the age of the individual from which you have taken them if you take fibroblasts from a neonate they stop dividing after 50-60 passages you take fibroblasts from an aging person for a person of 70 years or they stop dividing after 2-3 passages this number of passages is called the Hayflick number under the name of the person who discovered this event so the idea here was to see if we can screen the same library for microRNA that permit proliferation of senescent human fibroblasts basically we push cells to senescence senescent cells express not a marker of senescence like being enlarged and expressing an enzyme which is called senescent associated beta-galactosidase and then we search whether there are some microRNAs that push these cells to proliferate I don't enter into the detail of this screening these are the results so basically we found that if you see one it means that the microRNA has no effect we saw a lot of microRNA that push cells to senescence although they are below one but also we found a certain number of microRNAs, 39 microRNAs that push the cells to proliferate some of these are very very interesting because some of these they don't even they don't even require serum this is the most remarkable experiment I've seen in the last year that is this is a correlation of the effect of some selected microRNAs in the presence of 10% serum as the normal culture conditions and the complete absence of serum so for example you see that this microRNA pushes proliferation of cells that are senescence either with serum or without serum so think of a senescent cell put without serum you put a microRNA and this boom starts proliferating we have probably bypassed all the requirements for stimulation of growth factors with receptors on the surface we don't have an idea on how this microRNA is working this is something that we are studying yes no we can push them to proliferate for a few rounds but if we keep expressing this microRNA for example with the lentiviral vectors then they die by apoptosis so we push them dividing for a few cycles but we don't realize them which by the way for therapeutic purposes is even better this experiment were inspired by my personal desire of finding something that could push rejuvenation so this is a sign that I'm getting old and so this becomes one of the the big goals of research well I'm showing this because there are some microRNAs that we selected for in this screen that are exactly identical to the microRNA that we selected for in the cardiomyocytes screen for example there is one family that turned out all these family members they have the same seed sequence so the sequence that makes pairing with the targeted marinades and they all show up to increase proliferation of both cardiomyocytes and senescent cells it's called the 302-367 cluster you see these are the results for the cardiomyocytes you see that this cluster basically all the members turned out to be positive and this is a very famous family of microRNAs because it is absolutely required for keeping stem cell identity so basically an embryonic stem cell remains an embryonic stem cell until this family of microRNAs is expressed if you block the expression they start differentiating not only but if you force the expression of these microRNAs in other cell types you make this cell type becoming an embryonic stem cell in fact this is a paper published by the Morrissey laboratory last year, March last year in which he showed that this microRNA family if you make a transgenic mouse expressing this in the heart you have the cardiomyocytes keeping proliferating they also are not more cardiomyocytes they become an embryonic stem like cells and eventually the animal die so basically proliferation is not only a goal but the only goal but should be proliferation coupled with the permanence of the cell in an embryonic stem in a cardiomyocytes state another family that turned out is this 1792 family this is also a family which is known in the literature in the tumor literature because it's called the Oncomir-1 it was discovered as a series of microRNA that push cells to cancer cells to proliferate again this is another problem because if you if you want proliferation in the heart the least thing that you would want is that the same microRNA also push proliferation of tumor cells in the patients please in our case we work with one microRNA at a time so our goal is to find a biotherapeutic so something that we can inject in a patient with myocardial infarction in physiological conditions there are experiments in which it seems that for example with the three or two family there is no redundancy so if you block one member the other members don't compensate because they have slightly different targets despite sharing the same sequence in general terms however if you may plot a correlation between proliferation of cardiomyocytes and proliferation of senescent fibulas there is a very little proliferation and so we are very confident for example those we concentrate on like 590 1822 and 199 they work only in cardiomyocytes which is exactly what we want so something that is more specific for cardiomyocytes and all those that we selected don't push experimentally proliferation of fibroblasts so this is proliferation of cardiomyocytes this is proliferation of fibroblasts these are the top ones and you see that they don't work in fibroblasts basically we know something more now on how they work and most of them work through the activation of a pathway which again I think you are familiar with