'Leveraging genome engineering and stem cells to study rare pediatric CNS disorders’

What have your Ph.D. studies focused on?

“My research over the last few years has focused on developing and applying stem cell-based models for rare central nervous system (CNS) disorders, with the aim of uncovering mechanisms that drive these diseases. 

A central part of this work has been the use of pluripotent stem cells (PSCs), genome editing and forward programming approaches to generate disease-relevant cell types that are otherwise hard to obtain. This is both because we’re working on CNS disorders, where the relevant cells are difficult to access, and because the diseases themselves are extremely rare, making patient material scarce.

My thesis work has revolved around two different groups of rare genetic diseases affecting the central nervous system, namely leukodystrophies and lysosomal storage disorders (LSDs). The genes affected in these disorders are known, but much of the underlying disease mechanisms remains unclear, and there are no disease-modifying treatments available for patients. Until recently, human-relevant models for these disorders have been very limited, and much of the previous work has focused on neurons, while astrocytes, increasingly recognized as key players in neurological diseases, remain largely overlooked. That’s why we set out to create robust human PSC-based models, where we can gain insights into the underlying mechanisms and specifically the role of astrocytes in the disease pathologies.

Leukodystrophies are disorders characterized by impaired myelin development or maintenance, leading to white matter degeneration. My primary focus has been on the rare disorder Alexander’s disease (AxD), which is caused by mutations in the astrocytic protein GFAP. These mutations impair normal astrocytic function and disrupt the broader neural environment, leading to white matter abnormalities and progressive neurodegeneration, with most patients not living beyond childhood. Megalencephalic Leukoencephalopathy with Subcortical Cysts (MLC) is an even rarer leukodystrophy that typically begins in infancy. It results from mutations impairing astrocytes' ability to regulate ion and water balance in the brain, leading to white matter swelling, formation of cysts in the subcortex, and a gradual neurological decline and premature death. 

Lysosomal storage disorders (LSDs) are a group of rare metabolic disorders caused by defects in lysosomal function, leading to disrupted cellular function and multi-organ involvement. Among these, we have focused on neuronopathic Gaucher’s disease (nGD), which is caused by GBA1mutations, resulting in reduced activity of a key lysosomal enzyme. This leads to substrate buildup inside cells, severe systemic effects, and in the neuronopathic forms, early-onset neurodegeneration that often results in death in childhood. Impaired lysosomal clearance also causes secondary aggregation of alpha-synuclein, and carrying a GBA1 mutation is one of the most common genetic risk factors for developing Parkinson’s disease.

For both the AxD and nGD projects, we generated patient-derived induced pluripotent stem cell (iPSC) lines and isogenic control lines. This is done by collecting fibroblasts (skin cells) from patients, reprogramming them into iPSCs, and then using genome editing tools to correct the harmful mutation. This allowed us to compare diseased and healthy cells, where the only difference was the disease-causing mutation. For MLC, it was difficult to source patient material due to its rarity, so we used embryonic stem cells and introduced disease-causing mutations instead.

To track lysosomal aggregation in the nGD project, we used genome editing to develop a alpha-synuclein reporter iPSC-line. Following differentiation into neurons, this allows for the tracking of alpha-synuclein, which aggregates in many LSDs as well as Parkinson’s disease. This resource is now available to other researchers interested in the visualization of endogenous alpha-synuclein expression.

Using these iPSC lines, I mainly worked with 2D models using forward programming, where key transcription factors are overexpressed to generate cocultures of neurons and astrocytes. This allowed us to study how these disease-causing mutations affect these specific cell types and to investigate non-cell-autonomous effects, focusing on how astrocytes shape neuronal identity and function. 

With this approach, we set out to understand how GFAP mutations alters astrocytic function. By combining different cell types—healthy neurons with healthy astrocytes, healthy neurons with diseased astrocytes, and diseased neurons with healthy astrocytes—we could examine how the mutations affect each cell type and their surroundings. Using single-cell RNA sequencing on both 2D and 3D AxD models, we identified a previously undescribed neurodevelopmental phenotype of the disorder. We found that GFAP mutations impair the differentiation and maturation of both neurons and astrocytes, and that defective astrocytes alone are sufficient to impair neuronal differentiation.

Lastly, we also developed, at least to our knowledge, the first nGD brain organoid model. Using this we discovered interneuron defects and a severe imbalance in excitatory and inhibitory signaling. These findings mirror what patients experience in the form of myoclonic epileptic seizures, providing new insight into disease pathology.”

Can you tell us more about your thesis cover? 

