Dr. Pereira: Good afternoon. My name is Filipe Pereira, professor at Lund University and editor-in-chief of Cellular Reprogramming. I’m very happy to bring you a new episode of Reprogramming Stars, our flagship series capturing the findings, projects, and ideas of the leaders in cellular reprogramming.
Today, we have Dr. Lay Teng Ang, a Group Leader at Stanford University, USA. Dr. Ang was born and raised in Singapore and earned her Ph.D. from the University of Cambridge. She was later appointed a research fellow at the Genome Institute of Singapore in the Agency for Science, Technology and Research (A*STAR). Then, she moved to Stanford University as a Siebel Investigator and Instructor at the Stanford Institute for Stem Cell Biology & Regenerative Medicine. In 2024, she was appointed as an assistant professor in the Stanford Department of Urology and the Stanford Institute for Stem Cell Biology & Regenerative Medicine. Dr. Ang’s work focuses on understanding the mechanisms of stem cell differentiation and applying that knowledge to generate pure populations of desired cell types. Her lab has been supported by anonymous donors, the Stanford Diabetes Research Center, the Stanford Maternal & Child Health Research Institute, and the California Institute for Regenerative Medicine. In addition, Dr. Ang received several awards, such as the Catalyst to Independence Award from Additional Ventures, the Career Development Award from Bladder Cancer Advocacy Network, and the Inspiring Early Academic Career Award from the Stanford Faculty Women’s Forum. Dr. Ang, thank you so much for joining me today. It is a pleasure to have you featured as a reprogramming star.
Dr. Ang: Thank you so much for the opportunity. It’s my pleasure to be here.
Dr. Pereira: Your lab develops methods to improve the differentiation from PSCs, a major challenge since the discovery of iPSC reprogramming. How did you begin in the field of cell reprogramming?
Dr. Ang: In 2008, I received an A*STAR scholarship that led me to join Dr. Bing Lim’s lab at the Genome Institute of Singapore. At the time, his lab was focused on reprogramming and cell fate conversion. That experience shaped how I viewed cell identity—as something we can change if we understand the underlying rules. A few years later, I moved to the University of Cambridge, where I first learned how to grow and maintain human embryonic stem cells. These skills stayed with me and helped form the foundation of my lab’s work today. Ultimately, when I returned to Singapore as a postdoctoral fellow, I became interested in improving stem cell differentiation in terms of efficiency, reproducibility and maturity, which are often key bottlenecks in this field. My lab uses different combinations, doses and timings of signals at each differentiation stage to convert human PSCs into various desired cell types, while also blocking the formation of unwanted lineages. More recently, we have also been using transcription factor overexpression approaches to enhance cellular maturation and function. All these activities are inspired by my formative exposure to cellular reprogramming in Bing's lab and at conferences such as the International Society for Stem Cell Research Annual Meeting and Cold Spring Harbor Laboratory Meeting.
Dr. Pereira: Which aspect of reprogramming did you first investigate?
Dr. Ang: At Bing’s lab, we were focused on iPSC reprogramming, concretely on how to generate high-quality iPSCs from skin fibroblasts. His lab had identified Tbx3 as one of the factors that helped improve that process. In addition, he was interested in the function of microRNAs in controlling cell fate decisions and differentiation. Mostly, he explored transcription factors for iPSC and hematopoietic progenitor generation and microRNAs for differentiation, amongst other research areas.
Dr. Pereira: Great. Could you tell us about your paper from 2018 “A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells” (Ang et al., 2018)?
Dr. Ang: Sure. We are very interested in understanding how we can convert stem cell-derived human endodermal cells into highly pure populations of liver cells, while excluding closely related lineages like pancreatic or intestinal cells. In this study, we applied principles from developmental biology to make pure populations of liver cells. Specifically, we identified doses, timings and combinations of inductive and repressive signals across six developmental junctures. To generate liver cells, we used signals that promote liver differentiation and also blocked the formation of pancreatic or intestinal cells, which enabled us to obtain about 80% pure hepatocyte-like cells by day 18 of stem cell differentiation. These stem cell-derived liver cells could increase short-term survival in a severe model of liver injury. Building up on this work, we are improving the differentiation efficiency and function of hepatocyte-like cells.
