BIOENGINEERING OF THE GUT: THE ROLE OF ECM

Uh, welcome everyone, and, uh, uh, good morning, uh, on this side uh of the pond and uh good afternoon, uh, to, uh, Professor Pablo del Copi and good morning to, uh, uh, Professor, um, David Hackham, uh, from Hopkins, uh. Um, this is, uh, uh, the second, uh, webinar on basic science that, that we, we are doing now, uh, for YUSA. We've been, uh, running a series of, uh, um, webinars, uh, with, uh, more or less. Uh, once a month on, uh, mainly clinical topics, uh, and, uh, now, uh, we dive into basic science. So we had a very successful one on CDH and I'm sure this one will will be absolutely amazing. Um, the, the, the, today we're gonna talk about bioengineering of the gut, and we have two giants that are, are joining us. Um, my co-host, uh, Martin Lage is, uh, going to join us soon, so, um, but without any further ado, uh, I'm gonna introduce, uh, Paulo De Copi, who doesn't really need, uh, much of an introduction, uh, is a, uh, a friend and a colleague, uh, and for us in UPSA. He's actually just been elected as the incoming president, so that I would say is the biggest thing on his bio present for us. Paolo is the Nuffield chair of pediatric surgery and NIHR professor of pediatric surgery and consultant pediatric surgeon at Great Ormond Street Hospital in London, UK. Is the head of the surgery unit, stem Cell and regenerative medicine section, Developmental Biology and Cancer Program at UCL Great Omon Street Institute of Child Health. Um, Paolo, uh, comes from Italy, like myself, I'm from Padua. Um, he, um, got his medical degree in Padua University, but he spent time in, uh, uh, Boston, uh, uh, working with, uh, Anthony Atala and, uh, publishing a seminal paper on the, um, identification of, uh, amniotic fluid stem cells that was, uh, uh, published on Nature Biotechnology, but even more I would say on Metro magazine. I remember that day and Uh, it, it was very well publicized, uh, uh, worldwide. Paulo has more than, uh, has, has supervised more than 35 research fellows and PhD students, and, uh, he has been awarded various, various national and international grants, um, in excess of 30 million pounds, British pounds, so, uh, you can do your maths with and, and convert that to euros. Um, so his main interest is, uh, tissue engineering of the esophagus, and so, uh, Paulo, over to you. Tell us what you're doing, uh, in your lab at the moment, uh, and, and what the challenges have been, and, uh, uh, and, um, tell us all about your, your cutting edge research. Thank you very much, um, uh, Gusteau. Thank you, Professor Zanni and Professor Laque for the kind invitation to this, uh, webinar that, uh, uh, becoming more and more popular also, uh, among residents and, and registered. So, um, uh, I think this will be an opportunity hopefully to inspire some more registered to, um, to do research in this field. And uh I think uh Professor Lacker is connecting, which is great, uh, perfect timing, and uh uh he gave me a, a great pleasure to share this session with Professor Akam, which I always admire his work and uh uh he's certainly one of the pioneer in the field. So, um, I will, uh, start sharing my, uh, presentation, uh, uh, hopefully you will see it now and, um, uh, I will focus during my My presentation certainly on the role of the extracellular matrix because that's something that is very close to what we do in the lab. Um, I need to declare some conflict of interest, uh, uh, on the amniotic fluid stem cells and also on gels created from the extracellular matrix. So where I'm um talking to you today is actually from this new institute uh uh uh for researching rare disease in children, uh, which has just been opening during the pandemic here at Great Thomas Street Hospital. It's dedicated really to the um discoveries that hopefully one day will be translated on the opposite side of the street to the hospital and we are very fortunate to have, um, uh, this, uh, institute here. Which I know over in the United States and North America, um looks a small building but uh over in Europe it's certainly something that we go very proud of. So when we discuss about approaches for engineering, we, we have learned a lot from something that happened about 11 years ago. Uh, here is Kieran that was born with a congenital trache stenosis, uh, and, uh, at that time, after several operations, uh, he reached the point where a stent was eroding through the trachea into the Aorta and uh at that time uh something different needed to be done. So um Martin Ello that led the cardiothoracic team uh we took a desserized um trachea from a cadaver and uh uh we utilized that with cells derived from curran to transplant. Kieran is now 22 years old and he's doing well, is, uh, uh, an adult, so he's not anymore a gosh, but he, he does just uh follow up with the, uh, with the adult, but he's, he's doing extremely well. So we learned a lot about how to use mattresses, uh, to repair tissue or organs, uh. But we, we needed to do much more if we wanted to do something more complex and one of the issue that we really um come across with the um uh with the engineering is the fact of vascularization. So when you have a tissue or organ, you need to vascularize that and the trachea at that time, as a general surgeon, my only input as a surgeon was to, to wrap uh the omentum around the trachea. Uh, but if you want to, uh, iterate a gut, for example, this is certainly not enough, and that's why we were very fortunate to work together with Shaheen Rafi. Over at Cornell University that um uh is a uh an expert in the field of endothelial cell biology. And uh the idea of Shahin was that why we don't reprogram uh uh these cells uh back to more an embryonic stage and to make a long story short, using ETV2 as a transcription factor, uh, which is not expressed uh uh during the adult life, um, we reprogrammed the cells back to a benzo. Precursors and by doing that we noticed that the cells were able to grow much rapidly to form capillary for much longer in vitro and also to create vascular network. So those vascular networks could be profuse and we could use bioreactor to Um, uh, really demonstrate this, uh, like you see here with particles going from one compartment to the lower compartment through this network. But then, uh, the clue experiments was to take these, uh, endothelial cells and put it in a customized barrier reactor that we build in the lab where the cells can go from one compartment to the other but using human blood. This is human blood running through the uh. Endothelial network and you see there's no leakage. Here, the endothelium is in uh green fluorescence proteins, or GFP and you see the um uh um human blood running through that without forming clotting and without extravasation. So then we thought, well, this is maybe important for engineering and so in, in uh our customized barrier reactor, we try to use Use that to resize the vascular endothelium of a, an intestine. So you see here an intestine with this uh uh mesentery and uh the intestine that gets um repopulated by this endothelium and what we observe is that the endothelium was able to colonize all the peripheral vessels. So this uh overcome one of the main problem on uh regenima. In general in tissue engineering is about the revascularization. So you see here how the endothelium of the mesentery is completely covered by these ETV2 uh cells that get reprogrammed, and this is valid also when you only conditionally reprogram. So the cells are not always expressing ETV2, but they're expressing ETV2 during the repopulation of the organ. And then get switched off. These results doesn't change. So can we jump then to repopulate the old gut? Well, that's very difficult. We, I'm very fortunate to be part of the consortium uh that uh works um in Europe mainly but also across the Atlantic, uh, um, uh, on building uh a small intestine, uh, or trying to build a small intestine with, for children that have a short bowel. And we have learned a lot in the last few years working together, but we learned that it's also very difficult uh to do so. So we have uh been working on for a number of years now trying to uh vislarize