“It's been something I've had in the back of my mind for quite some time. I have a friend whose mom is a talented artist, and I had this idea that it would be nice to commemorate my Ph.D. journey through a painting that could also be used as the cover of my thesis. So, I commissioned this artwork from my friend's mother. I provided her with the rough idea of my vision and some microscope images of neurons and astrocytes that were generated in our lab. Then she had the artistic freedom to do what she saw from it. 

In a way, you can say that my cover is an artistic interpretation of astrocytes exerting their influence on developing neurons in their surroundings, and how that contributes to these disorders. I think it turned out quite unique and is a nice representation of the core theme of my research, blending science and art, and the complexity and beauty of the brain. 

And the final piece is an actual painting that I will hang in my home office.”

How did you end up doing your Ph.D. in Lund?

“I grew up in a small town between Stockholm and Uppsala. In high school I first tried an economics track but quickly realized it wasn’t for me. I switched to natural sciences and became really interested by biology, especially neuroscience and the complexity of the human brain.

After high school, I wasn’t entirely sure what to do. I worked, traveled, and explored my options before discovering molecular biology as a field. I applied to Lund University and moved here in 2015, about 10 years ago now, to begin my bachelor’s degree in molecular biology. I knew quite early on that I wanted to go into neuroscience and went abroad for a year to the US and studied cell, molecular, and developmental biology with a focus on neuroscience and biology.

I came back to Lund for my master’s studies where I handpicked the advanced courses that would fit the neuroscience path, I knew I wanted to continue with. I found it both very intriguing with the complexity of the human brain, and at the same time, two of my grandparents were suffering from quite severe neurodegenerative disorders. I felt like I couldn't really do anything for them, but maybe I could contribute to the field in some other way. 

When it came time to start looking for a lab where I could do my master thesis project, I came across a paper from the Stem Cells and Aging group led by Henrik Ahlenius at Lund Stem Cell Center that described a method for generating astrocytes from stem cells. I found the paper so fascinating, I had no idea that you could generate brain cells this way and reached out to the lab. I joined first as a master’s student and then continued as a Ph.D. student, working on what eventually became the focus of my doctoral thesis.”

What have you enjoyed most during your Ph.D. studies?

“More connected to the studies themselves, I’ve really enjoyed working with stem cells and the hands-on application of techniques like genome editing and forward programming. These kinds of methods are the reason why I went into molecular biology in the first place and why I wanted to join Henrik’s lab. Then, it’s also been especially rewarding to see years of work come together into a coherent story and see all the hard work pay off. And of course, I’ve been very fortunate to work in a supportive lab environment with great colleagues, which has been vital to my PhD studies.”

What has been the most challenging aspect?

“Time management and learning to say no, at least that was the case early on during my Ph.D. studies. In the beginning I was very eager to help and took on too many side projects, often at the expense of my own work. In the end, it meant longer days and weekends for me. Over time, I learned the importance of setting boundaries and prioritizing my research while still being a helpful colleague.”

What advice would you give future Ph.D. students?

“I’d like to highlight three things:

1. Find a good supervisor. It doesn’t have to be in your ultimate dream field or your biggest passion, but it should be an area you’re genuinely interested in, and ready to work with for the foreseeable future. You should also feel you can have open, respectful discussions with your supervisor. I’ve been lucky to have two excellent supervisors with complementary styles, and this has helped me to identify and shape my own approach to research and mentoring of others.
2. Identify your strengths and weaknesses. Set up a work style (and station) that works for you and helps you succeed. For me, I’ve learned that means staying organized, both in the lab and in the office, and blocking out focused time in my schedule, at times I know suit me best, for more demanding tasks. By trying to do this early, and finding what you are good at, or maybe not as good at, and thinking proactively as to what you can do to develop will be helpful. In line with this, I would also really like to say that the Professional Development Program (PDP) at the Center was really valuable, and I highly recommend it to all PhD students at the Stem Cell Center, not just for developing research skills but also for personal growth.
3. Step back from time to time to take a look at the bigger picture. It’s easy to get caught up in small details or setbacks. But research doesn’t always go as planned and sometimes experiments simply don’t work. Taking a step back helps you reframe problems, design better experiments, and stay focused on the broader goals.”

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

“I’ve already started transitioning into industry. I’ll be working as a scientist at a biopharma company near Stockholm focused on developing stem cell-based regenerative medicines. I wasn’t initially planning to move back north, but the position was exactly what I was looking for and was an opportunity that I just couldn’t pass up. Plus, my family lives nearby, so it feels like the right move both professionally and personally,” says Oskar.