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2015 STEM CELLS Translational Medicine Young Investigator Award Winner: Dustin R. Wakeman, Ph.D.



'Marriage of Approaches' Leads to Important Step Along Path to Parkinson's Therapies

Dustin R. Wakeman, Ph.D., is the recipient of the third annual Stem Cells Translational Medicine Young Investigator Award. Launched in 2013, the award fosters advancements in the field of stem cells and regenerative medicine by honoring a young researcher who is principle author of an article published in SCTM over the course of a year that is deemed to have the most impact and to push the boundaries of novel and insightful research.

Dr. Wakeman was first author on "Human Neural Stem Cells Survive Long Term in the Midbrain of Dopamine-Depleted Monkeys After GDNF Overexpression and Project Neurites Toward an Appropriate Target," published in SCTM's June 2014 issue. This manuscript reports on an important step in evaluating cell types and methods that will be effective for stem cell therapies in Parkinson's disease. 

Dr. Wakeman focuses his career on determining the long-term therapeutic value of stem cell therapy in neurodegenerative disorders. His interests include stem cell-based therapeutics, disease modeling, neural transplantation, and morphological and molecular changes in aging and neurodegenerative diseases including Parkinson's, Huntington's, Alzheimer's and more.

Dr. Wakeman gained expertise in stem cell culture and neural transplantation during his graduate studies at the University of California, San Diego (he received his Ph.D. in 2010) where he developed novel techniques for maintaining fetal human neural stem cells (hNSCs) with advantageous growth parameters, as well as helped standardize techniques for preparation and stereotaxic injection of hNSCs in a primate model of Parkinson's disease. Utilizing these innovative methods, he demonstrated for the first time the long-term survival of hNSCs and early neuronal fate specification in Parkinsonian primates. 

Dr. Wakeman joined Rush University Medical Center, Chicago, as an Instuctor in 2010. Now an assistant professor in the Department of Neurological Sciences, he has assembled a team of experts who have dedicated their careers to cellular transplantation in Parkinson's disease.  

Dr. Wakeman recently shared his views on his groundbreaking SCTM study and on the stem cell field in general.

SCTM: Please describe, in language intended for a general scientific audience rather than for stem cell researchers, what hypothesis you were testing in the research described in your award-winning paper?

Dr. Wakeman: In 2007 we published a paper where we demonstrated that fetal neural stem cells (cells that can generate multiple cell types found in the brain) can alleviate symptoms associated with Parkinson's disease in a non-human primate model. We transplanted these cells into brain structures where the dopamine neurons (the primary cells that degenerate in Parkinson's disease) are located, as well as where they project their fibers and release the dopamine neurotransmitter.

Our hypothesis at that time was that the local environment of the brain would inherently instruct the neural stem cells to mature into the missing dopamine neurons. Many scientists in the field thought that simply placing the cells in the location where they are naturally found would be sufficient to instruct the cells to mature and replace the damaged dopamine neurons. The local environment would still retain all of the signaling cues to finish the job on their own, and certainly there were a number of scientific articles using similar cells for other brain disorders to support this hypothesis.

What we discovered in that experiment was quite the opposite. As we predicted, the neural stem cell transplantation did in fact alleviate several of the functional deficits and restore behavioral measures back to normal. However, we were surprised to find that less than 5 percent of the transplanted neural stem cells had naturally matured into the dopamine neurons that we were aiming to replace.

The question then became, could such a small number of dopamine neurons actually be responsible for this robust improvement in Parkinsonian behaviors? We thought about this for a while and upon further analysis found that the neural stem cells were actually providing neuroprotection on their own through multiple mechanisms, but not likely through direct cell replacement.

At the same time, our collaborators on this project were diligently investigating a protein called GDNF that was known to protect and rescue dopamine neurons in the same model of Parkinson's disease. Shortly after our 2007 paper came out, they published an article demonstrating that GDNF could actually guide and protect fetal dopamine neurons that had been transplanted into the brain.

I distinctly remember my collaborator showing me this data for the first time and being very excited. We were discussing how our surgeries had gone earlier that day and any troubleshooting tactics we might need to employ the next day. As with many quality scientific discussions, we were enjoying an evening cocktail when we came up with the idea: Why not combine the two strategies and see if we could enhance the benefit seen from the neural stem cell treatment alone?

