Reprogramming Stars:
Dr. Diana Guallar
is an Assistant Professor at the Department of Biochemistry and Molecular Biology in the University of Santiago de Compostela (USC) and group leader at the Center for Research in Molecular Medicine and Chronic Diseases (CIMUS). The Guallar laboratory studies how epigenetic and epitranscriptomic cross talk is involved in the regulation of cell identity and plasticity in cellular rejuvenation and pluripotency. Her ultimate aim is to dissect critical pathways accompanying the loss of molecular fidelity observed during aging and in aging-related disorders that could be very valuable for clinical application.
Dr. Miguel Fidalgo
is an Associate Professor at the Department of Physiology (USC) and group leader at the CIMUS. The Fidalgo laboratory is focused on understanding how regulatory information encoded by the genome is integrated with the metabolic, epigenetic, and epitranscriptomic machineries to control cellular plasticity in the context of cell reprogramming and pluripotency, and how perturbations of these mechanisms could be associated with development and disease.
Introduction by Dr. Carlos-Filipe Pereira (Editor-in-Chief, CELLULAR REPROGRAMMING)
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. Diana Guallar and Dr. Miguel Fidalgo, professors at the Center for Research in Molecular Medicine and Chronic Diseases (CIMUS) in Santiago de Compostela, Spain. Their laboratories team up to understand epitranscriptomics and epigenetics in cellular reprogramming. Dr. Guallar did her PhD in Cellular Biology at the University of Zaragoza in Spain where she worked on endogenous retroviruses regulation in pluripotent stem cell biology. In 2013, she joined Dr. Jianlong Wang laboratory as postdoc at the Icahn School of Medicine at Mount Sinai (New York, USA). During this time, she became interested in studying the interplay of chromatin (i.e., epigenetics) and RNA modifications (i.e., epitranscriptomics) in cell reprogramming and pluripotency. In 2017, she returned to Spain thanks to competitive postdoctoral fellowships including a Marie Slodovska Curie, to explore novel aspects of cellular aging and rejuvenation using Yamanaka's model of somatic cell reprogramming. In 2021, she was distinguished with a Ramon y Cajal award, which allowed her to establish her own group at the CIMUS.
Dr. Miguel Fidalgo did his PhD in Molecular Medicine at the University of Santiago de Compostela, studying the role of kinases on adaptative cellular responses to external stresses. Then, during his postdoc also with Dr. Jianlong Wang, he shifted his research interests to understand how epigenetic and transcriptomic regulatory layers constitute a fundamental component of genome regulation in reprogramming and pluripotency. In 2016, he started his own group at the CIMUS where he has received funding from prestigious institutions in Spain including a Ramon y Cajal award. Dr. Guallar and Dr. Fidalgo, thank you so much for joining me today.
Dr. Guallar: Thank you for the kind invitation.
Dr. Fidalgo: Thank you for having us here.
Dr. Pereira: It is a pleasure to have you both as Reprogramming Stars. Your laboratories combine great expertise in several reprogramming and epigenetics and epitranscriptomics. Not so long ago, you have published a very nice article in Cell Stem Cell entitled “ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress” (Guallar et al., 2020). I find the concept of controlling reprogramming with RNA modifications very interesting. I will start by asking Dr. Fidalgo if you can tell us how your journey started in the cellular reprogramming field.
Dr. Fidalgo: After my PhD, I switched my field of interest to pluripotency by joining the newly created group of Dr. Jianlong Wang, who was expanding his pioneering contributions in protein–protein interactome networks essential for pluripotency. By chance, I was the person in charge of setting up Yamanaka's reprogramming system to address the function of some of the confident protein interactors of OCT4 and NANOG in embryonic stem cells that we were investigating in the laboratory. In those initial studies, we identified both barriers, such as ZFP281 (Fidalgo et al., 2012), and facilitators, such as the TET proteins (Costa et al., 2013), for the reprogramming process. We also identified, in collaboration with Ihor R. Lemischka's group, opposing effects of the mesenchymal transcription factors SNAI1 and SNAI2 during the final stage of reprogramming (Gingold et al., 2014).
Dr. Pereira: What motivated you to go to the United States for your postdoctoral work?
Dr. Fidalgo: Spain is not a country with an important investment in research for historical reasons. In fact, to have the opportunity to eventually establish your own research group in our country in academia, having a complementary training abroad is almost a requirement. It was not difficult for me to choose New York in the United States to do my postdoctoral work, as it combines both an excellent scientific environment with an exciting city for living where people from all around the globe interact.
