Activation of the Wnt signalling pathway is vital in the anterior-posterior patterning during neural development. In a new study, Stephane Angers and colleagues leverage previously developed selective antibodies against Frizzled receptors of the Wnt pathway to stimulate midbrain progenitor differentiation in human pluripotent stem cells. We caught up with first author Andy Yang and corresponding author Stephane Angers, Professor at the University of Toronto, to learn more about the story behind the paper.

Stephane (left) and Andy (right) in the lab

Andy, how did you come to work in the lab and what drives your research today?

AY: I was drawn to Dr Stephane Angers' lab when I took his course during my undergraduate studies. The course was about developmental pathways, and I was particularly intrigued by how the Wnt pathway plays many roles in stem cell biology. During that time, I also got to know a lot of the lab members through playing basketball, and we got along very well. They were extremely friendly and shared insights about the lab's recent developments, including work on pluripotent stem cells, functional genetic screens, and the development of a synthetic novel class of Wnt antibody, which is unique to what the lab has to offer. I would say that I was convinced to join the lab both because of the advanced research topics and the people. Currently, what drives my research today is a fascination with developmental biology; specifically, how a few cells in the embryo can generate all the cells in the body and possess all the built-in tools to do so. Dissecting these signalling cues and deriving these cells in vitro, specifically functional cells, and how to drive their differentiation, are my interests.

What is the background of the field that inspired your work?

AY: For me, my introduction to the field of stem cell research came during an undergraduate internship, which was part of a unique program offered by the University of Toronto called the Professional Experience Year Co-op Program. I was able to secure a spot to work as a research assistant for a full year in Dr Jeff Karp's lab at Harvard-MIT, which specializes in advanced biomaterials and stem cell therapeutics. The project that I was involved in focused on intestinal organoids, which feature a unique and rapidly dividing stem cell compartment, providing an excellent model for regenerative studies. I found both the model and the underlying concepts to be quite fascinating. Culturing intestinal organoids was my first-ever cell culture experience! From the internship, I also had the opportunity to attend seminars by world-class researchers and even pioneers in the field, which was truly inspiring. Listening to those talks really motivated me to continue learning about the field and to pursue my graduate studies in stem cells.

SA: I was always fascinated by the biology of stem cells during my career, and by the role of Wnt proteins as niche factors during differentiation. More recently, with the development of selective antibodies for the Frizzled proteins that are the Wnt receptors, and new antibody modalities that can activate Wnt signalling, my group has been interested in studying and manipulating Wnt pathway activity for cancer and regenerative medicine applications, and in further understanding their basic biological functions.

Can you give us the key results of the paper in a paragraph?

SA: Pluripotent stem cell-derived products are entering testing in clinical trials for various disease indications. One of the first of these therapies is the development of dopaminergic neuron progenitors for the treatment of Parkinson's disease. We were interested to see whether we could improve upon the current methods by deriving more homogenous and mature cells that can more faithfully represent the desired physiological cell types to improve engraftment outcome, and ultimately disease outcome in patients. Current protocols use small molecule GSK3 inhibitors to modulate Wnt signalling, despite the widely known fact that GSK3 is involved in a myriad of intracellular pathways. Off-target regulation of these pathways is therefore likely to negatively affect differentiation outcomes. By uncovering the signalling circuits and specifically activating Wnt/β-catenin signalling with novel selective molecules, we believe this can result in better differentiated cells.

Andy, when doing the research, did you have any particular result or eureka moment that has stuck with you?

AY: One of the most memorable moments in my research was bringing the patterned dopaminergic neurons to our collaborator for patch-clamp analysis, after a month and a half of differentiation. Observing the spike when the action potential was generated was truly amazing. That electrical response was the moment I fully realized my capability to differentiate pluripotent stem cells into functional neurons in a dish. Another significant moment was seeing the CRISPR screen results and realizing that the experiment was working as intended. It brought tears to my eyes, considering the immense effort and resources required for a genome-wide CRISPR screen in human pluripotent stem cells (hPSCs). A lot of careful planning and execution were involved because any issues, such as poor differentiation, contamination or staining problems, would be very costly, not only in terms of resources but also the amount of time involved. After months of work, it came down to a single moment of staring at the results of the screen on the spreadsheet. I was filled with an enormous sense of joy and relief seeing that the screen had worked remarkably well.

I was filled with an enormous sense of joy and relief seeing that the screen had worked remarkably well

Immunofluorescence image of dopaminergic neuron derived from hPSCs using FZD5-LRP6 agonist

Immunofluorescence image of dopaminergic neuron derived from hPSCs using FZD5-LRP6 agonist

And what about the flipside: any moments of frustration or despair?

AY: Being in a state of despair and feeling frustrated is part of doing scientific research, especially when differentiating hPSCs into neuronal cells. This process requires weeks of differentiation, during which cells can sometimes lift off the plates if the media are added too harshly or they may not survive for various reasons. Troubleshooting and delving into the endless cycle of optimising these protocols takes a significant amount of effort and time. However, these challenges are part of the journey. I am grateful for my lab members, especially Elli, Rony and Lillian, all of whom helped me through this process (experi)mentally.

Why did you choose to submit this paper to Development?

AY: We chose to submit our paper to Development due to its reputation as a leading journal within the field, particularly for its focus on stem cell research and developmental biology. A significant proportion of influential research articles in the field is published in the journal. We believe the uniqueness and significance of our work aligns well with the high standards and broad scope of the journal. Additionally, the wide readership can provide substantial impact and exposure for our research.

What is next for you after this paper?

AY: I remain deeply interested in the field of stem cells and would like to continue working within this area. After my PhD, I am planning to explore opportunities in biotech and the industrial sector, particularly with a focus in cell and gene therapy. My goal is to contribute to the translation of basic research findings that ultimately benefit patients. The advancements in areas such as stem cell-derived products and gene therapy are truly remarkable. The background of my research, which involves learning about advancements in clinical trials for Parkinson's disease, has fascinated me, especially knowing that the differentiated cells from hPSCs have already been injected into patients! I want to be actively involved in contributing to the future of this field, and I would like to take part in its ongoing development.

SA: My group is very excited to study how the Frizzled antibodies we helped develop to activate the pathway can be used in other contexts, such as in organoids, pluripotent stem cell differentiation or manipulation of the pathway in vivo. Based on our research, a next generation molecule is now advancing to a phase 3 clinical trial for the treatment of eye disease, and people in the lab are excited to use these reagents to probe the normal biology of Wnt signalling and evaluate whether manipulating the pathway could identify new therapeutic opportunities.

Finally, let's move outside the lab – what do you like to do in your spare time?

AY: I enjoy going on hikes, traveling and trying new dishes. I also love playing basketball and ultimate frisbee.