We hypothesized that adding GDNF to the experiment exactly the same way it had been used with fetal dopamine neurons would 1) enhance survival of the neural stem cell grafts, 2) increase the number of neural stem cells that matured into dopamine neurons, and 3) direct the outgrowth of fibers from these newly formed neurons into the area of the brain where they need to release dopamine to alleviate the behavioral deficits.
It was essentially a marriage of two completely different approaches to treat the same disease.     

SCTM: Can you explain why investigating this hypothesis is important to stem cell research?

Dr. Wakeman: There were several important reasons to test this hypothesis, specifically in a non-human primate model of Parkinson's disease. We really wanted to investigate and nail down this concept of using the fetal neural stem cell as a substrate versus the fully differentiated dopamine neuron.

As I mentioned earlier, there was a strong belief by many in the field that the brain had all of the cues to naturally instruct these cells to become dopamine neurons based on what it was missing. In this case, we are talking about midbrain dopamine neurons. Our previous paper showed quite the opposite, so we really wanted to see if we could push the system in our favor with the GDNF molecule that is so important for dopamine neurons in the brain and had been shown to have benefit in multiple animal models and early-stage human clinical trials.

It turns out that maturing these neural stem cells into dopamine neurons in the lab is a lot trickier than you might expect, so the obvious and most attractive approach was to allow the brain to do the hard work. Therefore, it was really important that we definitively investigate whether the primate brain retains this capacity.

It cannot be stressed enough how different the rodent and primate brains are on multiple levels and why testing these approaches in the non-human primate is absolutely essential prior to initiating any human clinical studies. This fact leads to the next major reason it was essential to investigate this hypothesis for the integrity of stem cell therapies in general. While I was performing these experiments in graduate school, I was also involved in a clinical fellowship through the Howard Hughes Medical Institute that allowed me to train in the neurology department at the University of California at San Diego. The program was fantastic, and I encourage any student to spend time with the actual patients that they are trying to develop therapies for in the lab. I was absolutely astounded by the number of patients that would ask the attending neurologist about an overseas stem cell treatment they had heard about and whether they could receive a similar treatment. Some of the patients actually went as far as meeting with these companies to discuss travel arrangements.

As I looked into these companies, wondering what they could possibly be offering as a "stem cell" treatment, it was immediately obvious that they were selling false hope to desperate patients with no substantive evidence to support their bold claims. They were using the exact same type of neural stem cells to treat just about every disorder associated with the brain, and it just didn't add up.

About the same time, there was a report from a Russian group showing that they had injected a young boy with what I can only describe as "brain mush," and tragically this child developed a tumor. This group was claiming that they were injecting neural stem cells, again based on unsubstantiated science.

With the rising boom in overseas companies marketing their stem cell treatments for Parkinson's disease, it became of critical importance that we address whether these cells could actually be coaxed into generating dopamine neurons and provide long-lasting efficacy in Parkinson's patients.     

SCTM: Briefly outline the approach you used to test your hypothesis.

Dr. Wakeman: The approach was actually very straightforward. I engineered the fetal neural stem cells to constitutively express GFP with a lentiviral vector. This was important, as we wanted to trace the fibers projecting from the grafted cells post-mortem to see if the GDNF neurotrophic support could enhance outgrowth and direct the axons to innervate the striatal target area. There were no antibodies at the time that could specifically detect differences between human and non-human primate cells. I spent a lot of time looking for them, trying to coax antibody companies to take on the task and meeting with experts in primate evolution like Ajit Varki, but at the end of the day they simply did not exist. Therefore, the GFP marker was critical for definitive post-mortem analysis.

On a side note, Stem Cells Inc. was successful in developing a human-specific antibody, Stem-121, which they eventually commercialized and is now available through Takara Bio. That antibody has amazing specificity with no background. Companies aren't usually given much credit for generating quality antibodies, but Stem Cells Inc. should definitely get some praise for that. It is a game changer when you think about commercializing human stem cell-based products and validating your cGMP final production lines for clinical use in large animal models. The FDA doesn't want fluorescent genes engineered into your cells. Stem-121 finally gave us the ability to visualize fine projection graft fibers in the non-human primate brain and allows for accurate biodistribution analysis.