Dr. Pereira: So, career development was your main aim. Dr. Guallar, can you tell us how your journey started in cellular reprogramming?
Dr. Guallar: Actually, for me, it was Miguel who taught me how to reprogram cells. Also when I arrived at Jianlong's laboratory a couple of years after him, it was just in time for the celebrations of the Nature article on TET function with NANOG during reprogramming, which resulted from the collaboration of Jianlong's and Jose Silva's groups (Costa et al., 2013). And I was excited to start learning how to reprogram myself.
I remember in my first experiment, I spent a lot of time looking at the morphological changes taking place after OCT4, SOX2, KLF4, and cMYC transcription factors (OSKM) induction. And I remember being worried to see that some cells began beating at some point, until I revisited Yamanaka's first articles to see that this could happen. I was very intrigued and happy to be a beginner in the reprogramming field.
Dr. Pereira: It is fascinating how we can switch cellular identity in such dramatic ways in a short window of time.
Dr. Guallar: Yes, the possibility of manipulating cellular differentiation programs, to generate any desired cell type is not only a major promise for therapeutic purposes but also provides a powerful tool to interrogate molecular mechanisms controlling cell fate changes for understanding development and tissue homeostasis.
Dr. Pereira: It was great to learn how you started in reprogramming, but it would be interesting to hear more about your Cell Stem Cell article, the main findings and the research avenues that this study has opened.
Dr. Fidalgo: When we started this project, it was well known that somatic cell reprogramming involves a profound rewiring of epigenetic and transcriptomic landscapes to successfully reach the pluripotent state. However, whether and how RNA modifications could also contribute to overriding somatic cell identity was incompletely understood. We found that the absence of adenosine-to-inosine (A-to-I) editing on RNA, which is among the most abundant RNA modifications in mammalian cells, was essential for mesenchymal-to-epithelial transition (MET) that occurs during the earliest phase of reprogramming toward pluripotency of mesenchymal somatic cells, such as fibroblasts.
Dr. Guallar: We identified that among the two enzymes, ADAR1 and ADAR2, which possess deamination activity of RNA mediating A-to-I editing, only ADAR1 was required for induced pluripotent stem cell (iPSC) generation. Indeed, we found that RNA editing by ADAR1 was in place to fine-tune the cross-regulation between innate immune response (IIR) and endoplasmic reticulum (ER) stress in mesenchymal-to-epithelial control allowing epithelial fate acquisition during reprogramming. Thus, our study not only delineates a novel regulatory layer that could be manipulated to improve somatic cell reprogramming efficiency but also highlights the need to further investigate other RNA chemical modifications as novel potential essential players to cell fate specifications for reprogramming strategies.
Dr. Pereira: Very interesting. You mentioned that there's ADAR1 and ADAR2. Is ADAR2 required in other cellular contexts?
Dr. Fidalgo: Well, at least in the iPSC reprogramming context, we found that ADAR2 was not necessary to reach the pluripotent state. Whether ADAR2 is important for other reprogramming systems is something that should also be explored, given its activity in protein recoding and other aspects of RNA biology regulation.
Dr. Pereira: Do we know the phenotype of the Adar2-knockout mice?
Dr. Guallar: Adar2-knockout mice are prone to seizures and die young. And these can be rescued just by introducing a genetic mutation in one of its targets: the glutamate receptor Gria2. This mutation recodes a glutamine (Q) to arginine (R), mimicking the constitutive editing of the Gria2 transcript, which is sufficient to rescue this very severe phenotype resulting from ADAR2 loss-of-function in vivo.
Dr. Pereira: It is impressive how RNA editing has such a dramatic impact, both in development and in reprogramming. As you know, I am particularly interested in immune cells. Do you think that A-to-I editing would be also required for other direct reprogramming processes such as transdifferentiation of fibroblasts into dendritic cells?
Dr. Fidalgo: Actually, it was during the review process in Cell Stem Cell when we were asked by the reviewers to test the universality of our findings with other reprogramming cocktails for iPSCs generation and also in direct reprogramming experiments, as was the case for example to induced neurons. Considering that reprogramming to a very distinct cell fate requires profound proteomic rewiring, a similar overload of the ER protein-folding capacity leading to ER stress could be expected. Thus, it would not be surprising to see that ADAR1 is also needed in other cellular reprogramming systems such as the generation of dendritic cells using fibroblasts as the starting somatic cell.
Dr. Pereira: It could be a general paradigm for reprogramming, right?