which is the hippo pathway so basically this is a main pathway which is involved in transduction of signals of mechanical signals of the cells and the pathway in a pathway has an activator which is called YAP-1 which is a transcriptional co-activator that when it is in the nucleus it drives the expression of proper referative genes but this activator can be deactivated by phosphorylation and there is a cascade of kinases in the cytoplasm that keeps this activator phosphorylated and inactivated in the cytoplasm we made an experiment by which we had a reporter-promoter sensitive to the presence of YAP and transfected this reporter into cardiomyocytes and then treated these cardiomyocytes with the proper referative mirror so this is the effect of proliferation of a series of mirror nas in this experiment and these are the levels of activation of this promoter you see that with a lot of differences from a microRNA to microRNA but basically most of them activate this YAP-mediated co-activation not only but if simultaneously we give the microRNA the proliferation but simultaneously we give an SI-RNA so we block YAP then we block this proliferation so it means that this is the pathway that is common to most of these microRNAs we now also know which are the targets at least for one of these this is called 199A3P which is the one we concentrate our attention on and one of the direct targets of this microRNA is the kinase called tau K1 which is upstream of the kinase cascade another target is beta TRCP beta TRCP is a ubiquitin ligase that drives the phosphorylity YAP for deactivation so if you block this you have more YAP and a third target is a cofilling 2 which is a protein which is involved in actin polymerization so if you have cell proliferation you must have actin polymerization this is work that we are carrying out in collaboration with Matteo and Ryan who are here I don't want to go into much detail on that at this moment but we think that this is the effect so basically what we are doing is it impacting on a physiological pathway for cardiomyocel proliferation I want instead to end up just two small stories that are very important in my view in terms of translation one is related to the possibility of having this working at the clinical level now you know the mouse heart is this big and so regenerating a few millions of cardiomyocytes in the mouse heart can be easy but regenerating billions of cardiomyocytes in a human heart can be much more much more difficult now the pig heart has more or less the size of humans and it has the same anatomy and physiology so this is usually in the cardiovascular field consider a very good model to test preclinical application before going to clinical trials and so we set up a system in which we have this is our collaboration with the group in PISA in which we have farm pigs in which we ligate the coronary artery in infarction and then in the infarct border zone we have ten injections of a navy vector expressing 199A so this one on which we concentrated our attention on this because it is the sequence is exactly conserved in all the species that are known in the microRNA databases and the results are really stunning in terms of infarct size this is how infarct size goes in the first two months after infarction so in day two the infarct is more or less the same but if you look at after one week or after four weeks you see that the animals treated with the microRNA have a much bigger smaller infarct size if you look at ejection fraction against more or less the same at day two by the week one those treated with the microRNA have a very significant increase in ejection fraction and what's most remarkable is that we follow these animals by magnetic resonance image is the best way to follow the function and anatomy of the heart in a very objective objective manner so these are two animals one treated with the control and one treated with this microRNA and each animal is seen at week one, week four and week eight and these are serious sections from the apex of the heart up progressively to the base of the heart this is the left ventricle and this is the right ventricle so if you see a week one in the control for example take this middle session, this middle session here you see that there is a big infarct here the infarct is contrasting red for better visualization you have a big infarct which is in the septu and the free wall of the left ventricle and basically this is an infarct that progressively over two weeks becomes a scar and you see the scar is very big the ventricle has dilated the walls are much more thin however if you look at the animal treated with 199 A at week one there is no big difference however week four you see that the scar has reduced a lot and week eight there is really very very little close up you see that there is very little scar you see the difference from here to here here it has almost disappeared this is a very reminiscent of regeneration I have also a movie which is very telling so this is a heart infarcted and treated with the control you see an almost dilation of the left ventricle thinning of the septum and the free wall of the ventricle this heart is severely dyskinetic, dysfunctional and if you look at instead this is a heart that was injected with the navy vector expressing the microRNA and you see that it pumps much much better it has not dilated there is much more contractile tissue there so this is a really very very exciting view of translation I show you the results with 199 A and now in the mouse