intestine, um, and we can create, uh, uh, intestine that deserize but contain still all the structure which is normally. Present uh in the intestine, and you can do that also with human and uh for who is interested, we're very happy to share protocols and so on. To do this in any of the pediatric surgery department that are connected today, we can visualize efficiently also human colon and you see the structure remains. So here is a colon that uh you see the, the crypt, you don't see the villa, of course, in the In the column, but you see the crypt maintain, and we can also this to understand the structure of the column. So uh in Grnobyl, there's an accelerator and we've done some experiments to understand a bit more the three-dimensional structure of the intestine and using this uh type of modality, we can then use mathematical modeling to try to restructure the intestine and actually here is some of the first few attempts that we've done about using the, the cell, small intestine in this case as a model to print out a new um small intestine. So you see here villi and encrypt present in the printed. Of course, we are surgeons, so for us it's pretty much the same, but actually scientists will tell you that there's quite a bit of a difference there. So which material to use to print? Well, this uh should be a material that allowed the organoids, which are really the stem cell of the intestine, to grow and to proliferate and eventually to differentiate. So we say, why don't we use the natural material, so the intestine and here uh is just some intestine, to get visceralized um in uh uh in, in vitro. We understand much more about the ECM now, so we, for example, see that there are cluster. Clustering of ECM. So you can notice here in the C figure that there are cluster of the protein expression of the ECM which is different. I mean, pretty much recapitulate what are the three gems layer, but we identify at least 4 clusters in which we have got. Liver and lung, for example, uh, endoderma organ, um, clustering together and, and some that are ectoderm clustered together as well. So we know that the ECM do not only contain information that are mechanical but probably also the biochemistry of this ECM needs to be investigated further. So we are being able to culture organize, derived from um uh the stomach, from the liver, or from the small intestine quite reliably um in this way. And actually, I want to highlight something that hopefully is coming out soon, which has been led by Giovanni Jobbe in the lab which has been using this type of technology to actually study COVID. So we saw initial and I'm sure many of you have observed that in children we had these GI symptoms in children with COVID, and in fact we are now able to see that there are some subtle differences between pediatric gastric organoids and adult gastric organoids that could actually explain why maybe pediatric is more susceptible to GI infection and COVID. And uh so, but the real aim is actually to grow these organoids to make a mucosa and, and indeed we focus now on the small intestine mucosa because that's what, what is really lost when you have a uh short bowel like the valvulus or in gastroschisis and work that we have done with Vivian. For the past 5 years, it's really focused on trying to grow organoids derived from children with short bowel syndrome to see if we could expand those organoids and maybe one day, for example, substitute the mucosa of the colon, so we don't need to rebuild the old intestine, but we could use the colon which is still there to have a mucosa that is absorbing and secreting. So the first trial of experiments that we did was to prove that we could expand separately duodenum, G-genome, and Aeo organoids and expand those uh uh while maintaining their signature and indeed that was The case. So we then uh focus on the genome organoids and we could specialize them uh to one, some of the characteristic um specific to the genome uh organoids. So we could expand and um And actually differentiate them. And so the final bit that we wanted to do is could we see them in D cell colon and D cell intestine and show there's no difference. And indeed that's what we've done. So this results human small intestine and human colon that was dearized and see that then with geojunal organoids. So, first of all, the D cell, small intestine, and colon do not def. They are much. So in terms of protein, there are very little differences there. Of course, we need to, we know the structure is quite different, but the composition of the matrix is not very different and that's important. So we see that this in again uh customized bioreactors and we were able to actually see that the cells were reaching some degree of specialization and who is interested. You can see more in the published work, but most importantly, we were seeing in the column seated some rudimental villi with newly deposit uh um metrics uh from the organoids that were seeded. So this is quite interesting because it means that hopefully in the future, we will be able to transplant the small intestinal mucosa in the column. So are we ready now to jump to more engineer organs? Well, as uh, uh, Augusto was kindly uh mentioned at the beginning, we are uh very much focused on one part of the gut which is upstream, which is the esophagus. And I'm sure I, I forgot some of the people involved in the work, but certainly, these are some of the, uh, of the key. Uh, people that, uh, have been, uh, really involved in the work that is trying to culture, um, esophageal cells, uh, outside the body and to, um, see them in deserized, um, esophagus. And this, uh, work is in collaboration, uh, with Paolo Bonfanti and at the Crick Institute and Julio Caso at the University of Manchester. So we have shown now for a few years that we can desorlarize the esophagus and we use bioreactor to resorlarize efficiently. We have seen over the years that some key elements are very important, so you need to co-see the fibroblast with the muscle cells. This allows the muscle cells to Migrate in the matrix and actually to differentiate and this is very important and uh uh I'm sure Professor Hakam would agree that actually fiber blast play, play a key role in many of the component components for regeneration. We use them bioreactor to somehow reprogram the cells to know what they need to do. So like contraction, and we can follow them up uh using luciferase. And we can also see that the bioreactor make the esophagus much stronger and thicker. I'm sure many surgeons would, uh, uh, be happier to suture the esophagus on the right than the one on the left that hasn't been conditioned in the bioreactor. So, uh, Paula, our group has, have focused, uh, their work. Also on isolating the epithelium and reliably uh culturing that and then using a preconditioner of the esophagus in the omentum, we can seed in the second stage, the epithelium to seed it. And in fact, the muscle can differentiate quite efficiently and what we have seen, and this is um the last um couple of experiments and I'm gonna show you is that we can implant now in animals. We've started with rabbit and we are now in the pig. Um, and what we can see is that we need to use a stent on the esophagus at the beginning. So you see here is a rabbit. So we know that the vascular supply is important. Here you see a, um, a patch of muscle wrapping, uh, the esophagus. And, uh, um, and then you will see a section that demonstrate how you need to um have a stent that helps uh the initial part of the structure uh of the esophagus. So we see here a CT scan just cutting. Through, um, and, uh, you will see the, uh, stent appearing. These are, uh, uh, PDS essentially stent are called ELA stent. We use it clinically and are absorbable stents so we can, um, uh, leave it there without, um, cutting. So I think uh um uh it's important to recognize the role of stem cells which in the last 5 to 10 years have really changed. We know almost as much as the stem cell of the intestine as the bone marrow nowadays, which is remarkable. There are still unsolved problems, so we are not ready for prime time, but certainly regenerative medicine is a clear a reality. So I