Dr. Pereira: What is the key aspect here? Is it cell purity, the differentiation time in which you capture cells for transplantation, or cell maturity?
Dr. Ang: Thanks for your question. We were one of the first to reproducibly generate stem cell-derived hepatocytes with such high purity. We are currently trying to improve their function and maturity, but the key aspect of this 2018 study is purity. When we compared our stem cell-derived hepatocytes with cells generated using other differentiation protocols, we found that our cells expressed higher levels of hepatocyte markers.
Dr. Pereira: And you tested these cells in a mouse model, correct?
Dr. Ang: Yes. It’s a mouse model of liver failure that lacks a key enzyme called fumarylacetoacetate hydrolase (FAH) developed by the Grompe lab (Azuma et al., 2007). This model is commonly used to study severe liver failure in mice and the engraftment potential of transplanted hepatocytes.
Dr. Pereira: My colleague here at Lund University, Malin Parmar, works on neuronal replacement therapies for Parkinson’s disease, and her group has found they have to transplant cells in the neuronal progenitor stage; otherwise, long-term engraftment is compromised. Do you find something similar with hepatocytes, despite their different biology?
Dr. Ang: We are testing which cells—immature or mature—are the most appropriate for improving liver regeneration, but in this FAH-deficient mouse model, it’s important that the cells we introduce express FAH. I speculate that we might need both immature and mature hepatocytes. Mature hepatocytes may be needed because they express functional genes to restore liver functions, and immature cells can potentially help quickly repopulate the injured liver because of their higher proliferation rate. We want to compare these populations by analyzing their regeneration potential and function across several models of liver injury.
Dr. Pereira: It’s important to know if you need more cells, more functional cells, or cells at different stages.
Dr. Ang: There has been a bit of controversy in the field since people transplant liver cells at different stages. Some papers have found that mature hepatocytes graft better, while others have shown that less mature cells or neonatal hepatocytes perform better.
Dr. Pereira: It was interesting to hear that you repress lineages to obtain a purer population. How many lineages do you repress? Do you do that at the same time you induce the liver lineage?
Dr. Ang: The differentiation process consists of six consecutive differentiation steps. At each stage, not only do we provide signals to generate our cell types of interest, we also repress the formation of unwanted cell types. To generate primitive streak cells, we suppress alternative germ layer lineages such as the ectoderm. Then, to make definitive endoderm cells, we block mesoderm formation. Subsequently, to generate foregut endoderm cells, we inhibit mid- and hindgut specification. Finally, to drive liver specification, we block intestinal and pancreatic fates. However, if any step is not efficient, it will affect downstream differentiation processes. This systematic methodology makes the differentiation more efficient.
Dr. Pereira: You need very patient group members to perform these roadmap protocols over and over again. Is this something easy to implement in the lab or to introduce to new students?
Dr. Ang: In this process, we apply some of what we know about liver developmental steps. But we often don’t know which doses, combinations, and timings we should use or if there are new signals that could control cell fate. That’s the difficult part, where we need to do more experiments to improve differentiation efficiency.
Dr. Pereira: Switching from liver biology to vascular differentiation, you published a paper in Cell entitled “Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses” (Ang et al., 2022). In a recent interview with Dr. Kyle Loh, your collaborator in this study, we discussed how deadly Biosafety Level 4 (BSL-4) viruses preferentially infect artery cells. Could you discuss the findings of this paper from your perspective? How can stem cell differentiation help in modeling viral infection? I think your approach was quite innovative.
Dr. Ang: Thank you. Despite progress in the field in generating endothelial cells, generating highly pure batches of artery- or vein-specific endothelial cells has been challenging. We tried to overcome this challenge and understand how to generate pure populations of artery and vein endothelial cells. One of the main findings of the paper is that PI3K activation promotes vein formation, while TGF-β signaling drives artery differentiation. Essentially, to make artery cells, we block PI3K to prevent vein formation. To make vein cells, we block TGF-β to prevent artery formation. By preventing the formation of these unwanted cell types, we can generate more than 80% pure artery or vein cells within a short period of time.
