Hello Friends 👋

In this episode of the Incubator: At the bench, we talk with, Dr. Augusto Zani, a pediatric surgeon scientist who discusses his work on fetal lung regeneration and the use of stem cell derivatives and extracellular vesicles for diagnostics and therapy. He shares his journey from surgical training to becoming a surgeon scientist and the challenges he faced along the way. Dr. Zani also explains his transition from studying necrotizing enterocolitis to congenital diaphragmatic hernia (CDH) and the unanswered questions in lung hypoplasia related to CDH. He highlights the importance of understanding the mechanisms behind these conditions and the potential of extracellular vesicles in promoting lung growth and regeneration. Dr. Augusto Zani discusses his research on congenital diaphragmatic hernia (CDH) and the potential use of extracellular vesicles (EVs) as a therapy. He also discusses the role of inflammation in CDH and how EVs have both regenerative and anti-inflammatory properties. Dr. Zani also mentions the potential use of EVs in other pediatric surgical conditions, such as gastroschisis and spina bifida. He concludes by sharing his excitement about moving his lab to St. Louis Children's Hospital in the U.S.

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The articles covered on today’s episode of the podcast can be found here 👇

Antounians L, Figueira RL, Kukreja B, Litvack ML, Zani-Ruttenstock E, Khalaj K, Montalva L, Doktor F, Obed M, Blundell M, Wu T, Chan C, Wagner R, Lacher M, Wilson MD, Post M, Kalish BT, Zani A. Fetal hypoplastic lungs have multilineage inflammation that is reversed by amniotic fluid stem cell extracellular vesicle treatment. Sci Adv. 2024 Jul 26;10(30):eadn5405. doi: 10.1126/sciadv.adn5405. Epub 2024 Jul 26. PMID: 39058789; PMCID: PMC11277482.

Jank M, Doktor F, Zani A, Keijzer R. Cellular origins and translational approaches to congenital diaphragmatic hernia. Semin Pediatr Surg. 2024 Aug;33(4):151444. doi: 10.1016/j.sempedsurg.2024.151444. Epub 2024 Jul 2. PMID: 38996507.

Biouss G, Antounians L, Aguet J, Kopcalic K, Fakhari N, Baranger J, Mertens L, Villemain O, Zani A. The brain of fetuses with congenital diaphragmatic hernia shows signs of hypoxic injury with loss of progenitor cells, neurons, and oligodendrocytes. Sci Rep. 2024 Jun 13;14(1):13680. doi: 10.1038/s41598-024-64412-x. PMID: 38871804; PMCID: PMC11176194.

Ganji N, Kalish B, Offringa M, Li B, Anderson J, Baruchel S, Blakely M, De Coppi P, Eaton S, Gauda E, Hall N, Heath A, Livingston MH, McNair C, Mitchell R, Patel K, Pechlivanoglou P, Pleasants-Terashita H, Pryor E, Radisic M, Shah PS, Thébaud B, Wang K, Zani A, Pierro A. Translating regenerative medicine therapies in neonatal necrotizing enterocolitis. Pediatr Res. 2024 May 28. doi: 10.1038/s41390-024-03236-x. Epub ahead of print. PMID: 38806665.

Figueira RL, Khoshgoo N, Doktor F, Khalaj K, Islam T, Moheimani N, Blundell M, Antounians L, Post M, Zani A. Antenatal Administration of Extracellular Vesicles Derived From Amniotic Fluid Stem Cells Improves Lung Function in Neonatal Rats With Congenital Diaphragmatic Hernia. J Pediatr Surg. 2024 Sep;59(9):1771-1777. doi: 10.1016/j.jpedsurg.2024.02.029. Epub 2024 Feb 28. PMID: 38519389.

Antounians L, Catania VD, Montalva L, Liu BD, Hou H, Chan C, Matei AC, Tzanetakis A, Li B, Figueira RL, da Costa KM, Wong AP, Mitchell R, David AL, Patel K, De Coppi P, Sbragia L, Wilson MD, Rossant J, Zani A. Fetal lung underdevelopment is rescued by administration of amniotic fluid stem cell extracellular vesicles in rodents. Sci Transl Med. 2021 Apr 21;13(590):eaax5941. doi: 10.1126/scitranslmed.aax5941. PMID: 33883273.

Zani A, De Coppi P. Stem cell therapy as an option for pediatric surgical conditions. Eur J Pediatr Surg. 2014 Jun;24(3):219-26. doi: 10.1055/s-0034-1378150. Epub 2014 Jun 19. PMID: 24945441.

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The transcript of today's episode can be found below 👇 

Betsy Crouch (00:02.388)

Hi everyone, welcome to At the Bench, the neonatal physician scientist podcast where we are incubating discoveries. I'm Dr. Betsy Crouch, co -hosting today with Dr. David McCulley. We are both neonatologist physician scientists and our co -host Dr. Misty Good is sitting this one out. David, do want to talk about our guest today?

David (00:20.918)

Thanks, Betsy. Yeah, we're very excited today to be talking with someone outside of neonatology. This program has been a great place to meet with physician scientists who are doing exciting work related to newborn medicine and newborn health and disease. And today we're excited to be interviewing Augusto Zani, who is a pediatric surgeon in Toronto. Augusto, would you mind just introducing yourself and telling us a little bit about the work you do and what your lab environment is like.

