In this interview, we caught up with Kristina S. Stapornwongkul, postdoctoral fellow at Vikas Trivedi’s lab at EMBL, to discuss their new paper on Cell Stem Cell. Their work shows that glycolysis has an instructive role during the early stages of development, defining cell fate. You can read the paper here.
Hello, Kristina! It is really great to have you with us. Could you tell us a bit about yourself?
Hi! I’m a developmental biologist and genuinely fascinated by the complex and beautiful process of embryonic development. During my PhD, I investigated morphogen-mediated Drosophila wing patterning in JP Vincent’s lab at The Crick Institute in London. For my postdoc, I then moved to Barcelona to join the labs of Vikas Trivedi and Miki Ebisuya. Here, I worked on the role of metabolism during germ layer specification and symmetry breaking.
How did you start working on this project? Where did the inspiration come from, or what was the background to this work?
I did my Master thesis in the Aulehla lab, which did some pioneering work in the field of developmental metabolism at that time. It was a completely new and fascinating concept for me. So even though I didn’t work on a metabolism-related project myself at that time, it really got me interested in that topic. After my PhD, I really wanted to explore a new field besides development. To join Vikas Trivedi and Miki Ebisuya’s lab, I therefore applied to several postdoc fellowships with a project to study the role of metabolism in germ layer specification and symmetry breaking.
Your work uses these 3D systems called gastruloids. For those of us who might be completely new to this, what exactly are gastruloids? How would you describe them?
Gastruloids are aggregates of mouse embryonic stem cells (mESCs) that recapitulate hallmarks of in vivo gastrulation, such as germ layer specification and the establishment of an anterior-posterior axis (AP). They are part of recent efforts to use stem cell-based models to study developmental processes, revealing the remarkable self-organizing abilities of these cells. The original protocol for gastruloids was developed by van den Brink and colleagues in 2014. By now, there are many variations of the protocol, depending on the research question to be addressed.
And, why did you choose to work with these gastruloids?
Mouse gastruloids are a robust and easy-to-use model system. We culture them in 96-well plates, allowing us to test many different conditions in parallel. Beyond that, I find stem cell-based models to be highly versatile tools for investigating the role of metabolism in development. Pluripotent stem cells can be easily genetically engineered, which means that we can take advantage of synthetic approaches to precisely manipulate metabolic pathways in space and time. In addition, their accessibility allows for real-time observation of metabolic and signaling dynamics, while controlled culture conditions provide the opportunity to study how various nutritional environments affect cell behavior.
Can you walk us through the main findings of the study?
Using an in vitro model for gastrulation (gastruloids), we found that inhibiting glycolysis promotes ectodermal differentiation at the expense of mesoderm and endoderm lineages. This effect is dose-dependent, indicating that germ layer proportions can be modulated by adjusting exogenous glucose levels. We found that the inhibition of glycolysis resulted in the clear downregulation of Nodal and Wnt signalling targets, which are absolutely required for mesoderm and endoderm specification. This suggested that glycolytic activity might be an upstream of morphogen signalling. To test this, we tried to rescue the phenotype by activating Nodal or Wnt signalling while inhibiting glycolysis. To my surprise, this restored normal germ layer patterning, even though glycolytic activity and overall growth were not recovered. This suggests that glycolytic activity is not an energetic requirement for signalling pathway activity but rather an upstream activator.
Thanks for breaking it down.
You’ve used gastruloids generated from mouse embryonic stem cells, and then confirmed the results in human gastruloids too. How was it working with two distinct gastruloid systems?
For the paper, the work on human gastruloids was actually done by LiAng Yao in Idse Heemskerk’s lab at the University of Michigan. However, I’ve since started working with this model system myself in Vikas Trivedi’s lab. The important part for robust patterning with this system is to confine the cells on so-called “micropatterns”. For that, we microfabricated PDMS stencils – a technique I learned from the fantastic Joana Mendonça da Silva. I particularly like that the human 2D gastruloid model starts as a pluripotent epithelium and then undergoes epithelial-to-mesenchymal transition in specific regions. This really mimics the formation of the primitive streak and captures the morphogenetic aspect of gastrulation.
And, what are some of the ethical considerations researchers should keep in mind when working with them?
That’s a really important point. Whenever working with human embryonic stem cells, it’s of course crucial to comply with ethical guidelines and respect the sensitive origin of the cells. While gastruloids are powerful models for studying early development, they don’t have the potential to develop into a full organism – in particular, they lack organised anterior neural tissues. However, there are other stem cell-based models that are more complex and more embryo-like. Currently, ethical guidelines are evolving alongside the science, and I think it’s crucial to stay engaged in those conversations, be transparent about what these models can and can’t do, and ensure that our work remains responsible.
As you mentioned in the paper, this study opens many possibilities to study how maternal metabolic states affect embryonic development. How much do we currently know in this area? And, how will this study help further our understanding?
We know from epidemiological studies that maternal diabetes or malnutrition can have long-term effects on offspring health, but we still understand very little about how these states influence early embryonic development at the molecular and cellular levels. Around 30% of human conceptions are believed to be lost during gastrulation, making it a particularly critical stage. However, studying these early events directly in humans is extremely challenging. Stem cell-based models like gastruloids offer a powerful system to mimic early development under controlled nutritional conditions. Going forward, I think this opens the door to systematically dissect how the nutritional environment impacts cellular metabolic states and developmental phenotypes.
What were some of the challenges you faced during this study?
One particular challenge was genetically manipulating glycolytic activity in cells. Initially, I thought I could simply overexpress one or two rate-limiting enzymes with an inducible promoter, and that would do the trick. However, due to feedback inhibition, metabolic bottlenecks, or enzyme saturation, this approach didn’t work. As an alternative, we designed a new genetic tool to increase glycolytic activity by manipulating glucose availability. This tool takes advantage of the fact that mammalian cells cannot utilize sucrose. It’s based on a membrane-tethered version of SUC2, a yeast invertase enzyme that breaks down sucrose into glucose and fructose, thereby increasing the concentration of these sugars outside the cell. By fusing a small aggregate of membrane-tethered-SUC2-expressing cells to gastruloids in low glucose conditions, we were able to rescue gastruloid development upon sucrose addition in a time-dependent manner.
Finally, what are your plans next? I hear that you are moving to a new position-tell us a bit more!
Yes, I will be starting my lab at IMBA Vienna this September! I’m very excited for this new role, and I am currently putting together a team. I hope that our work will help to better understand how the nutritional environment and cellular metabolism affect processes during embryonic development. There is more info on our website (https://www.oeaw.ac.at/imba/groups/kristina-stapornwongkul). So please reach out if you feel enthusiastic to join the team!
Thank you, Kristina, and all the best!