Nipah virus is one of the deadliest viruses on earth, with ∼59% fatality rate. It is known to attack blood vessels but there are no approved vaccines or treatments. Because Nipah virus must be studied at rare BSL-4 facilties, it’s also severely understudied. We’re very grateful to collaborate with Dr. Joseph Prescott from the Robert Koch Institute in Germany for this study. In summary, we discovered that Nipah virus mainly attacks artery endothelial cells, causing them to fuse and form large multinucleated cells. Our ability to generate artery- and vein-specific cells highlighted the identification of the differential tropism of Nipah virus.
Dr. Pereira: Was it known that TGF-β is implicated in arterial versus vein endothelial cell differentiation?
Dr. Ang: At the time, it was a new discovery that TGF-β can drive artery formation. The same year, two other labs also found that it promotes artery gene expression (Chavkin et al., 2022; Daems et al., 2024).
Dr. Pereira: It is interesting to think that TGF-β is present in the tumor microenvironment. Do you think this is something that could be explored in a cancer context?
Dr. Ang: That is a good question! I would have to look more into it. We generated some of the earliest artery and vein endothelial cell states. Whether that pathway translates into promoting artery endothelial fate in tumor vessels, we don’t yet know.
Dr. Pereira: Tumors utilize different strategies, such as angiogenesis or the adoption of blood vessels, to maintain an immunosuppressive state. It might be an interesting aspect to explore. But going back to the viral tropism, why do you think these viruses preferentially infect arterial cells?
Dr. Ang: We think it’s because arterial endothelial cells express higher levels of the entry receptor ephrin-B2 (EFNB2) compared to vein endothelial cells. When we knock out EFNB2 in artery endothelial cells, the entry of Nipah virus is suppressed.
Dr. Pereira: Building on this topic, you have a preprint from last year entitled “Metabolically purified human stem cell-derived hepatocytes reveal distinct effects of Ebola and Lassa viruses” (Prescott et al., 2025). Could you summarize the main results of this study?
Dr. Ang: In our preprint, we developed a new metabolic selection approach to further purify our stem cell-derived hepatocytes. By removing nutrients from the cell culture media like glucose, glutamine, and pyruvate, we could kill the non-liver cells or intestinal cells and make about 95% pure stem cell-derived hepatocytes. This highly purified population enabled us to study BSL-4 viruses, namely Ebola and Lassa. We infected stem cell-derived hepatocytes and profiled their transcriptomes to study the direct effects of these viruses. This is much easier to do with pure cell populations, because there are many neighboring and potentially unwanted cells in vivo. In this study, we found that Ebola triggers the activation of the integrated stress response (ISR) pathway, whereas Lassa does not seem to induce the same stress response. We checked public databases of nonhuman primates and discovered similar ISR gene expression in response to Ebola infection. Another difference is that Ebola virus downregulates the expression of many coagulation factors, while Lassa virus induces coagulation factor and gene expression. Together, these findings implicate hepatocytes in coagulopathy and immune dysregulation during Ebola virus disease and Lassa fever.
Dr. Pereira: You produced these highly pure hepatocytes through metabolic modulation. Could you generate them some other way?
Dr. Ang: There are other ways to purify stem cell-derived hepatocytes, but we wanted to find a new, simple way that does not involve equipment. Because we can just remove nutrients from the media, this strategy doesn’t need special equipment. It’s a very simple, cheap protocol that could complement flow cytometry approaches for cell purification. Following infection of stem-cell-derived hepatocytes with Ebola virus, we recently performed single-cell RNA sequencing, which is very hard to do wearing a positive pressure suit in a BSL-4 lab.
Dr. Pereira: In mechanistic terms, why do you think the ISR pathway is activated?
Dr. Ang: The integrated stress response (ISR) pathway is activated by multiple stresses, including viral infection, and can reduce protein translation and induce cell death as a means to combat viral infection. ISR activation can decrease protein translation, which can be an antiviral defense mechanism to prevent the virus from hijacking protein translation mechanisms. However, we are not sure. We only observe this in Ebola-infected stem cell-derived hepatocytes and not in Lassa-infected hepatocytes, even though both viruses infect the hepatocytes.