think many of the surgeons will be more and more involved in this aspect. So finally, a picture without mask, uh, I couldn't believe it. When we took it, we were allowed outside to take it and uh I, I could spend a long time, uh, thanking each and every one of the member of the lab. Of course, we eat only Italian, so that's why pizza and, um, and uh all the funding body. Uh, I would like to thank also my clinical colleague and thank you very much, uh, for the opportunity. Thank you, Paulo. That was, uh, uh, as usual, uh, an amazing talk with, uh, uh, a lot of uh uh new data also that you were very generous to share with us, so it's, uh, it's really interesting. Uh, I see that Martin has, uh, uh, joined us, uh, and just before, uh, I, I, uh, leave you the microphone to introduce Professor Akam, I want to say that we've got a good number of participants, but we are streaming actually also on Facebook and on YouTube. Um, good afternoon, everybody. Thank you, Paulo, for the excellent Talk and uh it's a pleasure for me also to be here. So, uh, let me introduce the second speaker of today before we start the discussion. It's uh Professor David Hackham. He's surgeon in Chief at Johns Hopkins Children's Center, obtained his Bachelor in Medical degrees at the University of West. And Ontario, then earned a doctorate in cell biology from the University of Toronto, uh, where he also did his general surgery training, and then, um, he completed a surgery fellowship, he did a surgical fellowship in Pittsburgh. Uh, in 2002. Then he joined the faculty in Pittsburgh until 2014, um, when he became the Garrett Family Professor of Pediatric Surgery at Hopkins University, where he is the surgeon in chief. Um, so Dr. Hackin's lab is focused on unraveling the molecular mechanism that underlined a lot of important diseases like any. IBD or trauma, and he seeks out actually, and that's, I think, is special to develop novel therapeutic strategy to prevent and reverse those uh processes. And it was actually his group, and a lot of people are aware of that who discovered that NEC is caused by increased expression of uh signaling of TR4 uh in the newborn intestine. Um, so, uh, David, before I tell your entire story, um, I just want to, which includes rodent models, um, intestinal stem cell isolation and culture, tissue engineering, and parents, and you've got funding through the roof. Um, but I think it needs to be, uh, emphasized that they are still the surgeon. And a great teacher. So one of your focus is also to be passionate, to be very passionate about training the next and the current generation of clinicians, scientists at all levels of training. And um so that's why it's a big honor for Yupa and all of us, David, um, that uh we um could get you on board for this session um today and uh for the session, Cool Kids to Basic Science. You're one of those cool kids, and uh the stage is all yours. Thank you very much. Those are very kind words. Uh, I'll never be as cool as someone like you guys with your cool accents and all that great people. Just pay no attention. So this is, um, this is my screen. Uh, thank you for the invitation. I'm glad I could move things around to be here. Um, Zoom makes everything both easier and harder. Uh, a couple of disclosures. Uh, first, I got my 3rd shot of, uh, Pfizer vaccine yesterday. So I think it's great, except I think my TNF, alpha, and IL-6 levels are through the roof, and, uh, uh, so I feel like I got hit by a lorry, um, or maybe even a meteor, but, uh, it is what it is, and, uh, uh, I encourage everyone to To get vaccinated. Um, It's a real pleasure, uh, following Doctor Ducopi, Professor Ducopi, uh, Paolo. I've followed his work for so many years. He's truly a leader in the field and did me a great honor, uh, when I was a leader of one of the American surgical societies, um, to be a, a featured speaker, and people are still talking about your talk, uh, as sort of, uh, the high water mark for that meeting. So, um, it's great, great for pediatric surgery, great for surgeon science. Um, but I was asked to speak about tissue engineering, so I will, uh, be focusing these remarks on tissue engineering, which I think will be complementary to some of the real groundbreaking work that Paolo, uh, presented. Um, Martin, a professor, uh, uh, Latcher talked about my passion for training, uh, and, uh, I, I know we all share that, uh, on this call, certainly. But I want to open just uh with this slide. These are the faces of the people that did a lot of this work. These are the surgical residents in my lab, uh that uh performed a lot of the studies that I'll be describing, and um I always put your name, your choice, cause I just gave this talk to the Department of Surgery. Uh, who, uh, are always interested in, in, um, looking for opportunities for research. But, uh, these are not the postdocs, the graduate students, or the MD PhD students who are wonderful and incredibly important. These are the, these are our brothers and sisters, general surgery, uh, trainees. And, and I think, as Paolo and others would agree, um, surgeons are well suited to do science as long as the environment is supportive and the question is relevant, and the environment is structured. Uh, I said I wouldn't talk about any scenes. All right, uh, David, I'm deeply sorry to interrupt. Could you go on, on full screen mode because this would like enlarge your presentation. Uh, yeah, and, yeah, there's no problem interrupting me. I'm gonna try it. It, um, sometimes it works better than others, but let's see. All right, can you see that's much better now. Perfect. Thank, thank you for, for that. I said I wouldn't talk about a netizing entercolitis, and I won't, except for the parts of the talk where I do, which are just at the very beginning. And so, uh, tissue engineering and, and our focus will be on the small intestine, uh, needs to solve a problem, and the problem is massive intestinal loss as occurs in kids. that have sustained NEC but also volvulus and other uh intestinal loss. We do hope to prevent this disease and to treat it. But, uh, the, the beginning, I need to, I need to just share a couple of neck slides before we launch into the solution, which is tissue engineering, because I want to put that in the appropriate context. Uh, it's always important to disclose that I do work with, um, companies, uh, especially since neck. is a disease that is largely seen in the setting of infant formula administration, the absence of breast milk. So I work with formula companies to develop formulas that are uh neck protective, as well as, uh, other companies that we hope will bring our discoveries to the clinic. I also have to acknowledge, uh, this book, and thank you to many, uh, that, um, contributed chapters, and also those that have purchased it. It, it's on Amazon, you can search on Amazon. It's not really a plug for the book, it's mainly a way of saying thank you. And that there's a one-stop resource um to go to for your trainees, for yourselves, for this, uh, for this book. And, and this is the pathogenesis right here at TLR 4 in the intestine and the dysbiotic microenvironment that we know these preemies have when they have formula. And it activates this signaling cascade that causes inflammation and intestinal distention, and, and, and neck. Uh, it is a bestseller. It's 1,248,610 on the uh Amazon bestseller list, uh, where I know you share with me your excitement for the movie to come out, and now that the last installment of James Bond is out, I think, uh, that, that actor um has time on his hands, and, and, uh, I'm just waiting for him to call for the screenplay. Uh, very briefly, what happens, uh, in our lab as we look at neck before we talk, uh, in a little more detail about tissue engineering, and I'm going to keep this to about 20 minutes, so we have time for discussion, is, uh, like all of you, these are the questions that we think are important. We're interested in what causes neck and, and as a corollary, why are preemies at particular risk for this disease? And is it, uh, is there some aspect of development that we can enhance the answer. is yes, and can we prevent it? Uh, and since breast milk