Once we had the cells engineered, it was just a matter of generating a large enough number of cells to graft a large cohort of animals. (I will discuss this in greater detail in the next section.) The other key reagent to this project was a well-validated viral vector to overexpress GDNF. Luckily, our collaborators had a strong preclinical program already in place utilizing AAV2/5-GDNF and had large production lots of the vector in house. This vector was being worked up for the FDA, so we already knew a lot about it as far as safety, biodistribution, etc. It would have been the exact vector we used clinically if the experiment succeeded.

The animal model we used was the MPTP-lesioned St-Kitts green monkey (African descent). This is a well-validated model for Parkinson's disease where the animals have a degeneration of dopaminergic neurons in the substantia nigra and subsequent loss of striatal projections and dopaminergic transmission. MPTP creates what we call a clinical phenocopy of the disease in primates with the characteristic motor symptoms associated with Parkinson's disease like tremor, akinesia, rigidity and postural instability.

As I mentioned, the actual experiment was pretty straightforward. We injected the GFP-labeled human fetal neural stem cells into the midbrain of the Parkinsonian monkeys and concomitantly injected the AAV2/5-GDNF vector into the striatum. The animals fully recovered from the surgery with no complications and we measured various functional parameters for one year to determine both safety and efficacy of our treatment.      

SCTM: Was there a specific methodological technique important to these studies?

Dr. Wakeman: I touched on this briefly earlier, but yes there was one major hurdle for this therapeutic program to be a viable option to treat a large cohort of Parkinson's disease patients, and this is true for any cell-based therapeutic in the regenerative medicine space. I had to develop a protocol to efficiently generate a large population of human fetal neural stem cells that had reliable batch-to-batch, test-retest consistency. For any cell therapeutic to get FDA approval there are a number of QA/QC release criteria that must be met and you have to generate large GMP-compliant master and working cell banks in order to meet your needs for a large patient population.

It took me about a year of culturing these cells in various formats and mediums to figure out exactly what parameters were ideal for large-scale expansion and cryopreservation. It turned out that they really like to be in contact with each other but not too overcrowded. To say the least, they are extremely finicky, and even small changes in the protocol could change the final product.

After the in vitro parameters were all worked out, the remainder of the experiment employed well-validated techniques. Although if you asked my collaborators that have spent the greater part of four decades refining surgical techniques, developing high-quality behavioral metrics, and characterizing the MPTP primate model, they might have a bit more to say about the importance of some of the other techniques.

As with any experiment, you get out what you put in.

SCTM: What does this mean for stem cell biology and its application?

Dr. Wakeman: The results of our study are important for several reasons. We demonstrated long-term survival (one year) of grafted human fetal neural stem cells and neuronal fate specification in the midbrain of MPTP-lesioned monkeys. Critically, we showed definitive evidence that fetal NSCs were not a viable candidate for treating Parkinson's disease, as they did not mature into sufficient numbers of dopaminergic neurons in the dopamine-depleted monkey brain.

This finding is important as several for-profit companies are currently marketing transplantation of fetal neural stem cells to Parkinson's disease patients without proper preclinical evidence for cell survival, safety or efficacy. Therefore, our rigorously designed preclinical study utilizing the most relevant model of Parkinson's disease definitively examined these questions and suggests that an inappropriate cell type was used.

A second critical finding from this study was that the degenerative primate brain is still a permissive environment for stem cell grafts and supports extensive fiber outgrowth long-term. Thus, changing the cell type for transplantation to a lineage-specified dopamine neuron would provide a feasible alternative to fetal neural stem cells.

Confirming the findings of the STEM CELLS Translational Medicine publication, we subsequently have shown that pluripotent stem cell-derived dopamine neurons do in fact retain their midbrain lineage and innervate the aged Parkinsonian non-human primate brain. Together, these findings are critical for identifying which cell lineages have the greatest potential for successful translation to the clinic.

SCTM: What's the best-case scenario that you would like to see come out of your study?

