In this HCA conference session, Dr. Silvia Sookoian presents two clinical cases to illustrate how single‑cell technologies transform our understanding of complex liver diseases. The first case follows a 53‑year‑old man with NAFLD that progressed to NASH cirrhosis, highlighting liver lobule zonation, hepatocyte and non‑parenchymal cell heterogeneity, and the role of scar‑associated macrophages, endothelial, and mesenchymal cells in fibrosis. Single‑cell RNA‑seq revealed distinct hepatocyte clusters, zonated immune cell patterns, and ligand‑receptor interactions (e.g., TGF‑β1, NOTCH3) that underpin fibrogenesis, leading to the identification of a lysophosphatidic acid receptor as a therapeutic target. The second case involves a 61‑year‑old man with COVID‑19 and mild liver enzyme elevation, using single‑cell transcriptomics to map ACE2, ACE2‑like, and furin expression across liver cell types, debating whether SARS‑CoV‑2 directly injures the liver. Dr. Sookoian discusses methodological considerations: the trade‑off between dissociative single‑cell profiling and spatial approaches, sampling biases introduced by liver biopsies, and the high cost and equity challenges for Latin American researchers. The talk concludes with a call for collaboration and open‑access data to reduce disparities in precision medicine.
Chapters
Welcome back to session 5. Good afternoon. My name is Holger Hain. I'm your host for session 5. I'm dialing in from Spain. It's already dark here. You can see by the, by the bad light source in my back. I would definitely prefer being in Brazil right now, but that's what it is. Together with Orit, who talked this morning, I'm co-chairing the Standards and Technology Working Group. And I can only repeat her words that you should want to feel free to reach out, whatever problem you have for experimental design techniques, reach out to us and we hopefully can help improving your experimental design and make your data quality as good as it can be. So today it's an honor for me to chair session 5. Last session of the day. Thanks for hanging in, but it's definitely worth it because we have 4 exciting talks in this session. As it is already tradition for the HCA conferences, we're on a very tight schedule, but that's therefore some housekeeping at the beginning. So we have 4 talks. You can again ask your questions in the chat. We have a Q&A session right after the talks. You can ask your questions in English, in Spanish, or Español. So cualquier pregunta, ponedlas en el chat y vamos a traducir todo. I will do my very best, at least. I will have some help from John and Julio to translate whatever comes in in Spanish or Portuguese. So without further delay, and to keep the schedule, I would say we start right away with the first talk by Silvia Sukhoyan, and it's about "From Cell to Tissue to Complex Diseases: Perspectives from a Physician." Christine, please.
Hello everyone from Buenos Aires, Argentina. I am delighted to share with you this talk, and I'm also thanking the conference organizers for the kind invitation to address this fantastic topic. What if I told you that single-cell research may help you understand the pathogenesis of complex diseases? I will share with you two clinical cases through which I will address the physician view of the power of single-cell research to understand the human health and disease. This is a case of a 53-year-old male with non-alcoholic fatty liver disease arterial hypertension, morbid obesity, and type 2 diabetes. Liver biopsy showed nonalcoholic steatohepatitis, which is the severe histological and clinical form of the disease. Since the age of 48, at the time of the first clinical consultation, patient gained weight and presents persistently elevated aminotransferase levels. Second biopsy, 5 years apart from initial one, showed the patient developed NASH cirrhosis. Nonalcoholic fatty liver disease is a condition characterized by abnormal fat accumulation in the liver, which can present hepatocyte injury and chronic damage, such as those that characterize nonalcoholic steatohepatitis, also known as NASH. The disease can progress into severe clinical forms including cirrhosis and hepatocellular carcinoma, and the disease pathogenesis involves a myriad of factors including genetic susceptibility and predisposed metabolic comorbidities. And despite more— two decades of extensive research in the field of NAFLD, there is currently no approved therapy for the disease. And diverse factors explain the low success rate in NASH drug discovery, including insufficient understanding of cellular function and patterns of gene expression. To move forward with this, let's review some key concepts of the liver anatomical structure. The liver consists of repeated anatomical units termed liver lobules. These lobules are hexagonal-shaped columns. Blood enters the lobules from the corners, termed the portal tract, and flows radially inward towards a draining central vein through the sinusoidal blood vessels. It is known that many liver functions are not uniformly across the lobules, a phenomenon termed zonation. More importantly, liver zonation is associated with diverse liver pathologies. For example, pericentral predominance is associated with fatty liver disease and alcoholic