Welcome to our newly created webinar series called ASGE Global Spotlight. This new series was created with our global audience in mind and at a different time from our usual offerings to make sure that you all have a chance to join live. These webinars will feature global experts in their field, and I am very excited for today's presentation. We have attendees joining us from all over the world, and the American Society for Gastrointestinal Endoscopy appreciates your participation. Today's event is entitled Repairing the Human Esophagus with Tissue Engineering. The discussion of this webinar will focus on the advances made in repairing the esophagus by using principles of tissue engineering. My name is Reddy Akova, and I will be the moderator for this presentation. Now before we get started, just a few housekeeping items. There will be a question and answer session at the close of the presentation. Questions can be submitted at any time, however, online by clicking the Q&A feature on the bottom of your screen. Once you click on that feature, you can type in your question and hit return to submit the message. Please note that this presentation is being recorded and will be posted within two business days on GILeap, ASGE's online learning platform. You will have ongoing access to the recording in GILeap as part of your registration. And now it is my pleasure to introduce our presenter for today, Professor Kulwinder Dua, who is a professor of medicine in the Department of Medicine and Pediatrics at the Medical College of Wisconsin. Professor Dua completed his training in India and his fellowship in the United Kingdom. The primary interest for Professor Dua is in advanced interventional endoscopy. He is an inventor and has several patents in FDI-cleared devices. Professor Dua has over 200 publications in peer-reviewed journals and is on the editorial board of several journals. Professor Dua has been involved with ASGE for many years in many different ways. And for his contributions to the field of GI endoscopy, he was recently honored by being inducted in the first batch of top endoscopists of the world with the title Master of the American Society of Gastrointestinal Endoscopy. Really we could spend the whole hour talking about Professor Dua's achievements. And we are very, very fortunate and honored to have him present today's webinar. And now I will turn the presentation over to Professor Dua. Well, thank you, Reddy Jakova, for that kind introduction. And thank you, Mustafa Ibrahim, if you are online, for including me as one of the speakers for this global spotlight. I'll be talking today on repairing the human esophagus with tissue engineering. I have the following disclosures to make, and none of these products will be discussed in my talk. I also want to make another disclosure that, as the name suggests, tissue engineering and so on, and somebody may perceive me to be a basic scientist. I'm not a basic scientist. I have a whole group of talented basic scientists working with me, and that somehow led to some of the discoveries that we have made in our interventional endoscopy work, and I'll be talking a bit about those. Now, in nature, there are a whole bunch of organisms that can regrow their organs. In fact, even regrow part of their bodies, such as the flatworms, the zebrafish, and so on. Plants can have a lot of their branches broken off, and they can grow them back. But as the species evolves and becomes more complex, like the human beings, our ability to regenerate drops, as shown over here, and described very nicely in this publication by Orlando. So the way the body can regrow, those potentials are lost the more complex the organism becomes. And with that in the background, at some point it was assumed that the human body has no capabilities of regrowing organs, except that when you remove an organ, it can hypertrophy but not regrow. Having said that, we did find that that's not true. And some of the clinical approaches in human beings where we use the principles of growth are, for example, transplanting exogenous stem cells in patients who have blood disorders, and these stem cells then program the host bone marrows, and we can then get our natural cells, or these stem cells then produce cells that are more normal than abnormal. So this is one of the ways you can start reinitiating the growth process in the hemopoietic system. Another area is that we have a lot of FDA-approved extracellular matrices in the market. For example, donated human skin like alloderm, the submucosal tissue of pigs called the SIS material. These are xenogenic, allogenic extracellular matrices, and when people have got extensive skin loss, you can apply this on the lost area and the skin can grow back. Or we have these non-biological meshes and biological meshes that we use to repair the anterior abdominal wall, and that initiates regenerative processes that can grow back the tissue. But when it comes down to growing a whole organ, like let's grow the whole liver, that is very difficult in human beings. And that, as I said, is based on the complexities of these organs that require many kinds of tissues to be integrated and crosstalking to each other, and that's where, as humans, we have lost our potentials, at least as far as the current science goes, in regrowing organs. However, some of the early attempts to regrow organs are shown over here. For example, people, when they have neurogenic bladder, scientists have taken tissue matrices, they have seeded them with pluripotent autologous urethelial cells, used this platform to cover the bladder defect, cover it with omentum, and the omentum supplies blood to that tissue-engineered graft and you can regrow the bladder. Similarly, the landmark paper that came out in Lancet, where in a young kid with tracheal stenosis, they were able to regrow the trachea using a cadaveric trachea, and so on and so forth. So there have been attempts made to regrow organs in the human body, but it's all very early and not ready for prime time. Now one argument people have is that why do you want to regrow the organ? Why not just resect it if there is a bad part of the organ, like if you have a lesion in the left lobe of the liver, you can resect the left lobe and you can live very normally with normal metabolic functions with the right lobe. So you have all these redundant organs, you can remove part of the pancreas, you can remove one of your lungs, one of your kidneys, you can cut out a piece of the small intestine or colon and do end-to-end anastomosis. So why do you want to regrow these redundant organs? And then when you have organs that are non-redundant like the heart, and even redundant organs like the liver, kidney, pancreas, lungs, you can actually transplant them. So why do you want to grow the organ? The reason is this, the oesophagus, which is what is the topic of discussion today. The oesophagus is a non-redundant organ. You can't cut five centimeters of the oesophagus and do end-to-end anastomosis, and neither can you transplant the oesophagus because of its complex blood supply. So in this situation, when you have a non-redundant and a non-transplantable