Augusto Zani (00:53.984)

Okay, hi Betsy and David. First of all, thank you very much for the kind invitation. I'm thrilled to be here on this podcast. I'm Augusto Zani. I'm a surgeon scientist. I'm a pediatric surgeon in the division of general surgery at the Hospital for Sick Children, Sick Kids in Toronto and associate professor of surgery at University of Toronto.

My lab studies fetal lung regeneration, specifically using stem cell derivatives. We actually do a bit more than that. And we started with that and then learned a lot about the power of exercise of vesicles. And so they can be used for diagnostics or for therapy. And so we've expanded on that on a bunch of perinatal conditions, so of course congenital and pharma hernia, is what we study the most, and that's why I said fetal lung regeneration, but also spina bifida, gastroschisis. So we use it as a tool, and we're learning a lot from that.

David (02:11.074)

It's really great to have you on the show today. And I knew when I first got to see you present at the CDH International meeting in Scotland that it seemed like you were doing really exciting work in the field that I'm really invested in, in studying congenital diaphragmatic hernia. But I've gotten to know a little bit more about the research that you've been doing lately with extracellular vesicles, studying a number of different congenital malformations as you just listed.

I was wondering just before we get into the work you're doing now, just could you talk a little bit more about your training and your background and really how you started to pursue a physician scientist career path?

Augusto Zani (02:53.598)

Yes. So I am from Italy, originally born in Rome and went to University of Rome, La Sapienza, where also started my surgical training. And then I was lucky enough to go for an experience abroad in Ireland, in Dublin. And very different environments. I could see that people really could learn with the hands-on experience, you know, the various skills and techniques and how to operate on babies and kids. And in Italy, it's quite different. In Italy you, as a surgeon, you learn by watching. And this is not really true. It needs to be much more hands-on. And so I, right away, discovered this world abroad and thought, okay, this is actually something that I want to pursue. I want to pursue a career in surgery. And I got into medicine in general because I was inspired by my pediatrician. He actually was an Italian and had studied and worked in New York City, the Albert Einstein Institute. And so I always thought, okay, this is guy that was a different kind of doctor from all the others that I met along the way. And so I loved biology, I loved anatomy and put everything together. So I set off to med school to be potentially a pediatrician. And then first summer.

Betsy Crouch (04:42.34)

Can you pause and incubate on that for a minute? What about him was inspiring?

Augusto Zani (04:49.662)

The way he really thought out of the box, his approach. It was an American approach. in this is of course the 80s in Italy and it was very different. Also the language that he would have, the way his office looked like and everything looked very cool. The books he had in the office, everything like really talking of another world, in fact was very inspiring. And the way he interacted with me or with other, I could see my brothers, he was different from all the other pediatricians that we had met before and after.

A lot of times, unfortunately, also now, I think it happens that it's the interactions between the physician and the family, especially with the pediatrician and the family, it's not with the actual patient. instead, I was on the hot seat, I was the one answering the questions, and it was very much more personal. So, all of that was very inspiring to me, and I thought, okay, I loved kids.

Augusto Zani (06:16.064)

This is me at the end of high school. You know, in Italy, like in the rest of Europe, you don't go to college first and then university after. You do everything altogether. You need to decide very early on what you want to do as a career in the future. So I went to med school and very early on I had the chance to go to Stockholm in the summer for an exchange program. And so I had this idea of becoming a pediatrician. But then I find myself with another six people, five girls and a boy. And of course, I was like 19 or 20 and the boy always thought he wanted to be a surgeon. And this is, you know, my friend who is actually in fact now a surgeon at the Cleveland Clinic in Miami. Since he was five or six, he always thought, okay, I'm to be a surgeon. So he said, okay, let's go and see some surgeries. I thought surgeries. Now, in fact, I have to say at the end of the experience, I was fascinated and was really incredible. But it wasn't really love at first sight. In fact, I sort of fainted the first day I was in OR. I really didn't crash on the floor, but I had to sit down. I felt dizzy. And it was like, my God, this guy was performing thyroid surgery. And he started, when you think of a surgeon, you always think of something very elegant. Instead, this guy, as in fact we a lot of times do, dissected the planes with his fingers. And so he put these fingers into the neck of the patient, was an adult patient. And I thought, my God, this was awful. However, that experience was amazing for me because this guy then who was doing oncology surgeries was taking the specimen to the pathology lab and it would be looking at the specimen, sorry, under the microscope. So I thought that this is like... wow, you basically a mix of the two worlds. I love biology, I love anatomy, and then you at the same time you have the anatomy and of course the manual skills.

Betsy Crouch (08:17.266)

That's not on the bedside. That's like bench to surgical point. Yeah.

Augusto Zani (08:20.51)

Amazing. I thought, okay, I want to become a surgeon. So I went back to Italy and said, I want to become a surgeon. It took me a few years to realize that you can be a surgeon fully dedicated to kids. And that's why I connected with the pediatric surgeons at University of Rome. In Italy, you need to write a thesis at the end of your studies. so we wrote a paper on glossophexy as an alternative to orthophexy for patients with respiratory distress following esophageal atresia repair. So the idea is that you anchor the tongue. The idea was that there was tongue instability like you have with pierre-robin sequence. Anyway, the funny thing is that I write this thesis, but also we wrote an abstract, which we sent to the to BAPS, which is the British Association of Pediatric Surgeons. The meeting was in London just a week after my graduation. And funnily enough, people, the co-authors, I was of course the youngest, but the co -authors apart from the senior author could not attend the meeting because either they had their spouses pregnant and about to deliver to people, and two instead could not speak English. So I thought, okay, I'm going to go and give it a talk and I go and see how it goes. And it was amazing. It was like a big, very well attended meeting with 500-600 people in the Royal College of Surgeons of England. It's a very formal place. And I remember I knew my slides by heart. It was the first time they were using PowerPoint, it was 2001. And I knew my slides so well that slide two or three I say to myself, good Augusto, you're doing really well. And then slide five, there's a blackout. And I say, my God, what happens here? I don't know. And so, but when they restarted, had this system sometimes in conferences they still have of the traffic lights. And so you needed to give the presentation when it was green or amber. And then they asked me, the president of the association was actually chairing that session, asked me, do you want to start again from scratch? And I absolutely not. I know it all. So I gave them my presentation and then they liked the paper and in fact they got then accepted.