prevents it, can we learn from breast milk, uh, how breast milk works? And the answer seems to be yes. Can we predict it? The answer to this appears to be maybe. And uh we're, we're integrating the uh health, uh, the electronic health record and, and Machine learning to come up with predictive models. And then the focus of the complications of neck, babies with neck have neurocognitive impairment, as most beautifully recently shown by Marty Blakely's group, significant lung injury, and for the purpose of this conversation, intestinal loss. And just one overview slide, and this is what's happening in the lab. Uh, you're very kind to mention our work on TLR4 dependent pathways. We think That many clues in the pathogenesis of neck will be found by looking at how TR4 signals in response to the abnormal microbiome and, and how probiotics and breast milk can influence the microbiome and, and shut down its developmental program. And just so people are aware, neck occurs in the presence of elevated TLR4 because TLR4 is a developmental gene. It's required for gut development in humans and mice. In every species we've looked at, piglets, monkey, and um the fetus was supposed to be born in a sterile environment, of course. And so teleol levels are very, very high. It turns on notch, it turns on wind, all these important genes for development of the gut. And so when the baby comes out prematurely, these developmental genes are still, uh, are still turned on. But now, the sterile womb is not so sterile, it's a dirty NICU, no offense to the NICU. But now these developmental genes see bacteria, unfortunately, they're also bacterial recognition genes. Don't ask me why, uh, I didn't design it that way, but these developmental genes have bacterial recognition roles, causing inflammation and neck, and so, uh, there are clues that link development. And disease, uh, pathogenesis. We're interested in the effect of diet and metabolism on that, uh, uh, uh, especially certain ligands. Prediction, uh, looks at maternal factors, machine learning, and then the long term complications, and we're going to focus not so much on the brain, uh, or on the lung for the purpose of this, but on the artificial intestine. And just one recent story, cause it's 5 days old, and so, I guess 7 days old. Time flies. Um. Uh, I, I point you to this paper that just came out as the first author, Mark Kobler, is a surgical resident. He's going on to become a pediatric surgeon. And, uh, uh, where we showed that this bacterial receptor, one of the things that it happens to do is it, it cuts down some of the enteric nerves in the gut, and especially the enteric glia, which are support structures that nourish the nerves, and their loss is critical, uh, for neck. And this finding uh was based on the observation. that we all make, that is, uh, every baby with neck first has abdominal distension and feeding intolerance, and, and essentially an ileus. And we all believe that this ileus is a consequence of neck. And so we thought, since it occurs so early on, and since Bell staging, bell 1, BL 2A is abdominal distension, feeding intolerance, maybe, which is a, a mark. and ileus, perhaps. Maybe ileus is a cause of neck, not a consequence. And in fact, that's what we showed. We showed that ileus occurs because TF4 knocks out the signaling molecules uh that allow the cells to work. Breast milk actually restores these, and we, um, identified a drug which we call J-11, which turns on these glia once again. Uh, and, and prevents neck, as you can see here, and it works in human tissue Xvivo very nicely. So, uh, that's, uh, uh, just a, a little bit of a neck story. And, uh, uh, this is a sort of what happens when those approaches fail. What happens when neck develops and as surgeons, we operate. And we resect the intestine, and now the baby's got short gut, and the work of uh Paolo and others, um, across the, the, uh, field and other fields are, are working on this question, uh, of developing an artificial intestine, which is a word we use, uh, and, and the rationale uh should be uh apparent, uh. And this is how that was just like a um a subtle Freudian slip. I did not mean to show it twice, but it's like advertising. If you just flash, you know, buy Coca-Cola, and you don't realize, or sorry, buy, uh eat pizza, and you don't realize that at the end of the talk, you'll be hungry and, and you'll order a, a, a pizza. So if I just flash that book, I'm just kidding. I didn't, I didn't mean to do that. Uh, so these are the components of uh the artificial, and I received no royalties, obviously, it's just for the field. I just wanted to say that. Uh, so the components are, uh, cells, scaffold, and the environment. And, uh, we talked, uh, uh, a little bit about some of these in the previous talk, Doctor, um, uh, uh, Paolo has really done a lot of, uh, important work in this. And I'm going to sort of touch on the stem cells a little bit on the scaffold, and not a whole lot on the niche, which Paolo, uh, described. This is our workflow, and, um, we do this either in mice or in piglets, or in dogs. Where we start with a um isolation of the stem cells and we put them on an appropriate scaffold, and, and the scaffold is critical as, as you heard about, uh, blood supply is essential. And, and we do use the omentum, and you'll see complementary work from uh our findings, which really support a lot of Paulo's work. And then we feel that we can bridge, uh, the, the ends and um have a, a blood supply to, to nourish and absorb. The stem cells we're talking about are not embryonic stem cells. We do not use embryonic stem cells. We use these stomatic stem cells that will give rise to the epithelium. And the more undifferentiated they are, they will give rise to all of the epithelial progeny, including enterendocrine and goblet, and panni, and the pana cells are so critical for supporting these stem cells. And uh these are the, the progeny. And uh we, uh, when working in human tissue, which we, we have to do, um, we work with induced pluripotent stem cells. These are fibroblast-like cells, which we differentiate into uh intestinal stem cells, and then mature intestine, uh, which, uh, um, led to a Nobel Prize, and, and that's the workflow. And so just a couple of Uh, slides to show what these structures look like. This is, uh, from the, uh, somatic stem cells. This is not IPS. You get these beautiful structures with each of the different progeny, you can do it from mouse and human tissue. Uh, just some, uh, videos here, you can see that down here, they do form these crypts, and they do shed into what is essentially a lumen. You can see that right there. And um uh there, there has been interesting, uh, this has worked on my surgical resident Doctor Laura Barton, who's now a pediatric surgeon in New York. And you can see the three dimensional structures, this cry is coming out at us, and they fuse together. They, um, under different conditions, they will form cysts where they secrete the material into the cysts. They'll differentiate and branch, but only so much. And we're very interested in, in how that happens. And we can modify this with various bacteria, that's a probiotic. Uh, it's um impaired in enteroids, uh, in the presence of the neck. And so, the scaffolds that we use are engineered. They're not 3D printed, but they are laser printed into a biometrix. And, uh, this is work in uh from our collaborators at Cornell University, where we get these delicate 3D structures that look very much like the native intestine. And uh you can see, uh, we can tubularize this, and we get these villi, and they're absorptive, and all they need is a blood supply. And they, they cover very nicely with stem cells, and again, this is the workflow. And so we need animal models to, to test this. And so I, I'm going to take you on a little bit of a zoological. For starting in mice, where uh this is the type of structure you get in the omentum, um, where just a cross section, uh, through some of these villi structures, and, uh, you can see, uh, that, uh, over time, we do indeed get, uh, a blood supply, which uh is shown here. Uh, we get the endothelial endothelial cell progenitors are