Kasra Khalaj, MSc, PhD - Best of the Best in Pediatric Surgery 2024

OK, so we move to the 3rd presentation of this last heat. We have Doctor Castro Calla representing AAP with human amniotic fluid, stem cell, extracellular vesicles, rescue features of lung development in human hypoplastic fetal lungs. So, let's see it. Thank you for the opportunity to present our work. It is well known that the lungs of babies with pulmonary hyperplasia, secondary to congenital diaphragmatic hernia, have the impaired triad of factors with impaired fetal lung growth, maturation, and vascularization. Although some therapies have been administered prenatally to treat pulmonary hyperplasia, none of these have been able to fully rescue lung development. In search for a regenerative therapy that could rescue normal lung development, we started studying stem cell derived extracellular vesicles, which are particles responsible for paracrine signaling, and in particular, those from amniotic fluid stem cells, or AFSCEVs. We have reported the main results of our studies using AFSCEVs on several rodent models of pulmonary hypoplasia. Now we have been investigating for the first time in the human. And understanding the clinical relevance in human pulmonary hyperplasia. I'm excited to show you all the novel human fetal lung eggplant model which we've now established at Sickkis. Fetal lung samples were harvested from terminations of healthy fetuses at 15 to 19 weeks of gestation, and these specimens were cultured as eggplants and treated with medium alone for the control group, or an inhibitor of RAC1, which is a rote GTPase that modulates lung branching morphogensis to induce pulmonary hypoplasia. We have confirmed that Ron expression is down regulated using CDH autopsy specimens compared to age-matched control. We have now switched to human derived amniotic fluid stem cells and characterized these EVs for size by nanoparticle tracking analysis, morphology using transmission electron microscopy, and Western blood for canonical surface markers following the International Society for Exercellular Vesicles guidelines. We've assessed the triad of dysregulative factors in pulmonary hyperplasia through the following outcome measures phenotypically, and we also investigated the changes induced by AFSCEVs at a cellular level by performing single nucleus RNA sequencing on all three of our groups using the 10x platform. I'll first walk you through our phenotype data. Here you can see encouragingly that fetal lungs treated with the RC1 inhibitor display histological features resembling those of pulmonary hypoplasia. And when we administered the human AFSEVs, we saw a restoration of airspace density with decreased parenchyma as well as branching morphogenesis marker FGF 10. We also see a restoration of key markers responsible for fetal lung maturation, surfactant protein C and potoplatin, when we administer human AFSEVs to these hypoplastic fetal lungs. When we looked at the vessels, we found that human FSEV treated explants had reduced medial wall thickness and restored vascular density. Encouraged by our phenotypic findings, we then wanted to understand the effects of this treatment that were being exerted on hypoplastic fetal lungs at the single cell level. With single nucleus RNA sequencing, we built an atlas of pulmonary hyperplasia, and here you can see a U map identifying all major cell types comprising of the fetal lung. Specifically, when we look at the epithelial cell populations in red and subcluster them, we found a unique group that we call the stressed 81-82 that had high representation in hypoplastic lungs that was down regulated after AFSCEV administration. This cluster had up regulated signaling pathways involved in cellular stress, death, and inflammation. We then looked at a cluster we call developing A1A82 cells, which have the highest proportion in hypoplastic lungs treated with AFSEVs. This cluster had high TGF beta and VEGF signaling. When we look at the fetal lung mesenchy, we see that human AFSCEV administration reduces the number of myofibroblasts in hypoplastic fetal lungs. And interestingly, in the endothelium, we see that human AFSEV administration can restore nuclei numbers back to control this cluster at the top, the vascular endothelial cluster, which is consistent with our phenotypic findings. And this cluster had increased VEGF signaling, which is a primary pathway involved in angiogenesis. So in conclusion, we are very excited as it is the first study to describe that human AFSCEV administration promotes the triad of factors, fetal lung growth, maturation, and vascularization in fetal hypoplastic lungs. And so moving forward, these results have direct clinical relevance as an AFSEV treatment represents a promising self-free approach to improving lung growth in babies with CDH. Thank you to our funding sources and thank you for listening. Awesome. Um, welcome, Doctor Kla. Thank you for this presentation. Um, I have one question cause this is, this is really promising, but, uh, my question is, what do we need to move this to the real clinic, um, patient? Like, how will we administer this and everything? Thank you very much for that question. It's one we get all the time. And um I'm really excited to share with you. Um, we've actually started um doing some work with the same source of these extracellular vesicles and a land model of uh CDH and this is in collaboration also in Belgium, but we've got our own, um, colony that we are working at with, um, at the University of Guelph here in Ontario. And so we're, um, we've been testing it so far, uh, pre-clinically and so this is obviously going to answer that question and, and so, uh, we've, we're also applying for, uh, further grant funding to support further studies in this lab model in order for us to really get this to the babies with CDH. Awesome. We have a question um from the audience that Su Yong Kim, uh, it's, in practice, will the stem cells be injected into the amniotic fluid? So, um, that's a really great question. Thank you very much for asking that. Um, we've tested various models, uh, routes of administration. We tried intratracheal and our animal models, I should, uh, qualify. Um, um, through maternal IV, etc. So far, uh, we've had a lot of promising, um, um, uh, results with the intra-amniotic injection. The ones that we're doing with the lam model are in combination with fetal, so they're administered, uh, in combination with, uh, uh, feto. And so, um, this would be, uh, a combinatorial-based approach, um, so far. But thank you very much for that question. Awesome. Great, um, phenomenal work. Thank you.