Dr. Pereira: Do you have different rates of cell death in the two systems?
Dr. Ang: Ebola virus progressively killed hPSC-derived hepatocytes; already 3 days after Ebola virus infection, most cells were detached. By contrast, Lassa virus induced minimal cytopathogenicity in these stem cell-derived hepatocytes.
Dr. Pereira: Do you think the Lassa virus becomes more invisible?
Dr. Ang: Interferon-stimulated gene expression is very low in both Lassa- and Ebola-infected cells. Both viruses are known to evade innate immunity. We are now trying to understand the functional relevance of the activation of the ISR pathway by inhibiting the ISR as we infect hepatocytes.
Dr. Pereira: Good reasoning. Do you detect any transcriptional differences in Lassa-infected hepatocytes?
Dr. Ang: Coagulation and complement genes are activated by Lassa virus, but not the ISR pathway. Ebola and Lassa are from different families of viruses, but both leads to viral hemorrhagic fever. Together with my virologist colleague, Dr. Joseph Prescott, we suggest that Ebola- and Lassa-infected hepatocytes could dysregulate coagulation factors and complement levels, which can manifest as coagulopathy and bleeding disorders in filovirus disease and Lassa fever.
Dr. Pereira: Very interesting. Would you like to share any current research projects from your lab?
Dr. Ang: One of our interests is to study how vein endothelial cells form. They’re harder to generate, in part because the intermediate progenitors and the signals that control vein development are generally less understood than those underlying artery development. Using PSCs, we found that efficiently generating vein endothelial cells in vitro entails a two-step process. The first step is to activate VEGF signaling to install endothelial identity. The second step is to inhibit VEGF signaling to increase vein endothelial gene expression. This finding was quite new and rather unexpected, as VEGF signaling has often been associated with endothelial development, but recent studies showed there are some paradoxical effects of VEGF in vein formation. We describe this in a recent preprint “Discovery of a primed endothelial progenitor that requires VEGF/ERK inhibition to complete vein differentiation” (https://www.biorxiv.org/content/10.1101/2025.10.11.681838v3.full.pdf).
Dr. Pereira: You also published a review entitled “Controversies Surrounding the Origin of Hepatocytes in Adult Livers and the in Vitro Generation or Propagation of Hepatocytes” (Pek et al., 2021), where you describe how liver biology can inform regeneration studies to generate PSC-derived hepatocytes. Can you walk us through the main stress points in the field?
Dr. Ang: I think the main controversy in the field lies in the different views on what liver stem cells are. Some think that liver stem cells are found near the central vein, in the central zone of the liver. Others think they are more randomly distributed across other zones, or that they are located around the mid-lobular zone. There is still some debate over which population is the most regenerative under homeostatic and injury conditions. However, there are more and more studies regarding this, so I’m optimistic that we’ll be able to shed more light on this topic.
Dr. Pereira: And I guess the location is important for function.
Dr. Ang: The current issue in the field of stem cell-based differentiation is that many cell types that are created from human embryonic stem cells or iPSCs are still immature. For example, the liver cells that we create from stem cells have some liver functions but are not completely mature; they cannot carry out all the functions of liver cells, including breaking down toxins and producing blood coagulation factors. The challenge is that, in humans, liver maturation takes place over years, and it’s really time-consuming to study in the body. So, we are excited about accelerating hepatocyte maturation in a Petri dish, including by exploring transcription factor overexpression approaches.
Dr. Pereira: In this case, are you more focused on functional maturation than lineage specification?
Dr. Ang: For this specific project, we are more interested in functional maturation.
Dr. Pereira: Is there any reason to believe that you would need to repress other lineages as well?
Dr. Ang: That’s a great point. We found that our immature cells express higher levels of certain transcription factors. We are planning to repress those to help increase maturation.
Dr. Pereira: Since maturation can be a vague concept, how do you define it?