attracted to the area and you get this fine network. This is von Willebrand factor. And uh uh that absolutely requires the enteroids, the scaffold alone doesn't seem to do it, and uh it's not as if the mouse itself uh is putting uh stem cells in, this is all donor origin, which is very important. Because of the potential uh clinical application uh for these. And, and this is just evidence that it's donor cells and that, that we recruit immune cells. And you can see, um, in, in a very over time in a bioreactor, you can get all these various tissues, you can get the epithelial cells, you can get the immune cells. Uh, etc. And, um, uh, I guess science thought that that shows promise. So, that's the mouse work. It's an epithelial, um, stem cell progenitor pool that grows on a 3D scaffold that gets a blood supply and works in a mouse. So, uh, there are obviously questions about the enteric nervous system, uh, uh, peristalsis, degree of absorption, scalability that, that, uh, are still open questions. But um we are not mice. Um, maybe we're a little closer to dogs. So we've used uh the same approach. Basically, we do a suave uh procedure. You can see this is the, the Lone Star that we use in our kids, not the exact same one, at least, I hope it's not the exact same one that I'm a little bit worried about it. But um it looks pretty similar. But we wash it, or at least we rinse it under hot water for, I hope we rinse it. Anyway, so you can see we're starting a suave and we do a mucoectomy, and then we implant. From the mucosectomy, our, our graft in here. And the advantage of doing this in a dog is that we can then perform. Um, proctoscopy, and, and you can see the, uh, uh, suture line here, and the graft, uh, proximal and, and how it looks like a very nice mucosa. What this gives us over the mouse is this is where this graft is supposed to be. It has an intact blood supply, where, uh, we are implanting a scaffold. In this case, it's absorbable, it increases the surface area, prevents stricture formation. If we just put the Uh, androids in there, it just scars down. And so that's, that's why you need a scaffold, and you get a little bit of a sense that the scaffold gives you, um, goblet cells there. We're perhaps a little closer, uh, in infants to piglets. And so, uh, using a piglet approach, this is a piglet that has had our artificial intestine, has a central line, and, and these are, um, Um, the piglet's friends. This piglet is supposed to be in the cage. So I'm not sure how, uh, the piglet escaped. Uh, the work in the piglets, uh, is, uh, shown here, essentially. And I'll just draw your attention down here where you can see we're implanting the scaffold with the androids, and we leave it in the bioreactor from anywhere between 2 and 7 days. And as Paulo said, the ability to sew this is absolutely critical for surgeons. And so, the design of the scaffolds, of course, has to have a 3D surface that allows incorporation, and adequate surface area, and vascular ingrowth, that's all fine as far as it goes. But if we can't sew it because it melts away, it won't hold a stitch, it's of no use to us as surgeons. And so, it's actually a, a real um bio mechanical engineering challenge, uh, which we work on with our Uh, biomechanical engineering colleagues, and, uh, you can see we have good success over time. Uh, I'm just gonna show you, uh, and we bring out stomas, and, and we've implanted this upstream of the stoma, uh, downstream of the stoma to assess the role of, um, uh, stool passage in enhancing or not enhancing. And uh in sio, this is a piglet that we've done a redo laparotomy. Uh, you can actually see this is a laser angiography, uh, that our graph is up here. In this particular case, it's not in continuity. And you can see it's got a beautiful blood supply. And so this is a segment of A couple of centimeters, you can see there of pig enteroids on scaffold implanted with a mesentery around it. It's um in discontinuity from the bowel. So even though it looks like the kid has the, the pig has an obstruction, the pig doesn't have an obstruction. And then we could put that into um uh continuity and, and, and absorb food. And this is the 3D structure. And this is what this uh looks like at autopsy and, and the kind of uh structures and the epithelial coverage that you can see. Um, before and after implantation, we want some degradation, but not too much degradation. And this is an area of ongoing concern. Uh, we need the scaffold for long enough to preserve the 3D structure to have an influx of myofibroblasts and other fibroblasts that are going to maintain the structure. It has to have elastic properties. And so this interface between scaffold and enteroid, uh, and, uh, blood supply is really, um, in my view, uh, the, the key, uh, unsolved, uh, or incompletely solved area. And you can see we get beautiful uh attachments and, and pockets where blood vessels, uh, have grown in. Uh, and, uh, uh, the porosity is, is, uh, one simple marker, and you can see these, uh, mini, um, Structures that we get. Doctor, uh, um, the copi beautifully illustrated the work that he has published on a decellularized intestine. I think this has great merit. We performed similar studies and, and it's very, uh, straightforward actually to uh decellularize and then to recellularize, which you can see, uh, right here. Uh, and I think that this actually It has great merit, and we've implanted this in, into pigs, and, um, and shown great structures that, that seem useful. But in, in the final minute or two, I want to turn back to the human application. And so if you have a child who's lost intestine, and you bring their intestine back together, after resection, the residual bowel will adapt. And that adaptation is very instructive uh to us, if we can Uh, uncover the signaling molecules that drive adaptation, we can achieve two things. One, we can simply enhance adaptation in, uh, kids with short gut by identifying pathways and augmenting them or inhibiting them. Using uh new drugs or, or gene therapies. Secondly, uh, if our goal and Paulo's goal is to develop an artificial intestine, wouldn't it be great if the Matrix liberated certain molecules. That actually enhance the adaptation of the epithelium on top of it. And I'm just going to give you, this is, uh, again, the type of uh refocusing that, that we need uh to bear in mind that others have talked about. This work is moving forward, uh, in multiple labs, and, and we're certainly learning from each other, and I'm learning from Paolo, and And others, Tracy Gripscheid and, and Mike Helmrath, and Jim Wells, and, and lots of labs really around the world that are doing this type of work, and many, many others. Um, but there are key barriers, at least the United States, uh, the FDA, uh, there are so many steps that may pose risk, not so much the intestinal cells, which we think would be patient derived or decellularized matrix. But, uh, and in fact, the same patient arrived, so we would take their skin fibroblasts and differentiate them and then re-differentiate them. But, uh, the steps of purification, um, require FDA compliance. And so we try and, um, model after industry standards whenever possible. There are ethical concerns, it's not cheap. And of course, you got to get the right patient. I always return to the beginning. These are the surgeons that, that did this work, uh, currently, um, who are, uh, working the lab and, and sewing intestines together and, and culturing stem cells and decellarizing matrix and feeding mice with neck and, and just uh having a good time. Uh, summary is here, the proof of concept, I think we've established, others have too, for the development of an artificial intestine. There's biocompatibility, absorptive studies are promising, and the, the work showing the transcriptomic analysis of novel genes uh to reverse short bowel. This is Uh, a broader view of the people that have done the work over many years, and the funding that has allowed it to happen. And, uh, um, uh, in happier times before COVID, uh, this is the type of uh picture we can take. Again, I, I'm truly honored for the opportunity to be part of this, uh, symposium