liver disease. Here I show you that fatty liver progresses to a severe disease in a zonated manner. Periportal predominance is associated, for instance, with autoimmune hepatitis., and zonation of liver expression is a key feature of other metabolic organs such as the intestine and the kidney. Then, single-cell technologies may allow the reconstruction of zonation pattern for the global hepatocyte transcriptome. Using single-cell RNA-seq, McPartland and coworkers studied the hepatocyte heterogeneity of human liver. Here we can see the proportion of cells that contribute to each cluster in the liver and the physiological conditions. For example, the author found 6 distinct hepatocyte populations comprising cluster 1, 3, 5, 6, 14, and 15 that were, as expected, enriched in albumin expression. Pathway analysis suggests that there are specific functions for each of the mentioned clusters. According to the periportal or perisondral zones. For example, cluster 6 seems enriched for expression of genes involved in lipid and cholesterol synthesis, and cluster 6 seems involved in cellular pathways of immune cell activation. The summary map of human liver from the work of Sonia McFarlane shows again zonation in the hepatic acinus.. Within the acinus are hepatocytes—you can see those in red—and non-parenchymal cells, for example, collagenocytes in green, endothelial cells, Kupffer cells, hepatic stellate cells, and liver-resident and infiltrated lymphocytes. Of note, the authors also show that liver immune cells are distributed in specific zonated patterns. And non-parenchymal cells are not merely bystanders. In fact, macrophages are important regulators for liver homeostasis and injury. For example, in the liver of our patient with NAFLD, we can see how infiltrating immune cells attack the fat droplets. And sometimes the inflammatory infiltrate is severe in terms of amount of infiltrating lymphocytes. Therefore, tissue-resident macrophages are regarded as the immunological sentinels of the liver. As mentioned before, our patient developed NASH cirrhosis, which is a serious liver disease. Here we can see large scars in the liver, uh, the operation, and we actually need to reveal what kind of cells lead to these scars. Hepatic stellate cells are major contributors of the myoblasts for the pool. Nevertheless, these cells are not the only ones contributing to liver fibrosis. In fact, macrophages, endothelial cells, and mesenchymal cells are also involved in the process of fibrogenesis. How then these cells of the fibrotic niche interact with each other? Single-cell transcriptomic profiling of scar-associated macrophages, endothelial cells, and mesenchymal cells show multiple ligand-receptor interactions. For example, macrophages express ligands that can promote mesenchymal activation, for example, TGF-beta-1. Similarly, endothelial cells can promote mesenchymal collagen production, for example, NOTCH3, and more importantly, interactome modeling of ligand-receptor pairs between subpopulations of scar-associated macrophages, endothelial, and mesenchymal cells in liver fibrosis provides a fantastic molecular framework for the therapeutic targeting of key pathogenic cell populations. This landmark study used single-cell transcriptomics to assess the non-parenchymal cell subpopulation in the fibrotic niche of patients with cirrhosis, and the author identified a scar-associated cluster of macrophages, trained to positive CD9-positive subpopulation that expands in liver fibrosis. Using single-cell RNA sequencing analysis, here the author also showed The reprogramming of liver non-parenchymal cells in mouse liver, and they identified two different subpopulations of cells characterized by low or high expression of TREM2. And these TREM2 macrophages derived almost exclusively from the NASH liver. In this study, single-cell RNA-seq was used to explore the hepatic mesenchyme in the fibrotic mouse niche. And the author identified three distinct populations of mesenchymal cells that present zonation across the hepatic lobule. And the author found central vein-associated hepatic stellate cells as dominant collagen-producing cells during injury-induced fibrosis. But more importantly, the author identified a lysophosphatidic acid receptor as a therapeutic target and demonstrated the pharmacological antagonisms of this receptor inhibits liver fibrosis. Let's move to the second case presentation. This is a 61-year-old male patient with also nonalcoholic fatty liver disease, mild arterial hypertension, that presents at the ward with fever, dry cough, headache, and mild dyspnea. And TCS scan showed bilateral lung infiltrates. The nasopharyngeal swab was positive for the new coronavirus, and the diagnosis was confirmed as COVID-19. Among other laboratory features, our patient presented mild elevation of aminotransferase levels, about 2 times the upper normal limit. But what could be the possible explanation of that? First, patients with chronic liver disease may be more vulnerable to the clinical consequences of COVID-19, including oxygen desaturation and hypoxemia due to severe pneumonia or the cytokine storm. Second, liver biochemistry abnormalities could be the consequence of drug toxicity. And there is a third potential but poorly explored