So for me again, very serendipitously, that was like seeing another world. So I went back to Italy, started my training, but really wanted to go abroad and I thought, okay, I do some clinical research here and there. I wasn't really a big fan of research as such. But then when I went to Dublin 2004, 20 years ago, I realized that actually the people that done research were the ones who could be in the OR and could operate. So I thought, okay, research sucks, but I'm going to do it because it's going to be the key to open the door of the OR for me.

David (11:37.58)

Can you say that one more time? I didn't quite understand. So, you needed to do research in order to be able to operate.

Augusto Zani (11:45.332)

Well, in order to be able to be selected to have then a training position in a good place, this wouldn't apply to Italy, but this was there in Ireland. So I thought, okay, something like this. And then again, I was in Ireland, the weather was crap. And then I can say crap, but I love Dublin and, but really.

David (11:56.674)

Got it.

Augusto Zani (12:11.4)

And then we flew to the BEPS meeting there that year was in Oxford. And I went to this meeting and I see that all the best presentations are given by people from the US. This meeting was very international. It's still not as much as in the past. And so I thought, my God, I want to go and do research in the US because that's where they do the best research. There was an Italian guy there who had just come back from Harvard and I asked him, Paolo, how did you manage to get to Harvard. And I just sent an email. And I was so naive that I believed that. So I went back to Dublin. And every day after work, I would go to an internet cafe where there was a bunch of people playing video games and me sending emails to big universities that I heard from in movies or so. Let's go to, I don't know, Yale. Nobody of course is answering apart from a guy that Dr. Dunn who's doing research intestinal regeneration in LA and the guy said you can come over. I don't have funding for your research for a salary for you but you can

do research for me and be at the bench and this could be, and this was 20 years ago. And I thought that would be awesome. He said, I'm going to a meeting, the AAP meeting in San Francisco. I thought, my God, yes, let's do that. So let's meet there. And when I went there, unfortunately for some reasons he had some personal issues. He didn't attend the meeting. And at the meeting I met another two people. One is an Italian guy who had left Italy 20 years before and he was in a Great Ormond street in London.

And that was Agostino Piero. And the other guy was Paul Losty. He was a surgeon at Alder Hey in Liverpool. Both offered me to do research. I met them. And this guy from the US, unfortunately, didn't show up. I also figured out in the US, I thought, OK, he doesn't give me a salary, but I can go and work in a fast food McDonald's or whatever, but you need a green card to do that. So, I even applied for the lottery and of course it's not a lottery that you win very easily. Anyway, I dropped the US thing and I thought, okay, Liverpool is the same latitude of Dublin. The weather is crap. So let's go to London. And so that's how I went to London. And Agostino at that time was studying and still in fact his lab is studying necrotizing enterocolitis. We didn't have many cases of NEC in Italy. And if you think, you know that of course the incidence of NEC is very variable around the world. And in the UK they had really very high numbers. And so I really opened the book.

Betsy Crouch (15:15.764)

Do you think it's

Augusto Zani (15:18.592)

I don't know. I don't know. I know that in Japan it's super low and maybe it's because moms eat a lot of sushi when they're pregnant, so maybe there's something there. But it could be that there's something also with the Mediterranean diet, as you mentioned.

Betsy Crouch (15:32.198)

Yeah, don't. Yeah, this puzzles me too. But it's not I, you know, we need Misty to tell us what what exactly it is about the diet. But yeah.

Augusto Zani (15:42.814)

So I ended up in London doing a PhD at Great Ormond Street. Again, I thought I'm going to do it so that then I can be trained properly in the UK. I was given a project whereby they wanted to start using stem cells to regenerate an intestine to attenuate the inflammation in a neonatal rat model. So it took me nine months to set up the model. It was like a nightmare, but I started really enjoying being at the bench and doing the experiments and sort of forgotten about the UR, although I was missing it a lot, but I really felt I was doing something with a purpose. I'm very skeptical.

Romans in general, people that are quite cynical and skeptical. I thought, this stem cell business, it's sexy now, it's fashion, it's not gonna work. So we inject this GFP labeled, minority of stem cells into the abdomen of these pups. And then we look for days, not hours, but days to look at us because of course it took us quite some time also to figure out what to do and how to do it. And then I saw that actually, yes, the cells were there. There were a few cells, but it's very interesting. I didn't believe they could do something. I was sure it wasn't. Instead I started doing experiments and especially I could see that they could survive longer. These pups that got injected. And then of course we did a number of tests looking at also the test of function and of course the levels of inflammation, of oxidative stress and from being a non -believer I really started believing in what I was seeing. And I thought, okay, maybe it's my hands. And so I had somebody who was helping me and this person being blind, completely blinded to the groups was doing the same under my supervision, making sure that the model was working, everything was fine. Again, we could get the same results and was like, my God, this really works.