and, and, uh, thank you for the opportunity to present our work. Well, thank you, uh, Professor Hackin for this, for this excellent talk, uh, and also Professor Decoy. Um, yeah, I think we're ready for, for discussion. I will, I will look in the chat, maybe Augusta looks at the chat and maybe ask the, the, the first question. So, so Paulo, you, you mentioned that you can sort of substitute the colon by Uh, replacing it with a different mucosa, like a small bowel mucosa. Does it matter where the colon lives? So could you actually take a segment of colon, interpose it, relocate it. Between two loops of small bowel, with this like epithelialize differently because the microbiome is then a small bowel microbiome, would that make a diff different, diff difference, or, or does it not matter? Yeah, thank you. Uh thank you, Martin. I mean, first of all, let me remark, I really enjoy, as always, the, the lecture of, um, uh, Professor Hackman, um, and And it's, uh, it's very complementary to what we are uh trying to do in the lab. Uh, regarding your question is you make a very good point. I mean, people have shown that you can transplant organoids from small bowel into the large bowel, at least in mice, and these organoids in a damaged column, um, can actually populate. I mean, Um, Hans Clevers has shown that Toshi Sato is leading really this, uh, this work and was published in, in Nature last year in which he was able to repopulate organoids in the large bowel. And, and you can think about, um, uh, doing that, for example, for this. Diseases um of the adulthood in, in the colon IBD is one of those, but actually if you think uh uh it's possible that the mucosa could be substituted to an absorb, uh, an absorber one like the jejunumalum. But you're making a very good point that actually the microbiome is still a problem in, in the colon if you don't interpose that to the small bowel. So it's possible, uh, as you mentioned, that actually if you transpose the colon and then you do a demm uh-huh, uh, you take away the mucosa, sorry, uh, of the colon, uh, a mucosectectomy, um, you can then substitute that with the small intestinal one that you have engineered. In the lab and then you transplant that into the column that you have interposed, but it's quite interesting that, uh, I mean it's not very well documented, but there are some reports showing that actually in some children with short bowel, part of the mucosa of the colon become small bowel. I mean there, there are biopsies that demonstrate that, so you can sort of promote that regeneration process a bit like uh Davis was mentioning before. Mhm. Thank you. Let, let, let me echo what everyone said. I mean, uh, um, Doctor Akam, you, you, you have, uh, given us, uh, an amazing lecture and especially very generous, uh, to, to share some, uh, new data, uh, with, with us. Uh, it, it's incredible. You, you, uh, are producing so much on the front of neck, and here you are giving us a, a half an hour lecture on something that is close but not that close, so I It's, it's, um, phenomenal. Um, if, if you go to our chat, uh, um, Francisco Bacconi from, uh, uh, France is asking, uh, um, something that I think you both alluded to which is the nerve system. Of course, we've, we've seen, we've all seen that, uh, everyone, uh, including, uh, Mike Elmrath and, uh, Tracy, Greg Scheid, everyone is starting, of course, with the epithelium. Uh, but we know very well that there's, uh, other self regulations, uh, and, uh, of course, uh, the ner nervous system, uh, too, and, uh, how far are we from, uh, uh, having, uh, really, uh, uh, a full package of, uh, cell populations, uh, uh, cell tissue layers that would represent the, the intestine. And then the other question still from uh the same person is uh about the tubularization procedure. How do you test the strength of the scaffold? Is for both of you? Right, OK, uh, I guess I'll go first. Thank you for the kind words and, and, uh, it means a lot to me. Thank you very much, and say hello to everyone in, in Toronto, and it's a it's my old stomping grounds. That's where I did my PhD and And my initial training with, with, um, with that incredible group there. Um So, um, the first question, uh, I think is very important, uh, the, the role of the enteric nervous system, uh, you know, it's, it, and I'm glad people are asking the question, right? It's like, it, it, it's pretty amazing that the cells, the epithelial cells self-organize and that they cover. And then it's pretty amazing that they absorb. And then it's amazing to get a blood supply. And yet the field wants, but wait, what about, you know, it's like my kids, I, I can never like make them happy. So I like when they're asking for more and more and more, cause it's, uh, you know, at least, at least they're asking and they're engaged. So that's good. Uh, if you read Tracy's work, it does look, but the barriers are that it's not just getting primordial um nerve cells, and we know the The uh ontogeny of our enteric nerves very well, uh, in mice, less so in humans, there are reporter strains, and we, we've generated knockouts, uh, and we know the, the subtypes of glia that are required for the subtypes of enteric neurons. This is important work, uh, that we can adapt. So I think now the foundations are there. The trick is being able to grow these cells at a stage where they can differentiate into mature neurons and glia. That they can connect and then it's not just the ENS but it's the muscle. If they have nothing to hook up to uh coordination parasy, it's very difficult. And so maybe you don't need nerves unless they have some immune or hormonal role as glia do. Um, if you can have, uh, a matrix that has some endogenous peristaltic activity, or if you can have an exogenous pump-like system, maybe the the role of nerves is, it can be subsumed by that. All the studies I've shown were relatively small, uh, you know, 345 centimeters. Um, which, uh, is sort of, uh, good, but not good enough. And so we are actively working on incorporation of, uh, nerves, uh, and muscle, uh, but in parallel looking at the other aspects of, um, pneumotic pneumotic and Hydrostatic devices for peristalsis. Uh, with respect to how to check for tensile strength, what we do is we, we do this X vivo and we have straightforward biomechanical, um, devices to assess, um, the breaking strength essentially. Uh, Paula, I don't know if you have a different view on the nerves. No, absolutely, uh, David, I, I would share exactly what you said, and, and that's one of the reasons why we focus on part of the intestine. So, 11 thing that we are getting more and more use is the fact that the organs that we may engineer will look very different to the organ we are used to have in the body. And uh, and it's possible that, uh, uh, you know, some mucosa that you would implant somewhere would help you overcoming some of the nutrition problem that you have with short bowel and, and, and that's exactly why we, we saw that very difficult um complete engineering of uh of, of, of the old intestine and we focused on part of that because of the different component. I mean, Um, uh, uh, Tracy, who is a, a, a, a very good friend and a collaborator on the European grant, and, uh, um, Jay Vacandi, I suppose I've shown it, you know, 20 years ago or more that, that you can put some cells derived from the intestine that used to be called organoid at that time, but contain also the mesenchyma, and you can have some structure that looked like the intestine. But actually the difficult bit is to make that structure working in an appropriate way, as you were saying. So I, I think it's very, very complex and, uh, and, and the, the way that we do it and I think um you know, uh I, I believe, I share that with, with you and the others in the call here is that we, if we understand mechanism, we go faster to patient. I mean, I always say also to To, uh, um, the clinical colleagues that come to the lab that actually understand the mechanisms don't make you more far away from patients, but we go faster because we understand what we can and we cannot do and also the regulatory are much happier to um help us translating something that we know