the the problem with the navy vector that I have is that this is very fantastic and I keep receiving I keep receiving emails after we publish this data of people saying well the typical email I receive is that I'm a 42 year old person I've been an athletic person all throughout my life I used to practice to run three times a week I have a very good I'm very fit I take much care of my my weight and the food the food I take however, six months ago suddenly I had a myocardial infarction I was immediately rivascularized but my ejection fraction now is 35% has been stable over the last two months my doctor says there is nothing that we can do for that can I please come there you inject your microRNA in my heart this is a typical because this is a typical situation you have these even young people who have nothing to do basically except trying to preserve the portion of the heart that they still have now even in this condition I would be very scary of injecting an navy vector that spreads a proper riferative microRNA because first I'm not sure what could happen after eight months after one year after five years and second I don't know if this vector has spread in certainly some spreads and I don't know if after one year there could be a tumor that has kept growing and so on so there are safety concerns on this so the best idea would be to find a way of injecting these microRNAs as simple with carriers that allow them to remain there for a very short time so basically what we started doing in the laboratory by now at the mouse level was to see if we can find some lipid formulation that permits us to do a single injection in the heart the moment of myocardial infarction and we found one which is based on the perfect, I mean by which these are the levels normal levels of microRNA 199 in the heart they don't change with myocardial infarction first and later they remain very very low if you inject a naked microRNA with these lipids then this concentration increases more than 200 times at day two but what is nice is that it remains there sufficiently high for at least 12 days and not only it is there for 12 days it's higher than endogenous but it's also there in an active form because we know the direct target so these microRNAs like these proteins here Homer click five and we see that they are down-regulated some of these up to 12 days so it means that a single bolus injection would be sufficient to have this microRNA remaining there the question is, is it sufficient also for function and in the mouse it seems so these are mice hearts left ventricle, right ventricle big scars in the controls if you inject this naked RNA molecule or this naked RNA molecule you see that these big scars are much much smaller so they are very significantly smaller if you look at functional parameter ejection fraction, fractional shortening they are all preserved so it seems that a single injection of this microRNA is able to boost sufficient proliferation at the beginning that keeps the heart functioning even if you look after eight weeks from the injection the picture that you see are these these are the needle track so where we have injected the needle and you can see these are probably inflammatory cell that pile up and these are small cardiomyocytes that are formed after proliferation so these cardiomyocytes are with the BRGU positive nucleus this is another picture which is very nice, you see they are smaller than the other because they have newly formed but they are there and there are many of these around so we believe that it is clinical scenario in which we have a patient and this patient has a myocardial infarction it is resuscitated, it survives there is no arrhythmia, it is brought to the hospital it is revascularized after three, four days from revascularization then scarring the position of collagen, fibrolysis and start to occur and just before that the cardiology goes back to the heart to the coronary artery of this patient inject a bolus of this microRNA the microRNA goes into the survived cardiomyocytes push them to proliferate and regenerate the position of the myocardium this has the advantage also that in clinical terms you don't have to have 100% regeneration but even if you have 10%, 20% regenerated tissue in clinical terms for the outcome of this patient after 10 years from the infarction this makes a dramatic difference so we are convinced that this should be a path that we can follow to go to the clinics whether we will be able to have these as drugs obviously in terms of applicability this is much better than apiastasis much better than stem cells because for stem cells you have to have these cells expanding the laboratory to be injected and here instead you can put these microRNAs as sort of drugs in vials and any interventional cardiology can inject them so these are just drugs to be injected apart from that I think that there is a sort of paradigm shift from this kind of evidence that is not necessarily we have to rely on stem cells to regenerate the heart or exogenous cells but probably heart regeneration can be achieved by stimulation of endogenous cardiomassile proliferation obviously microRNAs are a proof of principle that this can happen, can become drugs but there are equally interesting possibilities in trying to find growth factors that do this autonomously or in conjunction with microRNA and this is also what we have been doing that's basically all what I wanted to tell you and thank you for your attention if you have questions I'm very pleased to try to answer