Dr. Fidalgo: In principle, A-to-I editing mediated by ADAR1 could be essential at least in reprogramming contexts involving METs, although this will require further validations.
Dr. Pereira: It is impressive you not only demonstrated the requirement of editing for reprogramming but also went deep into the molecular mechanisms involved implicating components of the immune response and ER stress. How did you come about with exploring these two mechanisms?
Dr. Guallar: As it usually happens when you start a project, you don't know where it will end up leading you. In this case, the starting question was to address whether A-to-I RNA modification was important for cellular reprogramming, as Miguel mentioned before. And later, after analyzing the data we had generated, we realized that in the absence of ADAR1 catalytic activity, there was a sustained hyperactivation of ER stress, whose alleviation by genetic and pharmacological interventions was sufficient to rescue the effect of A-to-I loss during reprogramming. We then wondered why ER stress was activated and going back to the literature where IIR activation had been documented to be modulated by ADAR1, we decided to test if there was a direct link between ER stress and innate response in our model. And indeed, we found that the hyperactivation of IIR was leading to ER stress, all mediated by the double-strand RNA sensor MDA5 in the absence of A-to-I editing.
Dr. Pereira: I'm curious about the IIR. How did you express the transcription factors? Could transduction with viral vectors have had an impact?
Dr. Fidalgo: In our study, we mainly used viral overexpression of OSKM reprogramming factors. Although a previous elegant study by the Cook's laboratory had shown that viral infection could enhance reprogramming by the activation of innate immunity through the specific pathway of TLR3 signaling, in the case of ADAR1 loss-of-function, the hyperactivation of the IIR through MDA5 was detrimental to MET and iPSC generation. These findings suggest the existence of specific innate immune signaling pathways in promoting or restricting somatic cell reprogramming.
Dr. Guallar: Yes, and the fact that we confirmed the transient activation of the IIR even in a lentivirus-free OSKM transducing system points to an endogenous source of double-strand containing RNAs, which are Adar1 and Mda5 main targets, during MET. Given that endogenous retroviruses are A-to-I edited by ADAR1, and some of them get reactivated during reprogramming, it will be important to explore whether these parasitic elements are responsible for the transient IIR activation in cell reprogramming.
Dr. Pereira: There was recent article in cell linking activation of endogenous retrovirus, senescence, and aging. Have you seen the article?
Dr. Guallar: Yes, you mean the recent study by the Zhang, Qu, and Liu laboratories, right? In their study, the authors show that the activation of endogenous retroviruses (in particular HERVK) is not only a hallmark of aging but also a driving force of both senescence and tissue aging. This is in line with the study of the Izpisua Belmonte and Orlando's laboratories who recently showed that modulation of another retrotransposon family (LINE1) could promote cell rejuvenation and even life span extension in models of accelerated aging. So there is a direct link there, which would be very interesting to further explore.
Dr. Pereira: Do you think there is a link to A-to-I RNA editing?
Dr. Fidalgo: Well, given that Adar1 A-to-I main targets include transposable elements such as short interspersed nuclear elements (SINEs), this is something that we are currently exploring in the context of aging and cellular rejuvenation through reprogramming.
Dr. Pereira: For the audience of cellular reprogramming, it will be interesting to hear more about RNA modifications and reprogramming. Which RNA modifications are critical for cellular reprogramming in addition to A-to-I editing?
Dr. Fidalgo: Other RNA modifications such as methylation of adenosines (m6A) have been involved in reprogramming. For example, when we collaborated during my postdoc with Martin Walsh's group at Mount Sinai, we found that the transcription factor ZFP217 regulates pluripotency and reprogramming through the coordination of m6A mRNA deposition (Aguilo et al., 2015). Although until recently the role of m6A in iPSC reprogramming was controversial, recent studies suggest that it plays a cell context-specific function during cellular reprogramming, which could involve different RNA binding proteins defining positive or negative functions of this RNA modification. Given the wide variety of RNA modifications that have been described, I believe that it is just a matter of time that a more comprehensive picture of epitranscriptomics in reprogramming is drawn.
Dr. Pereira: What about the interplay between RNA modifications and chromatin structure or dynamics? Are those two mechanistically independent or intertwined during cellular reprogramming?
Dr. Guallar: This is a very interesting question that at this point remains unaddressed in the context of reprogramming, at least. In fact, when I was doing my postdoc, we were able to identify that the methylcytosine dioxgenase TET2 (Ten-eleven translocation 2) TET2 coordinated an epitranscriptomic and epigenetic interplay to silence endogenous retroviruses in embryonic pluripotent cells (Guallar et al., 2018). And recently, we have witnessed how relevant m6A modification of RNA is for transposable element regulation at the chromatin level. Thus, this is an exciting open research area that we would love to explore in cell reprogramming systems in the next years.
