Dissecting Dopaminergic Neuron Specification at Single Cell Resolution

What have your Ph.D. studies focused on?

“I have been a doctoral student in the Developmental and Regenerative Neurobiology Research Group, led by Malin Parmar. The group’s research is primarily focused on developing a cell replacement therapy for Parkinson's disease – a neurodegenerative disease where dopamine-producing neurons are lost. We study the development of these dopamine neurons to understand how we can generate those neurons to be used in future clinical therapies. Specifically, my research has focused on understanding the specification of different subtypes of dopaminergic neurons. This is interesting in our case as one subtype is more vulnerable to Parkinson's disease, known as, A9 dopaminergic neurons. The other subtype, known as, A10 dopaminergic neurons, is involved in cognitive functions, reward, and addiction but is mostly spared in Parkinson’s disease.  We still do not know why, or how, this division happens. 

We tried to understand this by using human brain organoids to model neuron development. These organoids are a complex model system that allows us to observe, study and manipulate the specification processes of defined types of human neurons. During my PhD, I worked on a project aiming to develop a protocol for generating organoids containing the dopaminergic neurons of interest. These models aim to mimic brain connectivity to simulate complex neuronal interactions and provide deeper insights into neuron development.

Then, we performed single cell RNA sequencing to analyze the cellular composition of these organoids. Single cell sequencing is an incredible technology that allows us to dive in depth into the identity of individual neurons, improving the level of resolution for these kinds of experiments in cellular biology. During my PhD, I wanted to learn bioinformatics in more detail, so I tried to handle some of the data myself while learning R and applying the knowledge I had learned during my master’s program.

We also did some in vivo studies, where we differentiated the cells in vitro and transplanted them into a rat model of Parkinson´s Disease, to further explore neuron differentiation and the acquisition of their molecular identity. I have also been working on a project where we combined dopamine progenitor cells with other cell types normally present in our brain, like glial cells and interneurons. By combining these cells, we found some interesting effects. They supported our neural progenitor cells as they developed and affected the maturation of our cells, so that we could study mature dopaminergic neurons in more detail. It seems that the interplay between the different cell types shapes their identity along the way, which has major implications when thinking of clinical applications. 

Although we still do not have the complete picture of how this happens, this is a big step forward in better understanding the process. Ultimately, the goal in the lab is to refine cell therapy products for future clinical trials. So, if we can understand more about how these dopamine neurons develop, we can improve the cell products used and in the end, better cells, will make better therapies.”

Can you tell us about the cover of your thesis?

“My cover artistically represents how dopamine progenitors differentiate and choose one fate or another, like becoming A9 or A10 dopaminergic neurons. Here, each cell is a koi fish. One of the reasons why I used koi fish is that I wanted to represent the complexity of each cell as uncovered by single cell sequencing and their trajectory during development. Furthermore, each cell is unique in its own way, which is why they have different colors. The way they split into two directions represents the process the cells move through during development while acquiring an A9 or A10 identity.

Then, in Japanese culture, the koi fish also represent transformation and perseverance through adversity, so I thought they would be a good representation of both the cells we worked with and the PhD journey.  The inspiration for this cover came from a painting in a secondhand shop in Hamburg, when I was there for the ISSCR meeting the past summer. 

It may be a bit abstract, but if you look at it from far away, I hope you see the fish as individual cells and the trajectories they follow during their development. I drew each fish using Illustrator and the seven colors used here come from the same color palette used throughout my thesis as well as in all my publications and illustrations.

For me, it is important that colors are associated with specific elements. I think this helps to tell a good visual story. So, I try to be consistent with colors, with specific shades for a stem cell or a neuron. I think it makes it easy to follow the whole way through.”

How did you end up doing a Ph.D. at Lund Stem Cell Center?

“I am from Italy, and I studied biotechnology and neuroscience in Pisa. I have always been interested in biology and passionate about science. Since I was in high school, I knew I wanted to study either biology or chemistry. Then, when I was in my last year, I discovered biotechnology. I always did better in these scientific fields and I was terrible at philosophy or literature. 

Choosing science was an easy, natural choice in that sense. I am the kind of person who can do stuff easily if I like to do something or am interested in the topic. Otherwise, motivation-wise, it can be hard for me to work on something I am not interested in or find interesting.