Betsy Crouch (18:13.426)

Yeah, it was rigorous.

Augusto Zani (18:15.52)

I was very fascinated by that. So I finished my PhD and decided that I wanted to pursue further training. And so I did my UK training in pediatric surgery. six years, you do a lot of pediatric urology too, about 50 % of the work is also pediatric urology. So from Great Ormond Street, then I went to King's College Hospital where you do a lot of HPP surgery. And then I was for a year in Belfast and then for two years the Royal London. These like four or five years it was mainly OR. There was no lab and I was really missing all of that. I thought, okay, it's, yes. . .

Betsy Crouch (19:00.99)

Can we stay there for a minute? Because I think that this is a hot topic, I would say, in pediatric training at the moment, which is just that I think to be a scientist used to be something that was more commonly a goal of doctors generally. And there's something about the way that we're training or the way that we're communicating about our science these days that is less winsome and less welcoming, I think. So you talked about your journey and you said that you said it well, but that you weren't that interested in science, something like that. And that transformed into a real captivation with your experiments. And you said that you spent five years in the OR and you missed being at the bench. So can you talk a little bit more about what was what was discouraging about being a scientist before you actually got to do it and then how your experience of doing a PhD changed your perspective on that.

Augusto Zani (20:10.016)

Right off the bat, can tell you that when we were in med school, they made us read scientific papers and it was like a nightmare. First of all, because you read something that you don't like. If you like reading in general, but I don't know, you like science fiction, they make you read something about, I don't know, something else. It was very boring and I was like, my God, this is really... I always loved reading and writing, but the scientific literature was so boring and I really didn't understand much of it. And it's so rigorous. Every word, of course, it's not like reading a novel, obviously. So that was already something that was a little hard. You when you read a review, maybe you understand, but you need to be an expert in that specific topic. Then you'll see the nuances you read between the lines.

And that wasn't really coming through when I was younger. And then one of the things that really I enjoyed the most was, of course, when I was at the bench, I did my experiments. And then I went to the meetings to present my results. And I was, of course, very, very scared. what really encouraged me to keep going was the fact that you would get a lot of feedback and good feedback. And people can be maybe nasty, pose a nasty question, usually it's against your supervisor, your senior author, not against you. So at the end of the day, I enjoyed doing the meetings and getting feedback and getting also, you know, people coming up with new ideas. And that was like, okay, want to know more. And so that was really enticing.

But when you do surgical training, and I'm sure it's also the same when you do your neonatology training, you're really fully focused. I mean, we take care of so many different types of patients that the subject's so broad that you need to need to focus on that. So I have to say, I was very sad those years. I mean, I was very happy to see my skills in surgery that were of course developing and operating more and more fancy cases. But the why and what's happening, it was like, you know, I was trying to, I was again an immigrant in the UK trying to break through there and my goal was to get a consultant position in London. I loved London. I felt like being a home, I lived there for nine years. So I thought, okay, I need to, I cannot get a position also where you have a lab. That's something that's very difficult. And there's in fact only a couple of places which would be mainly London and Alder Hey, especially in the past, where there would be academic, pediatric surgery done at the bench. So I thought, no, that's not for me. And so, but I felt a sense of some sadness, you know, going to conferences and presenting clinical papers, case reports and so on, seeing of course the science going through and thinking, okay, that was a very nice chapter on my life, but that's closed and now of course I'm gonna be a surgeon. And not knowing, because I have to say in Europe and in the UK, a surgeon scientist figure is not really that present. They're really unicorns. Unfortunately, they're becoming unicorns also in North America, but less so. And then after my training, everyone would go for a year abroad. And by that time I met my wife, who's also a pediatric surgeon, and she hadn't done the USMLE, which actually I had done, because I still had that American dream of going and training in the US, at least for a year. All the best TV shows on doctors are set in the US. ER, Grey's Anatomy and so on. So I absolutely wanted to go there. But okay, I had not done that. And at the same time, Agostino Piero had moved here to Toronto and had become the chief of surgery here. I was actually a proof of this emails with Cincinnati Children's with the program director there because I really was US bound and my wife, I was not even, she wasn't my wife yet. And so I was not thinking too much about her when this opportunity of Toronto came up and I thought, this is the best for me and for her. She can do maybe research for a year with Agostino. So she came over and she set up the mouse model of NEC for his lab. And I came over and I was the neonatal surgical fellow for a year. And during that year, I was also offered a local position in London. So I was like really...

You know, my goal was to get more numbers under my belt and go back to the UK with a better CV to compete for a consultant position in London. And instead, I was wanted a surgeon scientist. They had posted a job. I went for the interview and I was lucky to be selected.

And the dream was coming true because it meant for me to having my lab. Also at that time, I didn't know what the rules were here. The rules are that say you need to really show independence from your mentor. And we have a program here that's called the Scientist Track Investigator to give you start the funds, which quite generous, but just for three years. And then you need to show whether you can in fact be a scientist on your own. And I was lucky because in fact my scientific mentor was Janet Rossant, who had just stepped down from being the chief of the research institute here at SickKids. And for me it was just a name and then instead I discovered that she was like the real deal in stem cell research and a fantastic mentor.