clinical scenario, which is the possibility that the novel coronavirus may directly or indirectly cause liver injury. So here is the academic debate. SARS-CoV-2 may cause liver injury. These are molecular observations that could be in favor or against this theory. Some experts say that the low or lack of expression of ACE2 in hepatocytes explains that the novel coronavirus does not actually cause viral hepatitis. On the contrary, other experts believe that the level of ACE2 in cholangiocytes is similar to the alveolar cells of the lungs, indicating that the liver could be a potential target of the SARS-CoV-2. Let's see whether single-cell transcriptomic analysis helps to understand this issue. We examine the liver expression, a profiling of host receptors implicated in SARS-CoV-2 infection across the liver cell type from the McFarland database, and we focus on ACE2 AC-2, ACE2, ACE2, and furin. And the analysis showed that 3 human host receptors are expressed in the liver tissue. However, expression levels extensively vary across the cell types. ACE2 expressed the highest expression levels in cholangiocytes, followed by hepatocytes. And the other 2 receptors showed actually levels across all cell types, from hepatocytes to all cell populations. But other authors claim that the The pattern of expression of these receptors is not uniform across the different cell type databases. For example, cholangiocytes are the most, the ones that express the higher levels of ACII in all databases, but ACII expression in hepatocytes show large differences among the different studies. In summary, application of single-cell research is in the past of precision medicine. But single-cell transcriptomic is not the only one analysis we can use. In fact, you can appreciate here there are complementary molecular profiling technologies, for example, single-cell epigenomic and proteomic profiling and single-cell metabolomic profiling. So although the costs have reduced, it remains an expensive approach for some Latin American countries. So Collaboration is crucial to decrease the inequities in research and care of human disease, and open access of the large databases is also important. Thank you very much.
Thanks, Silvia. Now we have Silvia in the live chat for the Q&A. Welcome. Um, let me see, um, maybe I'll start with some questions. So the liver is a very highly donated tissue, right? And you were mentioning many single-cell studies, and with the single cell, we basically take their cells out of their tissue, bring them in solutions, we lose the spatial organization. So if you like think of an ideal scenario, would you rather go for the spatial, keeping the spatial context and let's say, so you would lose single-cell resolution, you gain spatial context, that would help you understanding better the function of that tissue and in disease?
Oh, thank you. This is a great question. Actually, the work that has been done in the Human Cell Atlas is fantastic regarding the liver, because we now realize how is distributed the population of parenchymal and non-parenchymal cells.. But in the context of diseases, I think that we do need to keep the special— spatial context, because sonation is key in all liver pathologies, particularly in metabolic diseases. This is very, very well demonstrated, not only at the transcriptomic level, but also at the epigenomic level, that sonation is the key and perhaps the answer to the pathogenesis of metabolic diseases.
Okay, great. And then related to the diseases, we sometimes have problems to get cells out of the tissue. And you've seen a lot of pictures with different degrees of disease stage. Do you think that there can be biases when taking cells out because some tissues might be more dense? So we might expect like a systematic bias when sampling from these later disease stages?
Definitely, definitely. You are in the key question that I've been— this question is being heard at least 30 years in my career when I started as a resident, and now that I am an old physician, I always listen to the same because most of the picture I show you in the presentation are the result of liver biopsy. That is a percutaneous liver biopsy. When you place the needle, actually, you don't know where you are going, but you can presume you are in the left, right, the lobule of the liver. But of course, sonation implies the whole organ. So, this is very challenging, particularly for studying sonation and particularly for studying metabolic diseases. Even when you take a surgical biopsy, which is larger in terms of quantity of tissue compared with a needle, even when you take this surgical biopsy, you need to go to very specific lobule. Generally, you use the left one. So this is challenging for understanding how the disease progresses. Because as you may know, the vascular system in the liver also is different according to the lobule. So this is something that we need to understand better right now because it's very challenging from the technical point of view.
Yeah, we have this problem in many tissues, but I believe in liver is particularly crucial. So have that in mind when doing the data interpretation. Good. Then for the sake of time, thanks so much, Sylvia. And we would move on to the next talk. Thanks so much.