organ, the only two approaches we can do is that when the oesophagus is so badly damaged, try to do something to heal it and preserve it rather than resecting it. Or if there is really a need to resect it, like oesophageal cancer, then try to do something to regrow it. So my discussion today is going to be on what are the regenerative medicine techniques of preserving the oesophagus, which otherwise will have to be resected. And if we do have to resect it, how can we grow the oesophagus back? So before we get into that, we need to understand what are the types of injuries that we are talking about. And we as gastroenterologists who also do endoscopic work, we know this very well. The injuries can be partial thickness defects, like when we do circumferential endoscopic submucosal dissection. Invariably, these patients will develop very tight oesophageal strictures. We keep dilating them, et cetera, et cetera. This goes on. And a point will come where we may have to resect the oesophagus and do a gastric pull-up, which means we could not preserve the oesophagus, although we were trying to preserve it. Another type of defect, which is not partial thickness, but it is full thickness defect, is a perforation. And this full thickness patch defect, it's only a patch of the oesophagus, not circumferential, are generally nicely managed endoscopically. But sometimes these land up with big abscesses, fistulas, and eventually these patients go for surgery. So although we tried to preserve the oesophagus, we may lose the battle. And last but not the least, we ourselves have situations where we cut the oesophagus out. And in these patients who undergo oesophagectomy, this kind of defect is called the full thickness circumferential long segment defect. And over here, our option are regenerating the oesophagus to preserve it, and we need to either regrow it. So regrowing it and regenerating are the two options we have in these situations. Now let us start off with partial thickness defect. When over 50 to 75% of the circumference of the oesophagus is lost, depending on the literature you read, almost 100% of these patients will develop refractory oesophageal strictures. Now we all know this, and we do this in our daily practice. When we have patients with strictures, we may use a bougie or a balloon and dilate them, and we cause all this epithelial trauma. We place stents in some patients, and the day we pull the stents out, it's almost like you pulled sandpaper through the oesophagus and look at the amount of mucosal injury. We use electrocortis for some of the resistant strictures, and look at the injuries we cause to the mucosa. And this is the practice we do for refractory strictures. What this does is that it basically goes back and leads to stricture formation. And because of this, we dilate, we traumatize the epithelium, we go back and develop a stricture, and we keep going in circles. And currently we are all focused on dilations, electrocortis, stenting, you name it. But have we ever thought about handling this part of the equation, which is something that is driving the stricture to come back? This is where tissue engineering is coming into play. Now, why does this happen? We need to understand from the publications on wound healing, why does this happen? So when you look at wound healing, and I'm giving an oesophageal patient who had a tight stricture as an example, once you break the mucosal barrier and cause either from dilation, epithelial injury, radiation, inflammation going on, or mucosal resection and submucosal dissection. Once you break the epithelial barrier, the first response of the body is that within a week, it leads to dense collagen deposition and fibrosis. And this leads to distortion of the organ. Epithelium, however, takes a longer time to regrow. It takes around three weeks to regrow compared to fibrosis that takes one week. So loss of epithelial barrier leads to fibrosis in one week and re-epithelization takes four weeks. So when we go back and scope this very patient, you find that you see normal squamous looking epithelium, but the organ has already been distorted. So in regenerative medicine, people are now focusing on how we can either delay the fibrosis and we know how injecting steroids and giving mitomycin are working. The results are very mixed, but more importantly, now we are trying to regrow the epithelium before fibrosis sets in. So in other words, if the mucosa is traumatized with mucosal resection or dissection or dilation, can we regrow the epithelium before fibrosis sets in, which means we can prevent fibrosis. So regenerative medicine is now focusing on hastening epithelialization before fibrosis sets in. And how has this been studied in animals is in this particular study, autologous pluripotent cells taken from the submucosal tissue of the buccal mucosa and skin, the keratinocytes from the skin, these were taken, these were suspended in phosphate buffer saline. The pigs underwent submucosal dissection, as you'll see over here, and the pluripotent cells were injected immediately into this area to hasten epithelialization. So when you inject autologous patients or the pigs own autologous keratinocytes, which are pluripotent or oral mucosal epithelial cells into the site of injury, it hastens epithelialization and epithelialization happens anywhere from between three to eight days. And in the animals where autologous epithelial or keratinocytes cells were injected to the site of injury, they could bring in epithelialization faster than fibrosis compared to the controls. And histologically, this was also confirmed to be the case where in those two pigs where they did not do this, there was significant inflammation going on while normal squamous epithelium developed in the pigs where they had autologous epithelial cells injected. So one of the suggestions that was made is that if you can take buccal mucosal cells from a patient and culture them and suspend them in phosphate buffer saline, that takes around about 10 days to do. And then you do endoscopic submucosal dissection. And at that time, you go and inject these autologous cells to the site where you removed the mucosa from that 100% risk of developing a fibrotic stricture. If you do more than three, if you do a 360 degree circumferential mucosal dissection, you can achieve some sort of preventative measure in formation of a stricture. This is another study where adipose tissue derived, autologous adipose tissue derived stromal stem cells were used in dog models. So getting adipose tissue from the subcutaneous tissue is very easy to obtain. These are again suspended in phosphate buffer saline, circumferential ESD is performed, and these are injected into the ROS area. As you'll see in the control group where no such injection was done, they developed a very tight stricture compared to the group where these autologous adipose derived stromal stem cells were injected. Now both buccal mucosal cells, skin keratinocytes and adipose tissue from the same patient are very easy to obtain. And therefore this, if it really becomes ready for prime time, may be something that we can do a week or so before