I think I actually managed to pull through these three years and basically start my dream job of being a surgeon in a high volume, high complexity center, like sick kids and having also a lab. But the rule was that I couldn't do the same thing of my mentors. So I had to drop necrotizing enterocolitis, which I thought, my God, this is like my life is to save the life of these kids. So I thought, okay, what is the other condition that really has so many unanswered questions? And there's so much that in fairness, also, Elke had done some research in years before with Prem Puri in Dublin on congenital diaphragmatic hernia. So I literally opened the book, I knew about pulmonary hypoplasia, but I never really dissected the lung. I'd never done anything on fetal lungs especially. And I opened the book and started reading and I had questions and I couldn't find the answers. And so I realized that actually there was a lot still to learn. And then from that experience of the stem cells, that was when I was doing my PhD, we could see an effect. There was a phenotype, definitely, but there were very few cells that could integrate into intestinal tissue. And at that time we talked about the paracrine mechanism and we thought of growth factors. We were really scratching our head. We had no idea what would be. And I started reading, now this is 10 years before, 10 years after, looking at papers.

We were talking about microRNAs and extracellular vesicles. I thought, okay, again, being super skeptical, I thought, okay, let's start the experiments using stem cell -derived extracellular vesicles and let's see if we can regenerate this hypoplastic lung, if we can rescue fetal lung development.

David (28:58.402)

Can we like start there and just talk about this a little bit? Because the transition from necrotizing enterocolitis to CDH I think is really interesting. And I'm curious like what question it was that you were wondering about in lung hypoplasia related to CDH that you thought like that there was a translation from necrotizing enterocolitis where it seems like there's kind of an injury, there's inflammation and cell death, as opposed to lack of cell growth in lung hypoplasia. Can you just talk about your thinking there or how did you see that that would translate?

Augusto Zani (29:33.386)

Yes. So I think that what we were seeing the most effects on were in fact on the epithelium in the intestine. And so I knew that pretty well. Of course, it was the LGR5 cells. We thought that the stem cells that we were injecting were not initially when we wrote the first grants, we thought we give the stem cells that will differentiate themselves and repopulate the epithelium.

This was our paradigm. I was absolutely wrong. And when I finished off my PhD, and I was actually lucky to publish this in Gut, took us a few years, but it was definitely something that made an impact, and especially not just on my career in terms of CV, but also on trying to understand the mechanism, which is the interesting part of being a scientist. You want to understand why things happen.

It was, thought that the stem cells cross -talk with the LGR5 cells in the niche and then they stimulate the repopulation through the progenitor cells. So I thought it must be the same also in the lung. And I was frustrated when I realized that in the lung there's not such a single population of cells from which, and the epithelium is very different, of course. So, you know, all these kind of knowledge that you have from med school was there, but really I was there more for passing a test or I had not really thought through and I thought again if we give the vesicles, it's like giving the stem cells, we're maybe influence the progenitor cell populations and then those will actually restarted the arrested lung growth. And I thought that was a good paradigm, which actually has changed recently, in my mind at least. And it's funny because you write things on papers and they stay there forever. And one of the things that we thought, we did experiments with different sources of extracellular vesicles from bone marrow stem cells versus amniotic fluid stem cells. And we could see better more results in terms of regeneration and rescue of, know, especially promotion of branch morphogenesis and cell differentiation with the ones from the amniotic fluid. And we thought, you know what, bone marrow stem cells, mesenchymal stem cells, stromal cells are actually very good with BPD, but BPD is an inflammatory disease. CDH, these lungs are not inflamed, and that's why maybe these other cells work better. Now we just actually came out with a paper a month ago, it took us a little while to publish it, but where we did single nuclear RNA sequencing and actually found out the fetal lungs of rat pups with CDH are full of macrophages and have an inflammatory profile. And again, my skepticism, I thought that the postdoc that did those experiments actually made something up. And then I went, we validated on the same model and I thought, okay, it's just the nitrofen model, it's a chemical. Then we went and saw the autopsy samples of babies that died with CDH compared to controls and it's full of macrophages. And then we thought, okay, let's see in the other models, in the rabbit, in the mouse, we see the same. So maybe now the paradigm is actually that there's an anti -inflammatory component, not just a regenerative component of the extracellular vesicles, but also an anti -inflammatory component. And so that story that I had in my mind that these vesicles were from cells that were not so much anti -inflammatory versus the others. Actually, it wasn't really the case.

Betsy Crouch (33:56.904)

So now what's your, I'm on the edge of my seat. So now what's your paradigm? If it isn't.

Augusto Zani (34:01.512)

My paradigm, I can show you, I've got a board there, but I know this is a podcast. I think that's the compression of the lung that actually starts an inflammatory cascade. This, of course, we're talking about congenital deformity, and there's compression that actually compresses also the heart, and now we're studying also the heart. And then the inflammation leads to an alteration of epithelial, and so impairment of growth, an impairment of maturation, an impairment of vascularization. And so that's why you then end up having vascular modeling. There's also something that is quite cool. We haven't really published on yet, so it's, which is EMT. see that we've done some sequencing studies and when the lungs are hypoplastic, there's a lot of EMT, when we give the vesicles, there's a lot of EMT. There's more, they switch back from mesenchymal to epithelial cells. And we're studying that in particular. But I don't think it's only the compression, congenital pharmacogenesis is much more than that. But I think that this is what we are working on.

Betsy Crouch (35:20.36)

So do you think the macrophages are responding to the compression, the changes in the pressure? How do you think the macrophages fit into the compression part?

