CAPS - Administration of amniotic fluid stem cell extracellular vesicles regenerates the lung epithelium in fetal rats with CDH at translationally relevant developmental stages - Kasra Khalaj

We have a return competitor, Doctor Collage from the Hospital for Sick Children in Toronto, Canada, is back for his second chance at the final title, and he will be presenting an amniotic fluid stem cell extracellular vesicles and how they regenerate the lung epithelium in fetal rats with CDH. Um, Doctor Richard Kedner from, uh, the Children's Hospital Research Institute of Manitoba, he is an expert in CDH and something tells me he might have a few thoughts to share, so we'll hear from him after. Hi everyone, thank you for attending my talk. Congenital diaphragmatic hernia is characterized by impaired fetal lung growth and maturation. This condition has unacceptably high mortality and morbidity rates. One of the biggest challenges for babies with CDH is that fetal hypoplastic lungs are immature, whereby there is an impairment of cell differentiation, especially in the epithelium. We study the use of stem cell derived excellular vesicles or EVs, which are the main mediators of stem cell paracrine signaling. In a proof of concept study, we recently published that amniotic fluid stem cell derived EVs can rescue fetal lung growth and maturation in several models of CDH at the pseudoglandular stage, which corresponds to 7 to 16 weeks of human gestation as indicated in the table on the left. To take the next step towards translating this therapy to babies with CDH, we are now investigating the AFSCEVs at translationally relevant developmental stages as CDH is diagnosed later, around 18 to 20 weeks, by investigating their effects on lung maturation at the stage where you can intervene, the canalicular and saccular stages. We derived the EVs from rat amniotic fluid stem cells, and to study CDH, we use the well established nitrophin model whereby we administer the herbicide nitrophin to pregnant rat dams at embryonic day 9.5. And as at the two stages that I mentioned, we isolate the hypoplastic fetal lungs and grow them on membranes as eggplants. Some of these eggplants receive the EVs, and some do not. We also obtain control fetal lungs from rats that receive control solution. To look at fetal lung maturation, we focused on the differentiation of key epithelial populations that make up the fetal lung, as you can see on the right here, alveolar type 1 and type 2 cells that primarily comprise the alveolus, as well as club, ciliated, and basal cells, which mainly comprise the bronchi and the bronchioles. We investigated their expression levels by looking at key markers of these cells using QPCR, Western blood, and immunofluorescent staining. The summary of our findings are on the right, whereby you can see in the red shaded areas that hypoplastic lungs compared to control at these two stages have a down regulation of alveolar type 1 and 2 cells, as well as basal and club. Remarkably, when we treat these hypoplastic lungs with the amniotic fluid stem cell derived extracellular vesicles, we are able to restore these primary markers, as you can see in the green shaded area, thus indicating that cell homeostasis can be achieved. These findings are important not only to better understand the CDH pathophysiology, but also because they further support the potential of an antenatal EV-based therapy for pulmonary hypoplasia secondary to CDH. And ultimately, this study puts us a step closer to translating this therapy to treat babies with CDH. Thank you for listening. Thank you, Doctor Kalais for another excellent presentation and also I'd like to take this opportunity to thank the organizers for putting together such an amazing event. Um, second time presenting today, Doctor Kalais, I think, uh, your work is one of the, the major new discoveries in, uh, congenital diaphragmatic hernia and, and lung development. And I think it's super important. I think most people will not be surprised that I say that because I think it is very important that we get better insight into what's happening before birth because we kind of know halfway during the pregnancy that these babies are going to be born with congenital diaphramatic hernia and abnormal lung development and then we're kind of waiting it out. So we're uh losing an opportunity to do prenatal interventions in these babies that, that might benefit them after they are born. I, I do have a few questions. From what I understand, you, um, use this as an eggplant model and you treated the eggplants, uh, uh, with the uh extracellular vesicles. Have you tried in vivo administration of these uh extracellular vesicles? Thank you very much, Doctor Kaiser for the question and also for the very encouraging comments. We have indeed performed, uh, some experiments looking in an in vivo rat model. Uh, we've, uh, tried different administration routes such as intraamniotic, tracheal, and so, uh, this is a part of a different project that, uh, we're actively investigating, and, uh, I can say that the results are quite promising. Uh, so stay tuned for that. Yeah, we will, maybe, uh, next year at the, the, the next best of the best. I have one more question if that's OK. I, I think, uh, it's also important to look at potentially safety profiles for these kind of treatments. The last thing we want to do is have a beautiful treatment that we can do in vitro or in vivo, but then having potential very negative side effects of target effects. Is there anything known about this for your treatment? Yes, thank you very much for the really great question. So, we are actively, uh, looking into, uh, collaborating with Doctor Jan De Prest, uh, looking at the safety and feasibility of this, uh, type of treatment in, uh, uh, in a lab model. And so, this is how we're going to look at, uh, translating this. Therapy. Uh, and so, this is something that we're actively investigating. Uh, we have also looked in the pre-clinical models which you've already seen the presentation for, but that's kind of the next step to really, uh, be sure that what we're administering can be safely, uh, given to babies with CDH. Thank you very much for the question. I don't know if you have any pressing questions from the chat, but if not, I have another one. Go for it, go for it. Um, so that you didn't find a difference in ciliated cells. Uh, that was one of the cell types that you did not observe a difference. Why do you think that is? Yes. Oh, thank you for that question. Um, multiple reasons. Uh, one is that, uh, When you think about the uh homeostatic uh or the cell composition of the hypoplastic fetal lung, um, what ends up happening sometimes is that there's this imbalance of different cell types, and so, uh, I think that this is one of the, uh, key cell types that, you know, is not being rescued that may end up, uh, uh, forming more of a, of a different, uh, uh, homeostatic, uh, uh, cell composition essentially. Uh, there are other types of cells that are, uh, being rescued and so we think that, uh, overall, the markers that when you look at the different markers that are expressed in branching morphogenesis as well, which is something that we looked at, these are key markers that are being, uh, rescued effectively. And so it's, it's not a matter of a specific cell type. It's more the, uh, greater, uh, uh, cell homeostatic balance that we're achieving with this treatment. Thank you for the question. Fantastic, uh, great presentation. That is the end of the round. Uh, we appreciate you presenting the second time. Uh, this is obviously some groundbreaking stuff. Thanks for coming.