Dr. Ang: In the adult liver, you have different subpopulations of hepatocytes. These cells express high levels of cytochrome enzymes, such as the P450 family of cytochromes. For example, cytochrome 3A4 (CYP3A4) is one of the most abundant cytochromes in adult hepatocytes. We are taking steps to increase its expression. When we overexpress specific transcription factors, we see a significant increase in CYP3A4, which is encouraging because this is a maturation marker present in adult hepatocytes. There are also other mature genes, like blood clotting factors, which are also important functionally.
Dr. Pereira: Looking at the future, where do you think the field of stem cell differentiation is heading?
Dr. Ang: I’m inspired by the approach you use, anchored transcription factor screens, to find new combinations of transcription factors to guide differentiation.
Dr. Pereira: Some people think that bioengineering is the future. Do you believe that or do you think this is a really complex tool to apply in a clinical setting? Is this something you see happening for stem cell-based regeneration?
Dr. Ang: That’s a good question. We work on stem cell differentiation using a 2D monolayer approach. We haven’t looked at the mechanical stress effects or factors such as hypoxia. Hence, the conditions that we use to grow these cells are not the same as in vivo conditions. Combining bioengineering with stem cell biology could introduce new variables for testing. We are collaborating with Dr. Ying Zheng from the University of Washington to use a microfluidics device to grow our stem cell-derived artery and vein endothelial cells, and then co-culture them with smooth muscle cells and fibroblasts to see the interactions. These mechanical cues could be important for sustaining endothelial cell survival. So, we are moving in that direction.
Dr. Pereira: You already mentioned several collaborations. Have collaborations outside of your field been important for your career?
Dr. Ang: Collaborations are critical, especially for multidisciplinary problems where no single lab has all the expertise and infrastructure. I had very rewarding experiences collaborating with groups from outside our field. For example, collaborating with Dr. Joseph Prescott at the Robert Koch Institute taught me a lot about virology. Without that partnership, I think we wouldn’t have been able to combine BSL-4 virology with stem cell biology to provide new insights into the arterial tropism of Nipah virus. I feel like I learned a lot through collaborations with other scientists. For example, I have also enjoyed collaborating with and learning from Drs. Kristy Red-Horse, Ying Zheng, Christine Cheung, Kyle Loh, Kyle Cromer, Filipe Pereira, Seung Kim, and many others who have been very generous about sharing their expertise and knowledge. Through them, I’ve adopted new lenses and tools to make my science more multidisciplinary.
Dr. Pereira: Certainly. Do you have any advice for young scientists that are starting their careers?
Dr. Ang: I can share something that helps me. Since science can be a long and difficult journey, it’s more fun to do it teaming up with the right people! I think it’s important to seek out role models who can genuinely inspire you and advocate for you when opportunities come up. I was fortunate to have the support of a team of mentors and collaborators, including Drs. Philip Beachy, Bing Lim, Kyle Loh, Thomas Graf, Joseph Prescott, Roel Nusse, Irving Weissman, Ravi Majeti, Ying Zheng, Kyle Loh, Joseph Liao, Seung Kim, and Kristy Red-Horse. It’s also crucial to invest early in communication skills to be able to explain your work to diverse audiences, including funders, other scientists, and lay people. I wish I had known that earlier. That will really amplify the impact of your work.
Dr. Pereira: Indeed, communication is everything. I would like to close the interview with some questions that are not strictly related to your research. If you could answer any scientific question regardless of your expertise or chosen field, what would it be?
Dr. Ang: I’m excited about why bats are tolerant to viruses deadly to humans. What mechanisms control that? I think they are not well understood. There is recent work reprogramming bat fibroblasts into iPSCs by the Zwaka lab (Dejosez et al., 2023). The platform could be useful to study differences between bats and humans in responding to viruses in terms of innate response or inflammatory pathway activation.
Dr. Pereira: Interesting. If you could have a science-related super ability, what would you choose?
Dr. Ang: The ability to see through problems or questions that others overlook—having an eye for hidden patterns to formulate key hypothesis and choose the appropriate experimental design. It would be great to have that super ability.
Dr. Pereira: That way you could tell what was really worth pursuing. Thank you so much for taking the time to join me today. It was great to learn more about you and your science.
Dr. Ang: Thank you for your time. I really enjoyed it!