Augusto Zani (35:28.092)

Yeah, so we're doing now, we have a knockout mouse model and where we actually knock out the macrophage in mice. And so there's no macrophages and we see less severe pulmonary hypoplasia. There's still some, but it's not as severe as. We're trying to understand now doing some special transcriptomics, you know, which one is the egg and which one is the chick, but it's not easy. Especially in fetal life, there's macrophages arrive at different stages, but it's kind of intriguing. It's very far away from how you fix a diaphragmatic hernia in the OR, but both are fascinating. For me, the fact that you can be in the OR and doing the surgery is great, but at the same time, trying to understand why this happens.

And maybe one day you don't need, you will still need the surgeon for the hole in the diaphragm, but you might have already a therapy that you can give in utero that actually attenuates the inflammation and promotes lung growth and differentiation and normal vascular development. That would be the dream.

Betsy Crouch (36:48.788)

And then to gear the conversation towards my favorite cells, the vascular cells. Because that's a mandatory part so far of all the half of the bench podcasts, which I'd like to take my prerogative to do. You've talked a lot about the epithelium in the lung. And so guess two different types of questions. It seems like the extracellular vesicles are having an effect on the epithelium certainly. But then you've mentioned the vasculature. So do you think that they are targeted toward the epithelium or do you think there's a vascular effect as well? how is that being targeted differentially?

Augusto Zani (37:29.376)

Great question. I actually have evidence that they do that. There's definitely the influence of the lung tissue at the multicellular level. What we've done, of course, you cannot get a piece of lung from a fetus with CDH and do experiments in the lab. So to avoid that, we've been able to take actually lung specimens, fetal lung specimens from terminations of normal babies between 15 and 20 weeks of gestation. And then we've induced hypoplasia by giving a chemical similar to nitrofen, it's also affecting, it affects Rac1. And so with this Rac1 inhibitor, we block the branching morphogenesis, but it has also an effect on the cell differentiation and also the level of the vessels. So we see a phenotype of vascular remodeling. And then we give the vesicles, so we gave the vesicles, of course, these are X -plants. So there's not so much of any, there is some anti -inflammatory component, but there's no macrophages, if you wish. But we do see that when we did, again, single cell transcriptomics.

We do see an effect on all the cell types, all the three lines of cell types, let's say. And so, including the endothelial cells and this work that is hopefully going to be soon a list on bioRxiv by the end of the year and then hopefully it's going to be published next year. But it's quite interesting. You know, the vesicles have, it's a cocktail of so many different things. There's of course lipids that we have not studied too much, but there's of course proteins and there are of course small RNA species. And so we of course have worked a lot on microRNAs, but now recently actually just come out of a lab meeting this morning, we have some very interesting data on long non-coding RNAs that seem to be also important for development has been studied. They have been studied mainly in cancer. But there's something very interesting. So we don't know really everything about really, we don't know what's inside the vesicles. We don't know everything about, for instance, these pi RNA and long known coding. It's really, but for sure there's genetic material that influence the target cells.

David (40:23.382)

Yeah, I think that I've asked you that before just how to try to figure out, know, which cells are receiving, you know, which components from the vesicles and how does that affect their development? And because I think you're learning a lot more about the mechanisms of abnormal development and normal development. And even though this seems like a relatively straightforward intervention for therapy, it seems like you're also thinking about how it's teaching you more about the mechanisms of disease and how you could intervene in a completely different way much earlier during fetal development. I wondered just a little bit more about that. Is that the way you think this will ultimately go or what's your thinking?

Augusto Zani (41:06.868)

Yeah, no, you're spot on. Absolutely. As I said at very beginning, for us, extracellular vesicles are definitely a tool potentially for therapy and possibly also for diagnosis. But also they tell us a lot about the pathogenesis and what actually is happening in these organs, in these developing organs. It's not easy to tease out what the players are. We've done functional experiments, mechanistic experiments where we have blocked some microRNAs with the antagonists. So you can see the effects. But again, it's like a big cocktail of quite many molecules. But it's quite fascinating. don't want to say that it's, again, this is the means being skeptical. I don't want to say that this is the panacea that will fix and cure everything.

But definitely there's something there that is worth it to study and will give us also a lot of information. One of the things that actually we're learning in the lab, again, going back to NEC, necrotizing enterocolitis, said to you, there was sort of a gentleman's agreement also with my now partner and at the time boss, Dr. Pierre, that I would not do research on necrotizing enterocolitis and that was good was also for me, for my career, but I always wanted to look into the brain of these pups when I was doing my PhD more than, well, now it's almost 20 years ago. And so we collected these brains and we started working on that. And we also published on that. And now, of course, NeuroDevelopment NDI is one of the most intriguing aspects of necrotizing enterocolitis. What we are working on now, and this is in collaboration with Estelle Gowda, who is the chief of Neurology here at SickKids, is on extracellular vesicles as biomarkers of brain damage in babies with NEC. Because again, I'm a surgeon. There is a baby with free air, neck free air. It's a no -brainer. You need to go to the OR. Or if you like drains, you put a drain. don't, but that's another story. But then there's a baby that maybe comes through and they're very sick. They have stage two necrotizing enterocolitis and we're sitting on them. They're not deteriorating. They're not failing medical treatment. And we're only looking at them and we're looking at their intestine. We're doing x -rays or ultrasound scans. We know very

little about their lungs and basically nothing about their brains apart from what we, unless they have of course hemorrhages and so on. But at the cellular and molecular level, we know nothing about those brains. And so what if we can actually use the vesicles, in fact cross the blood brain barrier to get information about those brains. And for me, surgeon, tell me if this is a baby that is suffering or maybe it's somebody that needs surgery earlier rather than later.