we do endoscopic submucosal dissection and have it ready and inject it right there and then itself after the resection. Now people have gone a little bit further and this is a kind of interesting study where in again porcine models, rather than injecting the cells, they used carboxymethylcellulose into which they had human placental stem cells mixed. It became almost like a jello and they gave it to the animal to eat it after submucosal dissection. And in this study they showed that even after administering these pluripotent cells orally to the animal, you do get some benefits of lowering the fibrosis response. So people have now tried techniques of even giving these products orally. So in other words, say for example, one undergoes endoscopic submucosal dissection and you have this kind of material ready, you can make the patient drink it for three or four days every day to see if these pluripotent cells can hasten epithelialization. Another concept that came up is that you inject pluripotent cells into the site of mucosal dissection. The pluripotent cells then have to grow pretty fast to become the epithelium. How about growing the epithelium outside the patient and you do the mucosal dissection and immediately apply the epithelium at the time of mucosal dissection. So you don't have to wait for pluripotent cells to develop the epithelium. You have applied the epithelium right away at the same time you did the mucosal dissection. And this is exactly what was done. They took again skin epithelial keratinocytes or oral mucosal epithelial squamous cells, cultured them for two weeks. They were then cut into discs of two centimeters. Three pigs were treated and three pigs were not. They had circumferential ESD and these do not require any suturing. They are very sticky. So you have to use an overtube, hold it with your biopsy forceps, go down the overtube and when the moment you touch the raw surface, they stick. And you do not have to cover the whole raw surface. You can apply one patch here, one patch here, and they connect very fast. So this is almost like immediate epithelialization after mucosal dissection. And what they found is that there was significant reduction in the fibrosis seen in the group who had these discs applied compared to the control group where there was significant mucosal constriction. I mean, almost touching 90% compared to 50% in the other group. Now fine, we talked about animals. What can we do in humans? And this is a landmark paper that was published in Gastroenterology. It is an open labeled non-randomized study, nine patients with superficial esophageal cancer who had more than two thirds of the circumference of their mucosa taken out with endoscopic submucosal dissection. And if nothing would be done, there's almost like 80, 90% chance that these patients are going to develop strictures. So what the authors did over here is that they took the buccal mucosal epithelial squamous cells from these patients. They cultured them for two weeks before they did the submucosal dissection. And they grew them into the discs like we described in that animal study and with an over tube. Again, as you'll know, immediately after the submucosal dissection, they applied these discs. So here is one disc, here is another disc. So you, as I said, don't have to cover the whole area and these will connect very rapidly. Luckily, because they stick pretty good, you don't have to do any suturing, nothing, just touch it to the mucosa and it'll stick. And only in one of the nine patients, a stricture developed. And re-epithelization was seen, complete re-epithelization was seen to have occurred by 3.5 weeks. So this is pretty promising. And this technique is now from animals extrapolated to human beings. But here is the big problem. I do mucosal resections, but I have a lot of difficulty asking my basic scientists to grow me the cell sheets because of regulatory reasons and so on and expertise. So you can argue that fine, you gave us this good example, but I can't do this. I don't have anybody who can grow the cell sheets in my patients. I don't have the expertise. So what was studied in Europe is that if you identify a center that does have this capability, you can take autologous buccal epithelial squamous cells and courier it to a center that can grow the cell sheets for you, schedule the patient to have ESD two weeks later. The center will courier it back to you and then you can do the ESD and apply it using the autologous cells of the same patients. And this was successfully studied in this particular study in 10 human patients, not animals, where engrafting was successful. Even if you identify centers who can grow these cell sheets for you, at least about a couple of weeks before you do the ESD. Others have done something very interesting. If you are very good in doing ESDs and you're going to do an esophageal ESD, go into the stomach, take out a flap of the stomach epithelium, do an ESD of the stomach and use that stomach mucosal ESD tissue and apply it to the esophagus. So you're basically doing two ESDs at the same time, ESD of the stomach mucosa to be applied to the esophagus and therefore you don't have to do any of the cell sheet business. People have used synthetic collagen that is available in the market and there are a lot of extracellular matrices. The problem with the extracellular matrices is that they don't stick to the mucosa as the mucosal tissue does and therefore you will have to have certain techniques of holding the extracellular matrix to the raw surface if you want to use it. So all of these are giving promising results. The bottom line is that if you lose the epithelial barrier, you're going to develop a stricture. Whether you lose the epithelial barrier from ESD or from dilations or from placing stents to prevent the stricture from coming back, you may want to immediately do something that can restore the epithelial barrier faster than one week, which is the time it takes for fibrosis. Now, taking this to the next level, we know that in people who have barrettes with high-grade dysplasia or superficial cancer, or in even squamous cell cancers, when you do Lugol iodine staining and you see an area that you're going to resect, you will find many other areas in the esophagus that may have metachronous lesions or dysplasias. So trying to focus on doing an ESD of one region and leaving a metachronous lesion at another spot carries no meaning. So this group, now these are in humans. I'm not talking of animals. This group from Pittsburgh, they have an interesting technique to do pan-mucosal stripping of the esophageal mucosa. They make a port by putting a G-tube in the patient. And from this, they have another endoscope on the top. They do a circumferential incision and they use a very coarse vein stripping device. So after doing a little bit of circumferential dissection, they catch the epithelium and they strip the whole epithelium out completely. And this is the whole epithelium taken out. That will take care of any metachronous lesions that somebody may have, like in a long segment Barrett's esophagus or scomaceal cancer. And this patient, I can