and we understand exactly, uh, every single mechanism. So that's why the journey rather than Um, uh, uh, getting more complex, get simplified, and we try to, to take out components if we can because that's, uh, put us safer and closer to the patient. Mhm. OK, thank you. There's this, uh, another question from Jose Mauchek asking about scaffolds. I, I bet that's for both of you, whoever wants to answer. Could the gut that is resected during surgery for NEC be used as a scaffold for stem cells? And he, he mentions like the cells are dead, but the scaffold remains, so would that work? So, I mean, if I can say from the D cell point of view, I wouldn't use that scaffold for a number of reasons. The first is that uh when you have ischemia or even perforation or necrosis, you, you, you, um, you know, you free in the tissue a lot of um of um both mechanical and Biochemical, um, results of the necrosis that does not really, uh, facilitate cell proliferation or cell seeding or anything and the structure, um, that results in the scaffold, um, it, it, it's not maintained, so I wouldn't, I wouldn't suggest using that scaffold, uh, for, um, for repopulation, but I don't know what David thinks. Uh, I love the question. It's very creative. I agree, uh, with Paolo. I think, uh, you know, there's neck, and there's neck and there's neck. So we're aggressive with neck because we think that it was surgery for neck, because we think that there's two reasons to operate on the belly. One is to remove the dead bowel, which clearly is dead and of no use. Uh, and the second is to, um, to protect the brain, you know, as the gut is releasing. Uh, molecules, and we've identified some of them, they cross the blood-brain barrier and they activate the microglia in the brain and they cause cell death, and that's probably, or at least they, they, and the cells are not neurons, they're oligodendrocytes, so you get a white matter injury. In humans and in mice, uh, that we've induced to develop it. And, and, and again, Marty Blakely to refer to his beautiful work in the Annals of Surgery, uh, neurocognitive impairment is higher, uh, in those, uh, patients that did not have an X lab, but were otherwise stratified, uh, suggesting that along with the disease. So, so with that mini diversion, I think the pendulum is going to switch, or maybe this is a society to help me do this, um. But uh I think the pendulum is going to switch, so we're operating a little earlier. Now that will come at the risk of having some unnecessary laparotomies maybe, but you know, a 1 kg kid can can tolerate the next lap pretty well generally. They release some fluid, maybe put a silo on. But you may be in an instance where you're removing bowel that's, that's a patchy, that you wouldn't, that isn't completely frankly necrotic, and, and maybe if you have the right setup, this is the bowel that you have in mind. And then certainly, but otherwise I agree with Paolo. Otherwise, uh, the stoma closure, uh, we have to be very careful when we're closing stomas that we don't take too much bowel. But especially if it's a shortcut kit, I, I will, I will compromise how beautiful the ends are to maintain. Uh, length, uh, but never compromised function, and so it's a, it's, it's a delicate balance that everyone on the panel faces every time they have a stone reversal a big neck. But if you've got a kid that may have a little extra, then maybe you could do that. I, I don't want to suggest we would operate for purposes other than to help the kid, but you follow me. So, the, the simple answer is probably not, but maybe there's some nuance there. Yeah, thank you. Yeah, oh, sorry, Augustus, just to add it on that there could be a very nice, um, you know, uh, understanding what happened to the SSRR metrics in this situation, and, and that will offer a lot of clues, uh, for regeneration as, um, David was mentioning before. Yeah, I, I totally agree with your comments and uh also with the comment that we maybe need to operate a little earlier. I really hope that uh your work and uh um the work done by, by, by others uh showing that these babies might not have a perforation, but their brains and their lungs are actually severely affected. They really, we pushed the surgeons not to put drains and uh possibly to go to the OR a little earlier. Um, there's a nice interesting, uh, question from Mauritz Markel, who originally comes from Martin's group in, uh, in, uh, Leipzig, but he's in, uh, Winnipeg, uh, uh, doing research. And again, uh, um, as you lined out the importance of TLR-4, so I think it's more for, uh, Doctor Hackem and immunological mechanisms in neck. Do you think intestinal organoids or the artificial mucosa can be able to fulfill the complex immunological tasks of the intestine, of the intestine and intestinal barrier. So again, we want more. We want not only that the gut works well, but also that it has a proper immunological function. Have you tested, uh, your, your, uh, Um, um, artificial intestine, uh, from this regard? Sure, it's a great question. Thank you. And, uh, it's really important because we, this is, I suppose, the fourth aspect of the epithelium. There's the nerves, there's the blood supply, and then there's the immune system. And, um, so just two points. So to your specific, and please say hello to, to everyone in Winnipeg, and I hope you're having a good time there. Uh, it's a, it's a great place. So, um, We haven't actually done immunologic tests on our graphs. Uh, we've done, uh, basic kind of cytokine release, but we haven't done Um, robust, um, assays for, let's say, antimicrobial peptide released from the panic cells, so that we, we've shown by immune histochemistry that we have panic cells, we haven't shown that their function as an example. So I would say we have not done that. It's a good idea. The second thing I would say though, is the role of TLR 4 in the epithelium. It's probably not human logic. It's developmental and coordination of the balance between proliferation. Through winter notch and death through caspase 3 mediated apoptosis. And so, telF4 has a non-immunologic role in the epithelium. But in the, in the myeloid compartment, lymphocytes and macrophages, the telF4 is TR4, and it recognizes LPS. And so, Uh, I think, um, that's how I would answer the question. I think we need an intact immune system because whilst I showed some work with, um, treatment of the graft with probiotic, I went through very quickly, and neck bacteria, that's really just in response to the epithelial signaling. It's not an immune response. And if we don't have an intact immune system, we won't have an intact barrier. And then it's why your question is so good, we'll just have bacterial translocation and, and we'll be back to square one. So, this is another area that we need to work on, and that's getting the right, and also, I would say experimentally, all our human work is in immunodeficient mice. So we're going to have to be smarter about that. This one final thing about the immune system, we showed in the paper in 2016, that the lymphocytes are critical in the pathogenesis versus protection from neck. And so if we take lymphocytes, From a mouse with neck and uh uh from the mouse bowel with neck and inject it into a naive mouse, the mouse will get neck. So lymphocytes, and these are TH 17 lymphocytes, and the human and mouse uh intestine has the same lymphocyte population. And then Akhil Marashwari has shown beautifully that macrophages are pivotal in the protection and induction of neck. And so there's no question that these cells are important just in barrier function in the neonates, and therefore probably in artificial intestine. Right. Then there's there's another question from Richard Wagner, also from my group, but he's in Boston in Patricia Donahoe's lab at the moment, and he's asking, did either question is for you both? Did, did either of you look into how stem cells are affected like a neck, gut mucosal stem cells. in patients, whether these uh change their monocular fate. So he's asking, where is the epigenetic imprinting of the affected parts of the gut, which could in turn inform which molecular pathways might be beneficial to