Giussepe Cala, PhD candidate - Best of the Best in Pediatric Surgery 2024

OK, so we continue with um another candidate from VBS. He's Giuseppe Calan, and he's from Ormond Children's Hospital with single-cell guided prenatal derivation of primary fetal epithelial organoids from the human amniotic and tracheal fluids. So let's hear it. Good morning, everyone. My name is Giuseppe Cala. I'm a PhD student in Professors the Coppi and Gerli teams at University College London. My presentation today will focus on a recent work that explores the generation of organoid models from amniotic and trachial fluids. Organoids are an advanced in vitro system, and these are grown from tissue-specific stem cells. Organoids can be derived from fetal or adult tissues and used to study tissue biology as well as diseases. However, in the case of fetal organoids, this can be derived only after the termination of pregnancy. Therefore, our questions are, can we generate fetal organoids without a fetal tissue sample? Can these organoids help prenatal diagnosis? Our answer is the amniotic fluid, and the amniotic fluid is recirculated by different developing organs, and it contains stem cells that have been broadly investigated in the past years. However, in our work, we analyzed the amniotic fluid in very detail by creating the first amniotic fluid cell atlas. In particular, we found that the majority of the cells present in the fluid are epithelial when observed at the single cell level, and also we found that these are shed from various developing organs such as the gut, kidney, and lung. We subsequently developed a methodology to isolate the viable cells from the fluid and then to culture them in 3D to make them form epithelial organoids of fetal origin, and these organoids display different morphologies. We then characterized the gene expression profile of the amniotic fluid organoids and found that they form three different groups that are the lung, kidney, and the intestinal organoids. For this presentation, I will focus mainly on the lung organoids. We are interested in studying congenital malformation, CDH specifically, and thanks to our clinical collaborators, we can collect amniotic and tracheal fluid from CDH fetuses undergoing tracheal occlusion. Also in this case, we were able to expand lung organoids from the fluid of these CDH patients and showed that they contain lung, epithelial ste progenitor cells. Furthermore, at the gene expression level, CDH organoids derived after fetal display less differentially expressed genes compared to the one derived before the fetal procedure. So they are more similar to the control organoids. This indicates that the fetal surgery is actually changing something, and this is reflected in our organoid model. Using organoids, we can also study the function of a given organ. In the case of the lung, we investigated the ciliary beating frequency, and we observed that CDH organoids seem to have an impaired differentiation and also function. Lastly, single-cell RNA sequencing enabled us to study in detail these organoids, and interestingly we found out that CDH organoids exhibit a very different cell type composition when compared to healthy controls. In summary, we showed for the first time that the amniotic fluid contains fetal epithelial stem cells that can form organoids in culture, and these organoids can be generated during pregnancy without using tissue samples. Moreover, lungs. id s can be derived prenatally from the fluids of CDH fetuses, and the potential of this technology is that it may serve in the future as a tool for functional prenatal hypnosis and also clinical decision making. I'd like to thank you for your attention, thanks also to my supervisors and all the people involved in this work, and I'm happy to take any questions. Amazing presentation. Organizer is such an interesting concept to me, something I'm not super familiar with. So is the overall purpose for them at this point for research and diagnostic purposes to look at each of these different cell lines and be able to look at it for research purposes eventually? Do you feel that they will replace organs or be able to be used in vivo, or kind of what's the overall? Dream for these organoids. OK, thanks for your question, Brittany. Um, so to answer that, uh, I'd like to say that the, uh, generally, uh, especially fetal organoids can be used to study development. So, uh, because it's difficult to access fetal tissue, organoids provide a different tool to study, uh, the epithelium of, of the fetuses. On the other hand, they have a really, uh, they hold the great potential for translational. Uh, medicine, um, this is, uh, to give you an example, uh, these organoids could be used, uh, to test drugs, OK, both, uh, known compounds, for example, that, uh, are already working in the clinics, as well as new compounds, um, and this, uh, the outcome of the, uh, of the organoids phenotype after the treatment with the compounds can be correlated with the clinics of the patients, so they, they have potential translational potential in this term. Rather than, uh, transplantation, this is at least the case for our system with CDH organoids. Yeah, that was gonna actually be my question is, what drugs or therapeutics would you think that would be avail would be testable on these organoids? Is there anything out there already that you're thinking, oh, this would, next stage would be, or is, is that, are we not there yet? Because a lot of this is already predetermined. I mean, is there therapeutics that could be applied. To these organoids, that is a very good point, Todd, and, uh, um, I can tell you that we, we are trying now to establish a drug testing platform. So our aim is to, is to test as many drugs as possible, but we are interested, of course, in those that can, um, for example, uh, enhance the proliferation of the epithelial cells, especially because we know that in CDH, uh, the lungs are hypoplastic. So we could try to improve the maturation of the lungs by testing specific drugs on the organoids and um subsequently in in the future I mean these drugs can be potentially implemented for example with the endoluminal tracheal occlusion procedure, OK, the same sys the same way as for example the nanoparticles could be implemented also therapies tested on the organoids. Are you making these commercially available? Uh, not yet, but we are, we are with a patent ongoing for, yeah, OK, thank you so much. That was phenomenal. Thanks, thanks so much.