I started getting into stem cell research because I did my bachelor's on murine stem cell differentiation and cortical neurons, similar to the topic I have been studying here. Before I started my master's, I went to Cambridge for an internship, working with breast cancer cells. While there, I found out that I like stem cells and neuroscience more. 

After that, I returned to Pisa and earned two master's degrees, one in neuroscience and one in neurobiology, from the University of Pisa and the Scuola Normale Superiore respectively. During that time, I did an internship here in Lund, in Malin's lab, and decided to come back for my Ph.D. So, I arrived in Lund about five years ago, right before the pandemic.”

What have you found the most enjoyable during your Ph.D. studies? 

“I was lucky to have a lot of freedom to explore many different things, develop interests, and learn new skills. I would say that I am still very much an in vitro person at the core, differentiating stem cells in the cell lab, but I have really enjoyed being at the interface between the wet and dry lab.

I also found traveling to be one of the most rewarding aspects of my PhD experience. My supervisor, Malin, encouraged us to travel for conferences which I enjoyed. It is great to exchange ideas with so many people, whether at conferences or visiting their labs, and others who came here to visit our lab.

What I think that I have enjoyed the most is this interplay or interchange of ideas both inside and outside our lab group. I also have enjoyed presenting my work. I know that not everyone is comfortable with being in the spotlight, and I do not necessarily enjoy it myself, but when the topic is our research or something I am passionate about, then I am happy to do it.”

What has been the most challenging aspect?

“One part of the PhD process is to accept changes, and this is something I felt was challenging. If I feel like I have control of a particular element, I need to know why it needs to be changed. If I am not convinced, it can be hard for me to adjust or change course. Trusting someone who knows more than me and accepting the change is one way to learn, of course.

Also, if you are like me wanting to work with wet and dry lab elements, it is important to try to find a balance between the two. Otherwise, it can become complicated and overwhelming. For instance, with bioinformatics, you need time to sit and think about each problem individually. You have to finish one before moving on to something else, and it can be difficult to plan how long it might take. But if you have a cell lab booked or need to collaborate with someone else, you have to be done by that time. You cannot always postpone it. While coding in R or Python is more of a lone wolf job, the cell culture lab is more of a collaborative environment, and it can be more difficult to set your time and pace.”

What are your plans following your Ph.D. defense?

“I will be in Lund for a few more months before starting a postdoc in Basel, Switzerland. I will still be working in neuroscience, but will move away from Parkinson's disease research and more towards basic developmental biology with some applications to dementia, schizophrenia, and Alzheimer's disease. The lab I am joining has similar research interests to what I have been working on, but it is slightly different, so I am bringing a lot of what I have learned here in terms of basic biology with me. The new lab is into single cell sequencing, bioinformatics, and computational neurobiology, so I will try to learn even more on that side.

That lab is also set up differently than what I am used to here. In Malin's lab, we are all working towards the same goal – improving cell replacement therapy for Parkinson's disease - whether it is with reprogramming, organoids, transplantations, or bioengineering. In this other lab, everyone has their own project, and they are all very different. It could be anything from brain evolution to Axolotl regeneration to brain organoids. We will be more unified by the techniques we use than by the questions we are asking.”

Any tips or advice for future Ph.D. students?

“I would advise them to find something that they like very early on, at the very beginning of their studies, and to learn the techniques and skills that are needed for that field. This way they have ample time to apply them in projects throughout their time as a doctoral student. I see many people in the final six months of their doctoral projects, and they suddenly realize that they have discovered electrophysiology or bioinformatics and are amazed by it. However, the time to explore these interests is limited, as the focus shifts to writing their thesis. The best way to identify your interests is to observe a wide range of people, their techniques, and whether those techniques align with your preferences.

For my future, it was clear from the start what I wanted to do, which I think is pretty unusual. Usually, people get to my stage and need to decide between industry and academia. Since I knew I wanted to be in academia, I planned certain aspects of my Ph.D. based on that. I knew I wanted to pursue an academic research career and that I wanted to understand as many different techniques as possible. So, if you already know what you want to do later, you can take some of the opportunities during the Ph.D. to develop the skills you will need for the future. That is what makes the Ph.D. so unique. You can tailor it to your interests and the skill set you want to develop.

And it is never too early or too late to start thinking about these things. If you find out that what you thought you wanted to do is not actually what you want, you can always change your mind. So, maybe it is also good advice to never close a door completely,” concludes Edoardo Sozzi.