guarantee you, will be a patient where 100% you're going to develop one of the most complex, bizarre, long stricture. So in fact, this study came out from Pittsburgh, from Badlack, who's a world expert on extracellular matrix. He is working in the surgery department over there. I know him very well. What they did is that they had five patients with long segment Barrett's and using the very coarse vein stripping device, they stripped out the whole mucosa. They used an expandable metal stent and they used a porcine extracellular matrix. This is small intestinal submucosal tissue called the SIS material. This is FDA approved. It is lying in every operating room. They use it for many reasons. So nothing is like experimental. So you take commercially available extracellular matrix. It is wrapped around the stent. After panmucosal stripping, it is positioned across the esophagus and the stent were removed anywhere from one to two weeks later. By that time, the extracellular matrix sticks and binds to the muscularis propria layer. And by four months to 24 months, repeated biopsies and endoscopic visualization showed normal K4 positive, K14 positive, disease free. That means no Barrett's, no dysplasia, scomas, mucosa, and none of these patients developed strictures. Point is again, restore the epithelial barrier as soon as you can. And this was again confirmed by another study. Sorry, this should have been 2014. That is kind of promising. There are many more studies. I don't have time to get into that, but point is the epithelial barrier is what was being the focus of regenerative medicine for partial thickness defects. When you talk about full thickness defects like patch defect, perforation, large perforation, stenting, clipping, suturing, fibrin glue, we as interventional endoscopy people know that these do work very well, but then the success rate, although it is very good, you may once in a while land up with a patient where you tried everything and it failed. Don't have to read all of this. In this particular study from a CT surgery group, patients are described where everything was tried. They developed bad problems, endoscopic interventions failed, and now they had no choice but to remove the esophagus. So they were trying to preserve the esophagus, but endotherapy failed. Now they are going for esophagectomy and therefore you are going to lose the esophagus. So what they did is that as one last chance, they used again pig urinary bladder extracellular matrix. Again, nothing very sort of experimental. These are available in the market and they had a lot of the diseased esophagus resected, but they did not do esophagectomy. They just covered those areas with extracellular matrix and they could, in all those patients, salvage the esophagus. Patients did not have to go and undergo esophagectomy and they were successful in preserving the esophagus. So for full thickness patch defect, endotherapy works very well in about 85% of patients, but on those rare cases where you find a lot of issues coming on, you may want to consider preserving the organ rather than doing esophagectomy. However, here is where the rubber hits the road. What if we have to do esophagectomy? I mean, we have around 500,000 patients diagnosed every year with esophageal cancer. Many of them who are non-metastatic may go for esophagectomy. We have a lot of patients who are also having non-neuroplastic problems like injuries from radiation, corrosives, trauma, kids born with atresia and fistula around 5 to 15,000 per year in the US. I mean, the esophageal cancer is worldwide. If this is the number in the US, you can imagine what will be the worldwide rate of patients who need esophagectomy for neoplastic and non-neoplastic organs. And don't forget, esophagus is currently not a transplantable organ. So, you have to do esophagectomy for cancer, atresia, other problems. Now, you can't transplant esophagus from a cadaver. Of course, there can't be any living donor for esophagus because esophagus is non-transplantable and non-redundant. So, you have to do a gastric pull-up or a colon interposition to restore luminal continuity. Now, whenever we do gastric pull-ups and colon conduits, very high perioperative morbidity, mortality in 5 to 10% based on which centers and how many they do per year, very high perioperative morbidities. You know, people develop food retention in the pulled-up stuff of the stomach. They have risk of aspiration. This is going to be lifelong, very poor quality life. So, unfortunately, that's the only option in these patients. The best approach from regenerative medicine would be that can we regrow a functional esophagus in these patients where we have to do esophagectomy? And how is that possible? So, going back to the drawing board bench studies in animals, this is from Badalak's group. What he did is that he took dogs. He resected five centimeters of the dog's esophagus, complete circumferential, full thickness, long segment. He used porcine extracellular matrix, again, commercially available, made them into a tubular structure. And in four dogs, he bridged the gap with this extracellular matrix tubular structure. And in five dogs, he populated the extracellular matrix with the dog's autologous muscle cells. These muscle cells have pluripotent potentials. And what was found is that in the dogs where the extracellular matrix was used without autologous pluripotent cells, all of them developed strictures. On sacrificing the dog, they found that there was esophageal regeneration happening at the edges, at this area, but the stricture developed in the middle. So, the stricturing process outpaced the regenerative processes in the middle because the pluripotent cells were being attracted from the normal esophagus down. And by the time the regenerative process could reach the middle part, the dogs had already developed strictures. But when you have the pluripotent cells already populated into the matrix, none of them developed strictures. So, this is an interesting thought process. So, if the defect is very small, you can just use the matrix by itself. If the defect is large, you may have to populate the matrix with autologous pluripotent cells. This group from France, what they came up with this concept, I'm just throwing in a few of these examples. I know the literature is full of hundreds of studies, but I'm just trying to show how people are thinking. This group in France said, you know what, when you do colon interposition, you carry the colon from the abdomen to the chest with its blood supply. When you do gastric pull-ups, you carry the stomach from the abdomen into the chest with its blood supply and anastomose it to the remnant esophagus. How if I grow the esophagus in the abdomen and then we take it with its blood supply into the chest? So, now what they're doing is that instead of pulling up the stomach or instead of interposing the colon, they are growing the esophagus first in the abdomen to be pulled up into the chest. And they use this animal model where again they used extracellular matrix. They populated the matrix with the animal's own autologous oral epithelial or skeletal myoblast cells. They