Dr. Pereira: This takes me to the next question: what are you doing now in your laboratories, now? Can you give us examples of your most exciting projects?
Dr. Fidalgo: One of our ongoing projects is trying to understand how dietary perturbations lead to aberrant epigenetic and epitranscriptomic regulation, which compromise the generation of safe iPSCs from obese donors, which are among the population in higher need for the use of regenerative medicine.
Dr. Pereira: So for instance, if you would induce in vivo reprogramming in obese or lean mice, would you have different efficiencies/outcomes of reprogramming?
Dr. Fidalgo: Recent studies in humans and mice have shown that diet-induced obesity can negatively impact induced reprogramming technology by not only reducing iPSC generation efficiency, and/or quality, but also resulting in the inheritance of “obesogenic features,” which can compromise iPSC-differentiated cell function. So, we would expect that in vivo reprogramming could be affected in obese mice, but this is something that, if I am right, is still waiting to be addressed.
Dr. Pereira: We're looking forward to seeing the results of that project!
Dr. Fidalgo: I hope we can share soon our exciting discoveries regarding the molecular underpinnings behind the impact of dietary perturbations in cell reprogramming.
Dr. Pereira: What about you, Dr. Guallar?
Dr. Guallar: In my case, I am interested in exploring how RNA modifications alone or together with epigenetics play a role in aging and age-related diseases. In vitro, somatic cell reprogramming can erase all cellular aging hallmarks, and in vivo, partial cellular reprogramming, by temporally controlled induction of the Yamanaka factors has been shown to ameliorate aging phenotypes by Dr. Sinclair's, Dr. Serrano's, or Dr. Izpisua-Belmonte's groups, among others. So taking advantage of reprogramming as a rejuvenation strategy, we aim at investigating how RNA modifications are altered with age and whether we can manipulate them to restore younger phenotypes. But I just started my laboratory as a principal investigator very recently, so I hope to be telling you more data and projects very soon.
Dr. Pereira: These are exciting times for cellular reprogramming for cellular rejuvenation with groups and companies dedicating massive resources. The next couple of years will take the field to another level.
Dr. Guallar: Yes, as you mention, there is a lot of interest in the field of reprogramming, as it can help understand and develop novel cellular rejuvenation strategies with the goal of improving human healthspan.
Dr. Pereira: Can you highlight your current challenges in studying cellular reprogramming? What do we need to improve to further understand the process?
Dr. Guallar: One of the main limitations when investigating RNA modifications is the lack of technology to investigate the co-occurrence of different marks in the same transcript to understand how epitranscriptomic cross talk can affect RNA biology in the context of reprogramming. But most importantly, for the majority of the RNA modifications that have been identified by mass spectrometry in RNA, there is no sufficient knowledge on which proteins are involved in their regulation or there are technical limitations such as the lack of sensitive and specific antibodies for their study.
Dr. Pereira: What about Nanopore technology? Does that help mapping modifications in the same transcript?
Dr. Fidalgo: As you mention, the use of long-read third-generation sequencing technologies such as Nanopore or PacBio has emerged as promising approaches that potentially can capture information about several RNA modification types simultaneously. However, according to the experts working in developing this technology, we are still far from accurately mapping more than two RNA different modifications in the same transcript yet. The technology is advancing very fast, and it will be key to fully understand the way in which epitranscriptomics is important during cell reprogramming, among other physiological and pathological contexts.
Dr. Pereira: I am curious to know how you are collaborating to push the field forward? What are the challenges and opportunities of your collaborative effort?
Dr. Guallar: Of course, collaboration is critical, not only in our field of interest, but for all aspects of scientific research. From the technical perspective, Miguel and I work with similar models, although we pursue different scientific goals, as we previously discussed. This is key to not only synergize at the scientific level but also to take advantage of shared human and material resources to push our projects forward. And of course, collaboration is not only restricted to both our laboratories, but each of us currently also collaborates with experts in obesity and/or aging both in our institute and abroad. This was exemplified by our ADAR study, where we benefited from the collaboration of experts in ADAR1 and IIR, such as Dr. Carl Walkley in Australia, or ER stress, such as Dr. Miguel Lopez here at the CIMUS. It is impossible to be an expert in everything, so collaboration is needed to connect different fields of study and look at data in a new light to really achieve breakthroughs.