And so with that, we started with the mouse model of NEC, and we have some very interesting data. And then now we are profiling the plasma -derived EVs from babies with necrotizing entrocolytis versus age and sex -matched controls.

This is where, again, the power of the EVs that you can play with and try and understand what happens, not necessarily just to fix things, but also try and understand what happens in these various organs.

Betsy Crouch (45:09.586)

Yeah, I think also another fascinating topic is the different sources of EVs. I was wondering what you think, you alluded to earlier the difference between the mesenchymal stem cell derived EVs versus the amniotic fluid ones. And I guess one targeted question is, you know, why for, you know, for CDH specifically, do you think the amniotic fluid ones are so effective?

Augusto Zani (45:39.104)

That's a great question. So when you go to these EV conferences that grow bigger and bigger every year, you realize that there people that study tomato -derived EVs or plant -based, know, bacteria EVs. there's EVs from, first, milk EVs are very popular also. Going, again about necrotizing enterocolitis. I think that motor fluid stem cells are multipotent.

Augusto Zani (46:07.552)

And they are less committed and that's why they have maybe a broader range of potential differentiation compared to the mesenchymal stromal cells, the more adult stem cells. I go back to that paradigm and maybe if we were to use the embryonic one, it would be even more. But it's, to be perfectly honest, it's difficult to really pinpoint because we don't know what causes the pulmonary hypoplasia. And if we knew that it was something specific, then I could answer the question more in a more scientific way.

Betsy Crouch (46:53.842)

And how in the future are you envisioning, like, how would you get the amniotic fluid, EVs, if that's your therapy of choice? I would smile if you want to.

Augusto Zani (47:04.352)

Yeah, that's, again, talking about changing paradigm over the years. One of my first slides 10 years ago was like, okay, you have a mom that has a diagnosis of congenital diaphragmatic hernia. They typically go for amniocentesis. You would get some of the amniotic fluid and then you will grow the cells, isolate EVs and then give it to the baby. And now instead, of course, the paradigm has changed in the sense that it would be much easier to have an immortalized cell line of, you know, off the shelf. It's more of a drug therapy where you know that the cargo is stable. There's very minimal batch to batch variability and you can give exactly always the same. And so it doesn't matter. It doesn't need to be autologous. So that's how I would consider doing that.

Betsy Crouch (47:53.332)

Cool, good luck. Yeah, I think that's a good paradigm.

Augusto Zani (47:54.88)

Thanks.

David (48:00.18)

I want to respect your time, but I also, you rattled off a list of other pediatric surgical conditions that you're thinking about using this similar approach with. I just thought it'd be exciting to close with just hearing a little bit more about the other etiologies you're interested in and how you think this extracellular vesicle work applies to them.

Augusto Zani (48:24.564)

Yeah, so I, you know, again, if you think of the inflammation and you think of gastroschisis, survival is pretty good. We're not talking about survival of babies with NEC or congenital diaphragmatic hernia, but definitely there's quite a number of babies that have complex gastroschisis and that having gut failure. And it's all about the inflammation in the intestine is floating in the amniotic fluid. And so one could think of treating that with the vesicles in different ways. I know that there are places in the States where they're now trialing after experimental work done in large animal models are trialing closing the defect in utero. And it would be good to do it while you also can spread a bit of EV base cream on these intestine. Same for the spina bifida, which is more of a, know, spina bifida, babies of course, the MOMS trial, we know that it's better to close them in utero. But then still there is quite a fraction, a number of babies that still do poorly. This is because again of the neuroinflammation. So this is something that we want to study more. But we definitely want to understand whether we can apply maybe a patch that can release, slow release of extracellular vesicles or other forms of topical administration. This is work that also in collaboration with people at Cincinnati Children's.

You know, the more I know that the problem for everyone who's a scientist is that you want to spread, you might end up spreading yourself too thin because you have so many questions and the more questions. And sometimes people knock on my door and we have a big group studying cystic fibrosis here because the Delta 508 gene was discovered here at SickKids. And so they said, do you think we can get EVs from the sputum of patients with CF. And yes, we do. And we actually just got a paper published and now we continue on that. And then there's somebody who's studying Rett syndrome and astrocyte derived EVs. And so we helped them with that. And so it's fascinating. It's great to see that other people are interested in the topic and obviously want to make use of the vesicles and we're very happy to of course help them as a lab. Bronchopulmonary dysplasia obviously, there's a trial using stem cells. I think the future will be cell free rather than cell based, but of course this is still for debate.

So there's a lot of potential applications, clinical applications in pediatrics, especially in perinatal medicine, as I mentioned. My lab, as I said, focuses mainly on CDH. So what we've done is we explored a lot the rat model and then the rabbit model and the mouse model. And now we started doing FETO in lambs. FETO, course, is what's on the market now. I'm biased because I worked, as I mentioned before, at King's College Hospital in London, where they built the FETO center. And I've been collaborating with Jan Deprest for a number of years now. And so the idea is to deploy the balloon, but before deploying the balloon, giving the vesicles. And so we have some interesting data that are coming up. And so that would be the first way to deliver the vesicles in the future. Then obviously the dream would be to give just a shot or multiple shots of EVs to a mother, just IV, to reach the fetal lung, but it's not that easy. For now we're trying with topical administration in combination with FETO.