put it in the omentum of the pig, wrapped around a stent to form a tubular configuration. And the reason they put it in the omentum is that it immediately establishes blood supply and matures. Then when that happened for two weeks, then they resected five segments of the esophagus and used this to interpose rather than the stomach or the colon. And they found beautiful results. The beauty of animal studies is that they then can sacrifice and get full histology. And they found a normal esophagus with all the normal layers. So, another concept, how you can regrow the organ and use it to be pulled up into the mediastinum with its blood supply. Here are the problems. I'm just throwing in this as a very quick slide. This is a kid who was born with tracheal stenosis. What the authors did over here, Dr. Elliott is a CT surgeon. And what he did is that he took a cadaveric trachea. Somebody who died, the trachea was taken and he decellularized the trachea to take away its immune generating response. So, you decellularize the trachea. So, now you only have a tracheal extracellular matrix. That cadaveric trachea that was decellularized and cut to the length of the kid's trachea was then populated. The cadaveric trachea decellularized what was populated with the child's own epithelial and mesenchymal cells taken from the bone marrow. This tissue engineering of autologous cells into the cadaveric trachea was done in a bioreactor in Italy. Once it was done, it was FedExed over to London and transplanted into the child. And it formed an organ. The cadaveric trachea may have been metabolized and bioabsorbed and the kid's own trachea grew back. Being decellularized to begin with, it did not generate any rejection response. Now, that's fine. That's a great landmark publication. But here's the problem again. These tissue engineering strategies are multidisciplinary, a lot of financial expenses involved. You need a lead time to grow the organ, regulation, surgery, and so on. So how about growing the organ directly in the patient and not doing any of this using the human body as a bioreactor? So I'm going to finish my talk by just giving a case that I had encountered. And we learned a lot from this case, but it has also raised a lot of questions. And then I'll stop. 24-year-old quadriplegic male presented with difficulty in swallowing, painful swallowing, neck pain, fever, chills. The reason he was quadriplegic is that about five years earlier, he was in a car accident that required cervical spine stabilization with a metal plate. And that probably was the problem now that the metal plate, the anterior metal plate, had eroded into the hypopharynx and the mediastinum. So the CT scan showed a large mediastinal abscess. And on direct laryngoscopy, you can see the cervical metal plate eroding into the posterior pharyngeal wall over here. And on a contrast study, all that you see is a big abscess and nothing else. This is a percutaneous drain that was placed in this patient. So this patient immediately had a multidisciplinary surgical approach. The CT surgeons, the otolaryngology, and the neurosurgeons were all in the operating room. They found a large defect extending from the hypopharynx to the upper esophagus. The metal plate was removed. Abscess was drained. A G-tube was placed. The patient continued to leak. Of course, the patient is having saliva now going again and again into the big mediastinal abscess. Recurrent abscesses formation, many surgeries, and all standard of care provided. At the end of the day, nothing is working. And the patient was referred to GI to see on compassionate grounds whether we can regrow this patient's esophagus. Compassionate ground becomes very important because you cannot do any of these studies without FDA approval. On endoscopy, immediately after crossing the hypopharyngeal region, you see a mediastinal. This is the upper esophageal sphincter, mediastinum, a lot of pus inside. Again, you see a lot of pus pouring. We found a small hole. We thought, well, that is where we can connect to the remaining esophagus. But that actually was leading to another big abscess cavity. So our guide wire did not go anywhere else. We used the catheter. We injected contrast to see if we can highlight the esophagus, but it was leading to another abscess cavity. So before the whole area gets distorted and the abscesses keeps destroying more tissue and may even be lethal to the patient, our first goal was to close the defect and reduce organ deformation by scar tissue because then we cannot reverse the scar tissue and regrow the organ. So you rather regrow the organ before the organ scars down. So what we did is that we took the thin scope. We entered the stomach from the G-tube stoma. We climbed up the esophagus and we entered the abscess cavity. We coiled a guide wire over here and then we came from the mouth. We caught the guide wire. We took it out from the mouth. Now we have established access from the mouth through the abscess cavity into the stomach. And the distance we measured from here to the upper esophagus sphincter was five centimeters. So five centimeters of the esophagus was circumferentially completely destroyed. So then over the guide wire we placed fully covered metal stents and these metal stents we had to use three of them. As you will see that this is the lower stent just above the lower esophageal sphincter. Telescoped into this lower stent is the middle stent that is also bridging the medial spinal cavity part of it. And because the defect extends up to the upper esophageal sphincter, we had to put the third stent across the upper esophageal sphincter into the hypopharynx, park it right at the interarytenoid area so that the epiglottis doesn't hit the stent when the patient swallows. We waited for a few minutes to make sure that the stent is not pressing the trachea. This was a little bit heroic. The patient had so much of damage to the hypopharynx that fortunately his gag reflex was not that strong. And now we have a non-biological stent that is maintaining the tubular structure of the esophagus. But it's a non-biological material. So how can we grow the esophagus? Either we can grow the esophagus in a bioreactor, get it FedExed over to us and transplant it, or use the human body as a bioreactor and stimulate organogenesis endogenously in the patient by itself. And we went for this option. So how did we grow the esophagus back or how did we attempt to grow the esophagus back? We have the three stents in right now. Alloderm is available in our operating room. It is commercially available, cadaveric, decellularized human skin. Through the neck incision that the surgeons were using to drain the abscess, we went back, we wrapped the stents with alloderm. And to attract patient's own stem cells to this site, we sprayed it with the patient's own platelet-rich plasma. Platelet-rich plasma has a lot of receptors that attract stem cells. So FDA-approved stents, FDA-approved alloderm, and patient's own plasma. Now what is the extracellular matrix? How does it stimulate regeneration? It is some tissue that has preserved vascular network. It maintains a three-dimensional