Dr. Pereira: There are also personality traits that are very good to complement each other, right? There are so many skills you have to master to be a good scientist. Can you tell me which kind of personality traits do you complement each other?
Dr. Guallar: In my opinion, Miguel is usually bolder and more willing to take risks such as exploring new areas of research or scientific approaches than me, so he definitely helps me to push my limits. And from my side, I think that I am more methodic and grounded, so I usually complement him by proposing a specific plan of action to get things done and reach specific goals.
Dr. Pereira: What do you think, Dr. Fidalgo?
Dr. Fidalgo: I agree with Diana. What can I say? She is not only my work colleague but also my source of scientific inspiration, and most importantly, my life mate and the mother of my son.
Dr. Pereira: It's very good to be rational about our best skills so we can take the most out of it, right?
Dr. Fidalgo: I think so, definitely!
Dr. Pereira: I would like to ask you about your ambitions and the vision for the field of cellular reprogramming. What will we be doing in 10 years of time? Which research will we be publishing in Cellular Reprogramming in 10 years?
Dr. Fidalgo: We are very excited about using epitranscriptomic reprogramming as a way to gain deeper insights in the molecular mechanisms governing cell fate decisions that could be important not only to solve some current challenges in cell reprogramming systems but that could also be relevant in other fields of knowledge such as obese- and aging-related diseases.
Dr. Guallar: Related to what will be done in 10 years, it is difficult to predict it since we are witnessing a technological revolution, for example, artificial intelligence among others, which makes it possible to address scientific questions hitherto unimaginable. In the context of epitranscriptomics, we hope to have enough tools that will allow addressing their relevance in all aspects of RNA metabolism and their contribution as a fundamental regulatory layer of cell fate decisions in cell reprogramming systems including in vivo approaches.
Dr. Pereira: Do you have any advice for younger reprogramming scientists who are starting in this field right now?
Dr. Guallar: Reprogramming is a very exciting field, and even though reprogramming has been studied for many years already, there is still a lot to learn and innovate in this passionate field of knowledge. However, I would strongly recommend that they chose a laboratory not only based on the quality of their science but also that can provide them with a good mentorship, as it was the case for us with Jianlong.
Dr. Pereira: Dr. Fidalgo, can you share one eureka moment, either an idea or a result that significantly changed career?
Dr. Fidalgo: It is difficult to find a moment throughout these years that I could define as a “eureka moment” per se. But for example, in our ADAR1 study, it could be the moment when we saw that alleviating ER stress not only improved reprogramming efficiency but also was sufficient to rescue the defect in reprogramming caused by ADAR1 loss, demonstrating the direct connection between these two processes.
Dr. Pereira: The power of designing simple experiments that give you the right answers. The experiments you have described clearly validated your hypothesis, giving you confidence on the molecular mechanisms. I would like to close this interview with some questions that are not particularly related to science. Dr. Guallar, if you were not a scientist, what would you be?
Dr. Guallar: In my case, I'm totally sure that I would have tried to be a journalist for National Geographic, which combines my passion for people and traveling the world, together with writing. In fact, if I think about it, most of these aspirations are also fulfilled while doing research.
Dr. Pereira: Dr. Fidalgo, if you were not a scientist, what would you be?
Dr. Fidalgo: This is kind of hard to answer because since I have memories, I dreamed about being a scientist. Although it is true that I loved to do sports, from soccer to all sorts of martial arts, so maybe I would have worked on something related to it. Perhaps training people, which is actually kind of similar to what we are doing now, mentoring and training future researchers, which is one of the most satisfying parts of being a scientist.
Dr. Pereira: What's the best piece of advice that you have been given to?
Dr. Fidalgo: During these years, I have been given a lot of excellent advice, so it is difficult to choose one. But probably the first one that comes to my mind is “you should go after what makes you excited and happy,” and “never stop asking questions.”
Dr. Guallar: In my case, the piece of advice, I think, was from my dad: “Don't be afraid of making mistakes.” And I remember this because when I was starting my PhD, it was so frustrating to mess up an experiment or to have a result that you didn't expect. And in the end, it's always better to learn from your failures than not trying in the first place, which you may regret in the future.
Dr. Pereira: It's an excellent piece of advice. Dr. Fidalgo and Dr. Guallar, thank you so much for joining me today. Thank you for your time. It was great to learn a bit more from you and from your science.
Dr. Fidalgo: Thank you so much, Filipe, for this opportunity. I hope to see you soon.
Dr. Guallar: Yes, thank you so much for having us.