mucosal regeneration if targeted. Very complex question, Richard, but um I hope I Transported that well. Do you understand what Richard was saying? It's a great question. Uh, Paolo, did you wanna, yeah, absolutely. Um, yes, I mean, uh, um, we, um, uh, we did a bit of work, uh, looking at organoids, uh, derived from, um, a patient with NEC, and, uh, I'm aware of some of the work has also been done by Augusto and Agostino in, um, in Toronto about uh um uh this. I mean, the, the, the question is that, um, um, I mean, from the organized point of view, you can certainly culture cells uh from patient with NECF from this uh resector segment and that's, that's going to intuitive because of course, uh, the intestine can regenerate. And, and the stem cell counterpart seems to be more resistant and more resilient to ischemic damage and, and to necrosis. Um, but we haven't looked, um, at that, uh, maybe David has more, uh, knowledge on the molecular, um, uh, path of, uh, of the stem cells of the intestine, uh, in case of NEC. We haven't, we haven't looked much into that. We are planning to do it, but it's not the data that we have yet. Thanks. Yeah. So, we, uh, we've shown that uh we can culture stem cells from neck. The, uh, the work on the epigenetics of neck and how that is maintained in cultured stem cells has been done. From Washington University in Saint Louis by Misty Good, and she actually has looked at that. She's presented it, I think she's published it. If not, she should be soon. And uh your, your question is um answered by her, and that is, yes, there are, there are epigenetic differences in neck that she picks up in the tissues. Um, whether she did it, uh, in the organoids, or whether she did it in fresh tissue, I, I, I think it was actually in the fresh tissue where she did epigenetic arrays, but it's the same basic idea that you're asking. And, and we've grown those stem cells and whether they maintain the memory or not, uh, Misty's work might shed a light, but we, we haven't looked at that specifically. But, but it's a cool question, right? Because if, if you knew that, then that may, uh, allow you to reprogram it. And perhaps even have a much better mucosal surface to it. Here's a question for Doctor Hachem, uh, um, apart from IPCs, uh, are there possibilities to announce the function of a cell or organoid repopulation, so like with gene correction, this is in the context of genetic engineering overexpression of the specific factors. Yeah, that's a great question. So, the, the, the answer is yes, uh, and, and, and then I'll, I'll, I'll give you the, the caution. Uh, so we published, um, some years ago, uh, it was a downstream molecule of PUA, which is P53 upregulated modulator apoptosis, PUMA, which when induced in stem cells, limits their proliferation, and so we, we used an adenoviruss at that point to knock it down and cells proliferated more. Uh, there are scaffolds that release, uh, uh, inhibitor of apoptosis, and in fact, uh, adding to the matrix apoptotic inhibitors enhances proliferation of the enteroids, and, and, um, we're developing scaffolds with additional molecules for that very reason. The concern is any growth factor, and essentially they're all growth factors at some level, has a malignant risk. And so for a kid, that's, that's something that we cannot convince a company uh to support us on. They're, they're worried about cancer, um, for good reason. And so we, we're not even sure what the FDA would say. But it's a great question and, and, uh, an area of, of hot investigation. Yeah, well, I, I don't see any other questions in the chat, but, but, um, um, may, may, may I ask you, Paula, when you, you talked about how to grow epithelium, um, that brought me to the question, like, in, in recurrent TEF, OK, we, we, we, we see these patients once in a while. And what keeps this re fistula open is the epithelium there, right? You, so you, you try to, when you want to manage that open surgically, it's, it's a very demanding procedure. So it, it's always smarter to try endoscopically to destroy the mucosa. You know, you know, there are people who do chemo cauterization of, of that. So do, from your research, is there any agent that you use? To decelarize parts of the body in a very smart way to solve this kind of problems, you know what I mean? So they sort of reverse everything, not build something up, but very smartly targeted, destroy it. Yeah, that's a very, very good question. I never thought about, uh, uh, Martin. Uh, um, I suppose we are more aggressive here in, uh, in London. Uh, probably because of our predecessors. So on, on recurrent fistula, we go back, uh, on surgery. But, um, um, yeah, I mean, uh, there are, there are some, uh, um, agents that may work. The problem is that these chemicals, uh, um, are not so safe to use. I mean, there are some, uh, enzyme that could be used, but uh those enzymes don't really work without the detergent essentially. And detergent, we know very well what they do to the esophagus. So, um, it's, it's a bit difficult to use them in a localized meta. I mean, it's a, it's a clever idea we can look at, but, uh, I, I, I don't have an easy solution to do, to use a detergent in a, in an easy and localized way. Without causing any damage to the neighbor cells, and, and that's, that's a real problem there. But you could think about creating a patch of epithelium, uh, that, that you could use to repair some of the fistula, and, uh, and that, that's something that we have been thinking about or for example, on EBE or in epidermolysis bullosis, uh, people have shown it for the skin. There was a Nature paper some time ago. Showing that you can take those cells and genetically engineer them and then creating patch of skin, but we have a problem, a huge problem in the esophagus, and we could try something similar on the esophagus as well. So that the patch of mucosa, I think may be uh something that could be useful there. That's too bad, Paula. I was hoping you had a magic solution for us, you know, when we have these kids that have recurrences, they're, they're a nightmare. Yeah, next time, next time. Yeah. OK I think we are well over the hour and, uh, of course this is showing you how much interest has been, uh, uh, what, what is really um good in a way to see that there's a lot of, uh, uh, trainees and young surgeons that are engaging more and more, and this is what, uh, really hope that we surgeon scientists don't become like pandas, uh, but, uh, there's actually more, uh, people that engage in Uh, and don't do research just to, to tick the box, uh, for, for their CV and so thanks a lot for, uh, your inspiring talks. Uh, um, this, this is really, um, a lot of food for thought, and very inspiring for, for the next generations. Well, thank you also from my side. I was uh quite amazed this time. We had a lot of discussion, a lot of questions from the, from the training trainees. So you're really inspired them. And, uh, well, thanks for being with us. We will put that on our YouTube channel and we cut that section out that David didn't want to share, which is totally fine. There's, there's no problem with, with, with that, but uh maybe the rest of the Webinar we can maybe we can share for the future generations, yeah, with pleasure. Thank you. Yeah. Oh, that's very generous. OK, and thank you also Gaya for setting this up. Um, thank you again like always we also, uh, put in the chat the Insta and the Twitter account. So I ask everyone to follow us on every social. Very good. So next webinar is in November on biliary atresia, laparoscopic versus open. Um, there's also to, like a lot of, hopefully a lot of good discussion. And yeah, that's from my side. Yeah, yeah, we have Professor Yamataka from, from Tokyo and uh uh Barbara Wilde Haber, who is the chief of pediatric surgery in Geneva. Uh, in Switzerland, uh, so it's gonna be very interesting. Uh, we're going back to clinical, um, we'll do some basic science for biliarytrisia sometime in the future. There's stuff to do there too. OK, thanks everyone, and, uh, really thank you for taking time from your busy schedules. Really appreciate it. Thank you. OK, thank you everybody. Bye-bye. Bye.