platform on which pluripotent cells can mature into site-specific phenotypic cells. It releases bioactive molecules that attract stem cells and it gets bioabsorbed and it does not induce any rejection reaction. Platelet-rich plasma, as I mentioned, has several growth factors that attract endogenous stem cells to the site. Now we could have actually done this. This is a study that I'm doing with my collaborators from overseas. We use tissue matrices and we populate them with human placental stem cells. I could have used this matrix, but this is not FDA-cleared. Anytime you play with human cells outside the human body, they have to go through a drug approval process. So we were not allowed to use this. And then we stimulated the bone marrow with granulocyte colony stimulating factor that releases the stem cells and the platelet-rich plasma attracted the stem cells to the site where we had all these placed. None of these other things that are used in animals we could use in this human because they are not FDA-cleared. Anyway, so this patient, after he got adjusted to the stent, we wanted to bring him back in six months to remove the stents. At that time, he was swallowing. We told him that after the removal of the stents, he can develop a stricture. He may still have a fistula. Nothing may have grown back. It may become a big abscess. So he decided not to give permission for us to remove the stent. Until four and a half years later, he came back with dysphagia, which was because of tissue growing into the stents where the membranes had ruptured. And now we had to remove all the three stents by using a technique called the stent-in-stent technique. And I will not describe that, but it's where you put a new stent into the other stent, cause pressure necrosis of the ingrowing tissue and take everything out. So when we took everything out and scoped him, many months after the stents were removed, as you'll notice, the hypopharynx grew back. The area where he had no esophagus, there was a new esophagus. There was some slivery pooling, some granulation tissue. And we found that the continuity between the hypopharynx and the esophagus and the pharynx was maintained. We biopsied the epithelium of the new esophagus. It was stratified squamous epithelium. When I talked to a regenerative medicine bench scientist, they said this does not mean anything because squamous epithelium can even grow on fibrous tissue. So you cannot say that you re-grew the esophagus. And of course, unlike animal studies, we cannot do full thickness, you know, resection of the esophagus to look at it histologically. So we did endoscopic ultrasound. We showed all five layers grew back, the mucosa, the muscularis mucosa, submucosa, muscularis propria, and adventitia. There were still some areas of fibrosis. And although structurally the esophagus grew back, we had to also prove that it was functioning. So we did an impedance motility study, found peristaltic activity and bolus transit on impedance. So my last few slides, this is the first human case of regrowing a long segment full thickness defect of the esophagus using off the shelf available FDA products. The patient is now 11 years out. He continues to eat orally and I see him once every so often. Question is, what did we do? We have no idea what we did. This is like from bedside going to the bench asking our basic scientists, Hey, tell us how this happened. And in our paper in the Lancet, there was an editorial on this manuscript and they recommend that based on the lessons learned from this first human case, we need larger animal studies using the same technique and then phase one and phase two trial. And if this, if these results are replicated, this will have a significant impact on patients requiring esophageal surgery for both benign and malignant conditions. So in summary, regenerative medicine techniques are very promising, not yet ready for prime time. They are coming in. Anything that is like today being looked at, I can assure you in the next three to five years will become prime time and stay tuned. They can be used for our patients with refractory strictures, patients where we do mucosal resections, big defects in the esophagus, and even patients with esophagectomies on the concept of either preserving the esophagus or regrowing the esophagus. Thank you. Professor Dua, thank you so much for that excellent presentation. The audience is ready for some questions for you. But first, as a reminder, questions can be submitted at any time online by using the Q&A box at the bottom of your screen. Our first question, Dr. Dua, is you mentioned a lot of different techniques. Are there, have there been any barriers or what are the barriers with the cell growth techniques, any regulatory concerns or barriers? Yeah, so they are country dependent. When I say country dependent, in the US, the law is that if you take a human cell and you manipulate it, it has to go through the whole process as though you are applying for a new drug to be approved. And approving a drug is a long process of trials and expense involved. So the problem we're facing is that when we take placental stem cells and culture it on a matrix and give it to this patient, it was considered to be a drug which is not approved. So now you're using a non-approved experiment on a patient. So the best I could get on this patient was to use FDA approved products that can be used for a human body on an off-label basis. So keep that in mind that if you have certain products that are available for the human body, but you're using it for an indication that is not FDA approved for that product, you can still use it as an off-label, but let your local IRB or ethical committee know about it. That I am using a stent that is actually approved for cancer, but I'm using it in this patient for a benign condition. I am using alloderm matrix, which is approved for the human body, but I'm using it as an off-label indication in this patient. So that way you can get ethical approval on compassionate grounds. Otherwise you have to get a rapid FDA clearance like this COVID vaccine. You see that it goes through that rapid process. So this may not be the case, say in India, in China, in Korea, in Europe. I don't know. So you have to go through the local board, your local hospital, ethical committee, clear it and use it. Thank you, Professor Dua. Yeah, it makes sense to, your answer makes sense to go to local individual regulatory boards. As far as the cost, I know you mentioned there's a lot of studies being done, but is cost another barrier? Are you seeing that perhaps industry is interested in this? Yes. So there is a company that has already, when I said that when you take human cells and you manipulate them, it is considered a drug. So there's a company that has taken adipose tissue from the patient and grown them on extracellular matrix. And from FDA, they got what is called as the orphan drug approval. So what that means is that they have now something that is looking for an indication, but FDA has approved it. And they, what they do is that, I think the company is based out of Texas. And they actually in the media had a big