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| 1 | ARDD | Jean-Marc Lemaitre at ARDD2022: Developing cell reprogramming-based st... | 856 | 31 | 37.1 | 22:30 | I'm ready to start the final session of the evening. And this is Sean Mark Limetra, who will tell us about reprogramming, I think. First, I have to thank Martin and Alex for this invitation, and also for the very, this very wonderful meeting. So for a million in my lab, we are interested in the cell plasticity, in development and aging. And we developed some reprogramming strategy to try to fight aging. And everybody now beneficiate of this famous hallmark of aging, initially nine, but now other. And what we know is that we can follow aging with this hallmark, and for sure they are involved in the root of the age-related pathology. In the lab, we focused on cellular senescence and epigenetic alteration. Why? Because simply because of my simple vision of what occurs in the cell when they stress. Cell experience during their life, many different stress, and stress generate damage. The cell has to repair the damage. And if she can repair the damage, she is able to continue to do the job. But we know that there is some hallmark, and we can observe what we call an epigenetic drift, with transcription, modification, and in per cell plasticity. If not, if she is not able to repair, we know that she can trigger a popposis, or senescence with this specific SASP, very delicious for the cell. So, all the on, we ask the question, is cell aging reversible? We decided to start from scratch. And we benefited from this famous discovery of Yamanaka with the four factors. He showed that we were able to convert an adult cells skin fibroblasts initially into a pre-epoton stem cell, raising the cell identity, and then a differentiation of these cells in any kind of cells opened the door to the cell therapy. So, we decided to use this cocktail, and we started from H-cell, and senescentral, the two types of cell we would guess to be present in tissue with the H, and we tried to reprogram then, and it didn't work at all with this cocktail. And some years after, six papers published, and one from Professor Yamanaka too, saying that celerars and senescence and aging are break and barrier to reprogram. So, because we were resilient, we have to be in aging, and we decided to try another cocktail, published the same year by Jemitom Son, replacing California's semi-inorganic and Lint-28 with the semi-efficiency of reprogramming on the other cell, but it didn't work at all. And we decided to mix the factors, and with the six factors, we were able to derive IPS efficiently from senescentral and from satanarian cells. And when we really differentiate them, we observe the fully-rejuvenated physiology, we reset the gene expression profile, we really don't get the telomer, we reset the pro-ephiative capacity, even from senescent cell, metocorea, physiology, versus reset, and the cells regain the ability to go back into replicative senescence. Even more, we compare the efficiency of reprogramming with six and four factors, and we observe that we are more close to human numbering stem cell with the six and with the four of Yamanaka. So then we explore the opportunity of this amazing technology. And with this technology, for sure, we can generate IPS with ascent with disease, and we develop IPS with premature gene syndrome, right? And also, for example, mesenchymol stem cell from osteoarthritis passion, and then to model the pathology after redefinition, and to retapitulate the pathology to eventually screen for molecules to prevent the disorder. And we were also involved in a cell therapy proof of concept from the degenerative and interpretable disk, and to make the proof of concept of the user of IPS to differentiate into progenitor of the cartilage and to be able to regenerate the interpretable disk. But in parallel, we have another question. Is it necessary to go to pre-potency to regenerate an aging cell? And we decided to develop in vitro and in vivo model to show this, and we developed mouse model. One reprogramming mouse model with one allyl of the reprogramming cassette, and another model with the progenitoring, the one copy of the gene forming the progenitoring. So initially, we start from skin fibres from this model, and contrary to what was shown before with the reprogramming into IPS, we decided to induce the expression of gene short time, short period of time, and we observed that at this time we decreased the DNA damage contrary to what was observed with the fully reprogramming process that generates stress. And we also decreased the senescence, we also stimulate diotophagy, but we did not switch the metabolism as it was described for the fully reprogramming into IPS. Then we decided to uneranged the expression by RNA sec, with these two models induced into OSCAN expression, and we identified 395 genes in common, which was unfortunately expressed by the induction of the four factors, early on. But what was really interesting is that we start with fibres, so effectively when we look at gene pathway trigger, we see one dealing, and epidermis development, for example, but interestingly we see also kidney development, we also stabilize differentiation, so many different functions of different tissue. So we decided to look at what occur in vivo in this model. And initially we tried to reproduce the publication of Ocampo in 2016 from the lab of Jose Pebel Montet, and we use similar mouse model, but no more ZIGOS project, so we are only living five or six months, and we use a NETEO ZIGOS project, so we are living two times more and more close to physiological aging. And we use this chronic induction, so today is the week during the life of the animal, and then we observe that similar increase in lifespan of our model, if we only induce today to this week during the life of the animal. Then because today's a week with one milligram per meal of dogs was highly expressing the cassette, we decided to decrease the level of induction, but not chronically, but during all the life of the animal continuously. And we observe similar effect on the lifespan extension. Then we would like to know what occur with only one reprogramming period, so we decided to trigger reprogramming during 2.5 weeks, but only in the life of the animal to see what occur. We zero point to milligram per meal of dogs, nothing occur, but if we increase a little bit at 0.5, we observe that there is an increase of lifespan of the animal in old age. So we induce at 2 months, and we have an increase in the life in the life. So we decided