David (53:05.364)

It's super exciting. It does seem like the thing that sort of spans all of those different topics is interest in like certainly mechanisms of disease, but mechanisms of development, regeneration, and suppressing inflammation that's happening because of abnormal development or something like that. It seems like that's where this approach generally is going to be teaching us a lot in all of the different diseases that you are talking about. I think one of the things that's so exciting about your work is that you span so many different fields. And I was just wondering, because you seem to have this big vibrant lab, could you talk a little bit about the people that are in your lab? Like, what are they training in? Are they training in clinical medicine? Are they doing developmental biology or cell biology? Or just what's the kind of focus of your group?

Augusto Zani (54:00.67)

Yeah, I think I'm very privileged to have a fantastic group of people that it's a bit of a mix. Some are, yes, developmental biologists or, I have to say, to me it's the most aspired to get into med school as it happens nowadays. It's not easy at least here in Canada. Some others are, there are some pediatric surgeons that again, because of the need for improving their CV and possibly getting into training, they do lab work. But what I'm really happy with is that they all get sort of bitten by the bug and they go back and they, unfortunately, change their life a lot because they can't just do research for the purpose of this, like something just to check the box and then instead they fall in love with it. And so I'm lucky I have to say all these fetal surgeries, my wife has also been helping a lot, being a pediatric surgeon she's done also a lot of the fetal surgeries. So we have collaborators also in other centers like London, Ontario with Adriana Buder, the MFMs at Mount Sinai. But otherwise it's my PhDs, my postdocs that really are doing a lot of work. And now the lab is changing face a little bit because I make now for the first time the announcement, but the lab is moving, is moving to the U.S. And so I, yes, so after 20 years, if I said 20 years ago, 2004, I got a sort of an offer. Now I'm going to be at St. Louis Children's Wash U from January 2025. So the labs, thank you, the stays here open for another, I think we have funding for another three, four years. But we are starting another level of research, I think, where I can see that the sky's the limit. So maybe it's me with my American dream come true, coming to the States. And yes, I have to say I need to learn how to play the NIH game. So far it was CIHR. So I see your faces. I can imagine it's going to be tough. Yeah, but it's a great opportunity. I know that's going to be a long learning curve, but there's been incredible support and incredible leadership in St. Louis. Colin Martin was also a surgeon scientist, recruited me and my wife. And so we're very, we're really thrilled to join the group there.

David (57:09.088)

Yeah, it's a fantastic institution. That's great news. Congratulations.

Augusto Zani (57:12.586)

Thank you.

Betsy Crouch (57:13.8)

Yeah, congratulations. And thank you for sharing with us here. You know, we like to end on something sort of joyful and fun just to emphasize that the physician scientists are people too. And so we were chatting a little bit before we began recording about your Italian roots and how that influences your lab to some extent. So if you would share a little bit more about how you get to incorporate some of your culture into the fun of the lab.

Augusto Zani (57:45.502)

Yeah, mean, being Italian, it's difficult not being a foodie because this is really part of our culture. I love eating different types, food from different cuisines, but at the same time also cooking, which is something that I think I always find relaxing when I go home. If I could do it every evening, my wife wouldn't be happy because she says I cook, but I don't clean up afterwards and she has to do it. But it's really great, great fun for me. And so in the lab, we have usually two parties, one in the summer, where we get to be in my backyard, then another one just before the holidays in December. And I learned this from Janet Rossant. She always made it potluck style. And then I sort of spun it to being an international potluck, so which everyone, because of course we are very lucky to have a lab that is big, but also with people coming from different parts of the world. The fun is that everyone cooks food from their own country, and so you get to know of course their dishes, and I think it's celebration of diversity.

Something I think privileged to come from Italy. Our food is amazing. We were on vacation in Italy for two weeks. The day we came back, we went for sushi because for two weeks we didn't have sushi. So we really love eating food from different countries around the world. And the same happens, of course, with the lab. And they like that. I thought, my God, well, it's not really my Italian culture that I host and I'm not cooking. I also cook, of course, or barbecue and so on.

But I'm asking my guests to bring stuff. It's not really very, my mother wouldn't be proud of that. But actually it's fun. Everyone enjoys it and it's amazing. Everyone gets a big doggy bag when they go home with whatever they like the most. So this is about the food. And of course I right away checked out what the food scene is like in St. Louis. Because that's one of the most important things for me.

David (01:00:13.654)

St. Louis barbecue, you'll become an expert, I'm sure very soon. That's perfect. That was great. Thank you so much for taking time to talk with us. It was awesome to hear more about your background and training and especially what's motivated you to do the exciting work that you've been doing now. I think it is awesome to hear from a pediatric surgeon, just like what questions they're inspired to learn more about and just the challenges that we all face about trying to develop a physician scientist career. I think those are shared among pediatric surgery physician scientists and neonatology physician scientists. And I think everyone doing this kind of work just faces the same kind of struggle. So it's just interesting to hear, you know, what's motivated you, what's exciting to you. So awesome to take time to talk with you.

Augusto Zani (01:01:04.5)

Thanks for inviting me. This was really fun and you don't get to talk about both science and personal life so much with colleagues or at conferences. So this was fun.

David (01:01:16.566)

Awesome. Thank you so much, Betsy. So thanks to our audience. We're looking forward to talking with you again. I think we'll be coming up with another show in about a month from now. And we'll look forward to talking with you more then. Thanks so much.