show of the first case they did in Mayo Clinic, patient having an esophagectomy for cancer. They took the adipose cells of that patient, couriered it to their headquarters, cultured it on matrix. And because it has got an orphan drug approval, they sent it back to the surgeons at Mayo Clinic, and then they are using it to regrow the esophagus back in that patient. That I suspect is going to be expensive. But when you talk about endoscopic submucosal dissection, just taking scrapings or a tiny biopsy from the buccal mucosa. And if you have a basic scientist in your university who can suspend these cells, culture them and suspend them in phosphate buffer saline, you can inject it at the site of the AFD. It only requires a week to do to culture them. That you have to go through your IRB approval. And that is not expensive. That is not expensive. Growing them into cell sheets is also not expensive. All that you need to have in your institute, a basic science lab, which has got what is called as good clinical practice clearance, which means that the product is grown in a sterile environment that can be re-implanted into the human being. Very interesting. That's a certification that the basic science lab has to get from the FDA over here. And once they get that certification, they can culture these and give it back to you when you do the ESD. Thank you. Another question is coming from the audience. Are there any injectables to prevent esophageal stricturing? I know this person has used steroids and it doesn't seem to be working that well. And the results with MMC have been inadequate. Any thoughts on that? The results of injecting steroids has been very mixed. It is so easy to do and it has got very low side effects. A lot of people are doing it without even knowing whether it works or doesn't work. So currently, what can be done is that if you have extracellular matrixes available in your operating room, SIS, alloderm, whatever is available in the local country, one option is that try to figure out a way that after you have done, say, a stricture dilation and there's a lot of mucosal trauma or you have done submucosal dissection and a lot of mucosa has been stripped out, can you apply the extracellular matrix right there and then itself and whether you want to then apply, put a small stent to hold it as was done by the Pittsburgh group for pan-mucosal stripping. Because culturing the cells from the buccal mucosa adipose tissue, suspending it and all may not be readily available in many centers, but tissue matrices, you go to your, talk to your surgeon and you'll find that they have a bunch of them lying in the operating room. You can get them and you can make it into a tubular structure, take it down the esophagus and put a stent in the middle to hold it against the wall because this matrix tends to fall off. You want to try and innovate, put a clip on the top, maybe a clip in the middle, a clip at the bottom. So these are all like trial and errors. Immediate epithelialization is the goal, whether you do it with a matrix, whether you want to grow them into cell sheets or whether you want to inject pluripotent cells. I was also thinking, and this has not been done, is to, you know, we used patients' own platelet-rich plasma to attract stem cells. So how about after we do circumferential ESD, we spray the area with PRP, which is very easy to do. I mean, we, before we do any, the way we get PRP is that we take around about 80 ml of blood, we centrifuge it and from 80 ml of blood, you will get 2 ml of platelet-rich plasma. And then you put it in one syringe and in another syringe, you have thrombin and calcium. And the moment you inject the two syringes and the two mix, they form a very sticky gel that can stick to the raw surface. So these are various tricks, but as I said, all experimental as yet. Those are very good, good insights. Thank you, Professor Duat. Another question is regarding the prefabricated stem cells, metallic stents, step-by-step. Your technique, would you prefer to do your technique with the prefabricated stem cells, metallic stents, step-by-step? Yeah. So remember the slide I showed with my collaborators from overseas, they have tissue matrix, which is SIS, and they have used human placental cells and they could beautifully grow it on that matrix. So now, if that structure is wrapped around the stent and deployed, you have all the ingredients in them. And therefore, whether that will be a shortcut to achieve something that we achieved in a long cut manner. But again, that will have to go through a lot of regulatory processes. Even just playing with human stem cell is under strict scrutiny in the US. So if everything comes prefabricated, if there is say a company or somebody tells me, hey, you know what, I have a stent wrapped with alloderm, which has got human placental stem cells. Do you want it? My answer will be, yeah, definitely I would want to try it. But whether I can get permission to try it is a different question. Yeah, thank you. But you can try it in animals, definitely. But you see the problem with animals is that I only gave you a fraction of the studies out there in the literature, just as a proof of principle, just for that. But in animals, they have grown the esophagus in so many ways that now I'm lobbying with my IRB that why should I do animal studies? Look at the literature. I don't know what is the hurdle that this animal data is not being translated to humans. I think the bench scientists and the clinicians have to cross talk and carry this science to humans. Because in animals, every permutations and combinations have been done and shown that it can work. Yeah, they're very interesting. The gastric stripping for post-esophageal ESD reconstruction. Would you say that is ready to be experimented in humans? Yeah, so that technically speaking is not, you know, like I would say a big regulatory issue. That is more of consenting the patient issue. So one of the things that you're going to do is you're going to do ESD of the stomach and the patient needs to understand that can cause a perforation and cause bleeding. But if that doesn't happen, the whole length of the gastric mucosa that you have stripped out, which you are now using it immediately to cover the esophageal mucosa is all like the same patients, same cells, and there is nothing like you're giving an exogenous epithelium of another patient to this patient. So if you're using autologous tissue from the same patient into this, this will be a standard IRB approval. It does probably may not have to go to the FDA approval pathway because see, once you take the gastric epithelial cells out of the patient and keep it in a bioreactor for two days, then it becomes a drug. Then you can't use it. If you are doing it at the same time, you're cutting it out. For example, using the platelet rich plasma of the same patient was not an issue for me because it was the same patients platelets that I gave it back to the patient at the same time. I didn't take it out. I just like spread it back. So I think it can be done. And I just would recommend that you run it by your local ethical committee that you are