Meet Mark Denham: Getting rid of what we don’t need in stem cell-based therapy technology
The “Behind the Science'' profile series takes an in-depth look at a scientist or group within the Nordic EMBL Partnership. In this article, we meet DANDRITE group leader, Mark Denham, and learn about the advances he and his group have made in Parkinson’s disease research.
Dr. Mark Denham began his dive into stem cell research as a PhD student at Monash University in Melbourne, Australia. At that time, the stem cell field was new, and pluripotent stem cells had only been available for a few years. Stem cell research was connected closely with understanding human development. While the field has now grown extensively and includes the use of stem cells as tools to study, e.g., disease mechanisms, Dr. Denham explains that his interests in stem cells initially took root from the developmental biology perspective: “My passion is to understand development, specifically how the nervous system develops, and then use that knowledge to generate cell types from pluripotent stem cells.”
In a nutshell: cell therapy technology for Parkinson’s disease
Dr. Denham’s group at the Danish Research Institute of Translational Neuroscience (DANDRITE) has been working exclusively with human pluripotent stem cells, with a main goal to generate the types of neurons needed for the treatment of Parkinson's disease (PD). Dr. Denham explains: “The main project that we've been working on over the last seven years since I started at DANDRITE, is to develop a stem cell therapy for PD. For this, we take a different approach at generating dopaminergic neurons.”
Parkinson’s Disease is a neurodegenerative disease affecting millions of people around the world. In PD, dopamine-releasing neurons in the basal ganglia, an area deep in the midbrain responsible for motor function, either die or are impaired in function. This results in a loss of motor function, balance, and coordination. One highly anticipated avenue of treatment is to restore the lost motor function by replacing the missing neurons. Dr. Denham’s group uses human embryonic stem cells as a starting point to generate new neurons. He explains why:
One of the key advantages of pluripotent stem cells is that they can generate any type of cell in the body. And that's the attraction to them, for cell replacement therapies or for disease modeling. The other one is that they're undifferentiated, meaning that they can grow and expand to an unlimited degree. And so those two main characteristics are what is so attractive about pluripotent stem cells for generating dopaminergic neurons. Simply put, they represent a very early stage in development.
Goal-setting: the aim and the hurdles
From the day he first set up his lab at DANDRITE until now, Dr. Denham’s major aim has remained the same - to use undifferentiated human pluripotent stem cells to generate the specific cells needed for treating PD.
The stem cell field is a competitive one with numerous academic labs and some companies around the world with a similar goal, and there are challenges. Dr. Denham explains:
The current state-of-the-art, cell-based approach for PD is to differentiate stem cells into a dopaminergic progenitor. Then you need to transplant these progenitors into the brains of patients or animal models. Importantly, you can’t transplant a mature neuron because it doesn't survive the transplantation. Mature neurons, with long axons, don't integrate well into the host circuitry.
Researchers are trying to improve axon guidance and maturation by overexpressing specific factors in the brain, but a second challenge arises. The identity and quality of the progenitors are difficult to control. Dr. Denham explains:
With the current methods, you get less than 5% of the grafted progenitor cells becoming dopaminergic neurons. Instead, cell types such as serotonergic neurons that you don't want are made and have a negative effect on the graft function. In addition, batch-to-batch variation causes reproducibility issues. Then come the hurdles of scaling up and thoroughly testing each batch along with freezing down huge quantities of these progenitors and doing a lot of quality control testing. This is essentially what they're doing for the current clinical trials. And so moving that towards an actual therapy is going to be a costly process.
Hurdles aside, Dr. Denham finds promise in cell-based therapy because “even this low percentage [5%] causes some recovery of motor dysfunction in PD animal models, with the expectation that similar recovery would also be the case in patients."
While the current state-of-the-art is impressive, there is a need for improvement. And that's what Dr. Denham’s group has been working on. He explains, “if we can improve upon this state-of-the-art, and have a much higher outcome of dopaminergic neurons, then our approach will have a lot of benefits. You won't have the cell types that you don't want. Reproducibility will be much higher, and costs of production will be much lower.”
Breakthrough: quality and quantity
Going from an embryonic stem cell to a dopaminergic neuron, there are many choices that a stem cell has to make. Differentiation protocols try to promote these developmental decisions by pushing cells down a particular pathway, but it is difficult and inefficient to mimic development outside of a living organism. There are four steps in an average differentiation protocol, and each step may have only a 20-80% success rate, resulting in a very low outcome of the desired cell population.
From this perspective, Dr. Denham and his team went back to the drawing board and asked themselves: “if you look at a pluripotent stem cell, and then say, for a cell therapeutic approach where we just want a dopaminergic neuron, one specific type of cell from an embryonic stem cell, then what are the characteristics of a pluripotent stem cell that we actually need?”
And the simple answer is:
The undifferentiated aspect is really critical, but the pluripotent aspect is actually not so important if we just want the embryonic stem cell to do one thing. So, we decided to try to restrict the development of the cells by basically preventing them from differentiating into unwanted cell types. Then they could only become one specific type. And we generated, what we call lineage-restricted, undifferentiated stem cells.
How does one prevent cells from making mistakes in a differentiation pathway? Dr. Denham explains: “In the human genome, there are roughly 20,000 protein coding genes as well as non-coding RNAs. Of the 20,000, approximately only 14,000 are ever used in any one cell type. Of course, there are also a lot of housekeeping genes.”
Key to their clever approach is that only a portion of genes are actually involved in dictating and maintaining a specific cell type, even between types of neurons. With that perspective, they realized that
There are definitely genes that we can get rid of. Then we would block or prevent unwanted differentiation. Using the CRISPR-mediated gene editing approach we deleted genes that are critical for generating the cell types we don’t want. For example, we don’t need some of the genes needed to make the spinal cord. And so those genes that we knock out, aren't involved in development for the dopaminergic neuron.