to look at what occur at 8 months, close to the end of the life, but what we observe, what was absolutely amazing, that we change immediately the body composition of the animal and during all the life. So in fact we maintain the muscle mass, and we decrease the increase of fat with the age, but during all the life of the animal and only for short induction early in the life. And this modification of the body composition is associated to an improvement of fitness as a measure by this rot out test and the middle-wing time on the wheel and also the grip test, the increase the fitness, the strength of the muscle mass. Then we decided to look at age-related pathology, and we started with the osteoarthritis, and what we observed in that treatment early in the life, 2.5 weeks, at 8 months of the animal we see that there is a decrease of fat degradation. And the connection with the sub-chondral bone was also involved, and we observed that there is an increase of bone volume and also bone thickness of this part of the knee. And if we look at osteoporosis on the TBR cortical region, we have also an increase of the bone volume and due to an increase of bone mineral density on the cortical regions. Then we look at the structure of the tissue, of different tissue. We observed that, for example, in the kidney, there is an improvement of the microarchitecture of the kidney. When we ate the mice age, there is an increase in size of the glomeruli, and also an increase of fractional mesangular array, but we observed that with the early induction, we preserved the structure of the glomeruli and the surface array. Similarly, in the spline, with the edge, there is a sort of distortion of the surface between the two parts, the white part and the red part. This is the marginal zone, and it's possible to evaluate this and to use a score, and we observed that we maintain the distortion, we preserve the distortion of the separation between the two parts. And I like to say yesterday that fibrosis is aging, so we looked at fibrosis, and the early treatment, prevent fibrosis in kidney, in spline, and also in lung, and there is a tent for the liver and the earth. But the last tissue, and not the least, the skin, and it's absolutely amazing because you can observe this is the same magnification. So definitely, we observe that all the layers of the skin are improved, late in life, with the early treatment of reprogramming. And even more, if we share the mice, there is an improvement of the air recovery by an early treatment of 2.5 weeks. So because of the distal effect, we guess that probably epigenetic mechanism has to be involved. And what we decided to do, not to develop clock, because we were on the progeria model, and for other reasons that we will find out later. We start to identify this epigenetic drift by a metronome analysis on Illumina Bids, with the 250,000 CPG reference. And we identified differential methylation site related to aging that correspond to this epigenetic drift. And then among these sites, we look at what is differentially, what sites are differentially methylated early on after the treatment, and the consequence at 8 months. And what we observe is that at 8 months, when we treat early on, there is differential methylation site among the aging differentiated methylated sites, but it's specific for each organ. So you understand it's complicated to have a clock in this condition, but anyway, what was absolutely amazing is that almost 100% of this site differentiated at 8 months, among the after the treatment, were reversed in a methylation. The methylation status is the same after the induction. But what was more amazing is when we compare to the site targeted early on, the methylation targeted early on, there is no overlap. So it means that the reprogramming initiate an epigenetic mechanism, but this on methylation site, but it's not maintained, it just propagated. And at the end, the site differentiated at 8 months, they recover a yogurt for epigenetic information. So finally, we observe that this early induction of reprogramming is organ specific, and it's initiated by transient OSP-CAM expression, for sure, to 0.5 week at 0.5. It's not maintained, but propagated. It's restored your full epigenetic information on the aging DNS, the animated site, but it also increased longevity, it improved body composition during all the life of the animal. It also improved the tissue structure, it prevent fibrosis, and also some age-related disease. So we believe that it's a new opportunity, probably to treat and to prevent aging. So this landslide to acknowledge all the people of the lab, especially Olivier Millevais, Contain Allé, Nora Leborn, Paul Benzadoul, really involving this project and other who left the lab, and also involving this project and also all the platform and the financial support we have. We also put the position to work on reprogramming. So thank you. Thank you so much, Jean-Marc. We have a couple questions. Thank you very much. Very nice presentation. You showed this progerian model. Do you have any data on the world top? And also how can you separate what is from the partial reprogramming and what may be an effect from the toxic high clean itself? Yes, when we do with the toxic clean itself, there is no effect. And you can see also with the modification of the metabolism we observe this morning with this toxic clean. We see that on mitochondria we have no modification, no change at all. And we also did experiment on the world type mice. And this is the result. We know that there is also an increase in lifespan. And this mice live to time longer, but we also treat at two months of the life of the animal and we observe an effect late in the life. Very cool. Thank you. Thank you. Your docs have taken effect on every cell. So my question I guess then is what do you contribute the effects on? Is it on an cellular level of every cell being reprogrammed to a level? But on the other hand, your new data on the opportunity clocks maybe is we are suggesting that it will be an effect on some type of a progenitor or stem cell pool that then propagates further down with time. How do you see this? For sure, we we the past is present in all the cells when we program program typically all the cell, but for sure we don't know if there is some cells with more efficient action on the tissue. But we didn't develop single cell analysis so each time we look at global on the tissue, but we observe that there is a modification in microarchic architecture and so on. So we will exother is a global tissue effect. Thank you so much. Thank you. And... | ↗ |