doing this in a patient because what if the patient has a gastric perforation and dies? So you need to get a consent and you need to get some sort of clearance locally. Otherwise it should be doable. Yeah. Thank you, Professor Duarte. That's great to hear. Another question is regarding the post-surgical distal esophageal fistulas. Does your technique compete with vacuum aspiration treatment or how do you think your technique could be applied here? So a good point over here. So my flip question is that, yes, the regenerative medicine technique is not recommending that it is a replacement to endoscopic vacuum therapy. What it is telling us is that after endoscopic vacuum therapy has failed, is not working. And now you're going to do an esophagectomy because you tried your stents, you tried your clips, you tried vacuum therapy, nothing is working. The defect is so large that now either the patient is going to be on TPN or a G or J tube feeding or esophagectomy with colon interposition. It is at that point where you can consider regenerating rather than esophagectomy. Regenerating to preserve the esophagus rather than esophagectomy. So regenerative medicine in failed endoscopic vacuum therapy is the step that we do not instead of endoscopic vacuum therapy. Thank you. Thank you, Professor Duarte. Another question is what are the chances of growing the cells on a resected part of the colon that it has been previously transplanted? Can this work on patients who are already treated in order to increase the quality of their life? So if the colon has already been interposed, see when what happens is this, when pluripotent cells or stem cells are placed at a particular region of the body, what tells the stem cell that it has to become the esophagus? Because that same stem cells, if I put say in the uterus, it will become the uterus. So they have to be local signal, signaling pathways that the stem cell gets from the structures around it that, hey, you need to become the esophagus. We don't want you to become the trachea because you are parked in the esophagus. So the remnant esophagus may be sending some signals and the location is a very complex science which none of us understand. Some signaling pathway tells that stem cell to become the esophagus. Now imagine you have an interposed colon lying in the chest and you put a stem cell. What will tell the stem cell to not become the colon? What will tell the stem cell to become the esophagus? That will be a bit confusing. And one of the problems that can come up is that when people do esophagectomy and we try to grow the esophagus, the lower part is going to be in continuation with the stomach and the lower part may turn more like a stomach tissue and the upper part may become more like an esophageal tissue based on the signals the stem cells are getting. So if you already have a colon interposition done and on the colon interposition platform you're trying to grow the esophagus, I think there'll be a bit of confusing signals coming to the regenerative process of the stem cells. I'm just speculating. I'm not sure. Thank you. That's a good answer. Thank you. To reduce strictures after extensive mucosaectomy, is reducing fibrogenesis more important or re-establishing mucosa? Yeah, so this is just like sort of a philosophical statement. If the esophagus perforates, the first response of the body is to preserve life and that will be to close the hole and that's why fibrosis happens within days. Fibrosis happens within days to preserve the life if there's a perforation so that people don't develop mediastinitis and so on. While epithelialization doesn't preserve life, it brings in a barrier so that that area where you have fibrosis doesn't become a chronic ulcer. The epithelial barrier then maintains stability. Unfortunately, the epithelial barrier takes a longer time, three weeks to develop compared to fibrosis that develops within a week. So to delay fibrosis is where mitomycin C and steroids and all of those are tried. While to hasten epithelialization, we don't have any good medications. We try to implant the epithelium right away. We try to put matrices over there. We try to inject pluripotent cells. Those are the only options we have and none of them are ready for prime time. Now one thing also, Dr. Badalak was in Milwaukee in the recent past and he's a world authority on tissue matrices and he told me that now tissue matrices are coming out in the form of liquid gel material. So we only think of tissue matrix as a sheet of paper like a matrix which is more flat extracellular matrix. But now they can take that same matrix and they can make it into a powder form that can be then applied as a liquid. So one of the things he was saying is that next time when this powder and the powder reconstituted into a gel or a liquid consistency comes out, you can spray it on the mucosal resection site and you can see if that makes a difference. So I'm sort of very interested to see if that works. Very insightful insights from Dr. Badalak. Thank you, Professor Dewa. Any concluding statements for the audience today? I thank everybody. I know it is good morning, good afternoon, good night, past midnight for people from wherever they have logged in from which part of the world. But I am very thankful that they did log in. It's nice to have. Unfortunately, the platform that we are, we can't see each other face to face. But I know that what I talked today is the future. It probably won't help us in our day to day practice as of now, but it is worth knowing that what may be coming in the future. So I'm sorry if what I talked today may not help your practice today, but it will definitely be helpful tomorrow. Thank you, Professor Dewa. I know that was a great presentation. Very, very, very insightful. As a final reminder, please do check ASGE's calendar of events as we will continue to feature relevant sessions to our global spotlight series such as this one. Our next webinar for the series is entitled African Esophageal Cancer Consortium or AFRIC and the ASGE opportunities members. That one will be held on Thursday, April 29 at the same time. This will be a good opportunity to learn about the work of ASGE and AFRIC Consortium in East Africa in different ways you can get involved. And in closing, again, Professor Dewa, thank you so much for this excellent presentation and thank you to our audience for making this session interactive. We hope this information has been useful to you. And with that, we'll conclude our presentation for today. Thank you. Thank you, Randy, for having me. And anybody has any questions, I have given permission to share my email. You can write to me directly. Thank you. I will go ahead and add that to the chat. I just shared Professor Dewa's email. If you want to reach out to Professor Dewa, kdewa at mcw.edu. Thank you again. Thank you, Professor Dewa. And thank you all. Enjoy the rest of the day or evening.

repairing human esophagus

tissue engineering

regenerative medicine

esophageal defects

pluripotent cells

tissue matrices

extracellular matrices

epithelialization

tissue regeneration

esophageal strictures

preservation of esophagus