This approach produces a new kind of engineered stem cell population that is undifferentiated, but no longer pluripotent, i.e., lineage restricted. Dr. Denham explains: “By knocking out four particular genes, we get a dramatic preference towards making dopaminergic progenitors. But also now we're no longer worried about what else these progenitors are going to do in the brain, because we have restricted their potential.”
The results of the group’s study show that when grafted into the brains of rats that have lost dopaminergic neuron function, there is a large increase in the production of dopaminergic neurons. But what is even more impressive is the speed at which the animals regain specific motor functions: “Within only eight weeks motor function behavior is restored, whereas previous work has shown a timeline of five months until recovery begins.”
Dr. Denham attributes the marked improvement in recovery to better quality of the cell population in the graft: “I think the really key part of our method is that we get rapid behavioral recovery, which carries huge benefits for patients. Also the reproducibility is much more stable.”
The next challenges for Dr. Denham and his team are to make further improvements in the quality of the cell population, and to take up the question of quantity.
We've got some further improvements that we can make as we only knock out four genes so far. We can also work with additional genes to make it even better. And then I think we can improve the purity even more, even though this is really good, better than anything that's out there at the moment, I think we can improve a bit more. And we can also now start to do experiments where we can ask, what's the minimum number of cells that we need to put into a patient brain? We're getting a fast recovery, so maybe we can put in less?
Translation: from proof-of-concept to potential for the clinic
Dr. Denham’s start in the infancy of stem cell research during his PhD followed by his move into the Parkinson's field gives him a 20-year perspective in the field: “To be able to go from a proof-of-concept to something that actually has a huge potential to go to patients, I think, is pretty good. I’m really happy with it.”
Dr. Denham and his group are now well-positioned to start translating the discovery, which will require forming their own start-up company or partnering with an existing company to move the work toward clinical trials: “Taking this towards the clinic, which is definitely our goal, whether it's on our own or with a company, the questions are different than in the basic research realm. Initially, it's about proof-of-concept and safety studies. And then efficacy and GMP production aspects.”
Dr. Denham also explains that there is wide potential for this clever approach beyond Parkinson’s Disease “The big picture is that this concept can be used for generating any cell type that could be used in a cell therapy, e.g., spinal cord injury, potentially, or even for disease modeling.”
Collaboration: team-work at DANDRITE and beyond
Being at DANDRITE and in Aarhus has meant that Dr. Denham had key collaborators in the same building.
Sadegh Nabavi’s group has been patch-clamping our neurons to help show that they're active, and that they have the normal properties you would expect. And, for the cell transplantation work, we collaborate with Marina Romero-Ramos’s lab, just on the ground floor here. They normally inject viruses into the brain to generate PD models. So they have the injection and behavioral setup established. We just said, instead of putting in a virus, can you put our cells into the brain?
And, thanks to the Nordic EMBL Partnership, CRISPR technology was developed by Emmanuelle Charpentier, formerly at the Laboratory for Molecular Infection Medicine (MIMS), DANDRITE’s sister institute in Umeå, Sweden, with perfect timing.
Career: connecting the beginning with the future
As a university undergraduate, Dr. Denham took classes that piqued his interests and landed in biology. The developmental, rather than disease, aspects fascinated him: “Understanding how a single cell can develop into an entire organism, but not just that, how it happens reproducibly is fascinating. Studying those events and understanding the genes involved in that process is exciting.”
And then near the end of his Bachelor’s degree studies, he found an institute for reproduction and development at Monash University that was associated with the hospital, and their stem cell projects lured him to a PhD position: “I really liked studying development biology, but I also wanted to do something that had application as well. And that's where I saw the stem cell work as being something that had potential to lead to a therapy.”
Since he started as a group leader at DANDRITE seven years ago, Dr. Denham has led a group of about six people. Being a group leader has allowed him to lead and do the research he is passionate about.
Dr. Denham works to keep his group focused and motivated: “I think the best way to try and do that is to show them that the work that we are doing is excellent. Show them that I am excited about it and that we’ll get there as a team.”
Research-wise, Dr. Denham has set a vision for the future of his group: “I’ll define my lab by lineage restriction and begin to look at other lineages inside, and perhaps outside, the central nervous system, while continuing to improve the approach for PD or diseases of other organs.”
With the Nordic EMBL Partnership annual meeting just around the corner, Dr. Denham will have a chance to share his story with a diverse group of scientists, maybe already making those connections for taking his undifferentiated, lineage-restricted approach outside of Parkinson’s disease.
As a final thought for the wider community, Dr. Denham shares: “Stem cell therapies are progressing faster toward clinical trials, and the work we are doing will significantly help that, help to transition not just to a clinical trial but to a potential therapy. Our work has the potential to not just help Parkinson’s patients, but also those with many different diseases."
Brief career summary
Dr. Mark Denham was initially trained as a developmental and stem cell biologist, completing his PhD in Stem Cell Biology at Monash University in Melbourne, Australia in 2005. Dr. Denham then moved to Lund University in Sweden as a Postdoctoral Scientist studying Parkinson’s disease. He then spent five years at the Melbourne Brain Centre, University of Melbourne as a Research Fellow before returning to Scandinavia for a Senior Research Fellowship at Karolinska Institutet in Sweden. In 2014, Dr. Denham began his career as group leader at DANDRITE, heading the Stem Cell and Translational Neurobiology Lab and was very recently awarded a grant from the Innovation Fund Denmark to support the translation of his cell therapy technology for Parkinson’s disease.
Group Leader & Associate Professor