Dr. Wakeman: The best-case scenario is two-fold.  The first, and probably less likely, outcome in an ideal scenario would be that these bogus overseas clinics offering fetal neural stem cells for Parkinson's disease patients be shut down. I would like to see the FDA take a stronger stance on a variety of "stem cell" treatments being offered in the United States, as many of them are based on little to no scientific data.

Most of the treatments offered in the U.S. are centered on hematopoietic and mesenchymal stem cell treatments, and they are being offered for just about any ailment you can imagine. The serious companies that are invested in these therapies are utilizing the proper FDA approved routes, and this is no trivial matter. They have a lot of money and resources invested in these treatments so it is unfortunate that other companies are operating, probably illegally, and not being properly regulated or held to the same standards as the companies that are using the approved route to the clinic. 

The second ideal outcome is very much becoming a reality, and that is the use of pluripotent stem cells to generate large numbers of authentic midbrain dopamine neurons to treat Parkinson's disease. There are several world-class groups with extremely active programs making huge strides toward this goal.  There are groups in Japan, Europe and the United States that get together for an annual meeting called GForce-PD where they present unpublished data and basically spill their guts to each other for the greater good of the field.

It is a rather unprecedented idea to tell your scientific competition exactly what you're doing; however those involved have adopted the format as a way to make significant advances in a much smaller amount of time, learning from each other's triumphs and failures.

There is no reason for all of us to independently test each hypothesis. This way we can weed out the ideas that don't work and focus on making improvements to the successful approaches. Therefore, the best-case scenario is already underway, using the results from our study to switch gears and focus on a more relevant cell source.

SCTM: Let's turn the spotlight on you for a bit. Why did you choose to go into stem cell research?

Dr. Wakeman: I certainly didn't know right away that I wanted to be a stem cell guy. I was doing a research rotation in my first year of graduate school, working for a brilliant physician-scientist, Joe Gleason, looking for polymorphisms in families with Joubert Syndrome, searching for disease genes. I was finishing up my rotation and I asked Joe if he had any suggestions for my next rotation. Joe had already peaked my interest in neuroscience, so I was pretty sure I wanted to stick with neurology. He started talking about stem cells and a professor he knew back at Children's Hospital in Boston during his clinical training. That's how I got into contact with Evan Snyder, who had just moved his lab to San Diego. He also recommended Rusty Gage, but Rusty didn't have any rotation openings at the time, so I rotated with Evan and it worked out.

I think the hook for me was that I really wanted to work on something translational, and at the end of our meeting Evan mentioned this transplantation project he had with Gene Redmond at Yale and that I would have to travel to St Kitts. I jumped at it, and it turned out to be a great opportunity.

Maybe it wasn't the safest project for a Ph.D. student, but it seems to have worked out for me.

SCTM: Can you talk about your training, any mentors who might have influenced you and what motivates you today? 

Dr. Wakeman: I think I could write a book about that. I earned my Ph.D. from the University of California at San Diego, which is pretty much the ideal place to be for a stem cell biologist, particularly one that works with the brain considering the large number of outstanding labs at UCSD, Salk Institute, Scripps, Sanford-Burnham and the new Regenerative Medicine Center that would make any stem cell investigator drool with envy. Southern California is a powerhouse in the stem cell field for a reason, and it was pretty awesome watching it all come together.

When I joined Evan Synder's lab, human embryonic stem cells were still in their infancy and just starting to be explored in depth. My rotation project in Evan's lab was actually investigating new medias to support undifferentiated pluripotent growth.

It's amazing how far we have come since then, and yet how that area of defining a basal medium for growth is still a hot topic and of huge commercial interest, especially as these therapies are starting to hit the clinic in human trials. Clearly the academic atmosphere in San Diego was great, but the clinical domain was of equal stature. I was fortunate enough to train with some of the best neurologists in the field. Guys like Doug Galasko and David Song blew my mind with their clinical accuracy and ability to connect basic research with their clinical practice. I'll never forget my first day with Dr. Song. After asking about my dissertation project, he simply says, "So you're using the MPTP model; that's not Parkinson's disease."

I really had to reevaluate what I was doing and how I was going to approach modeling disease in my career. He reminded me that my goal is to treat real patients and to address their entire array of symptoms. In this case, he was reminding me that motor symptoms are just one array of deficits in Parkinson's disease and not to neglect the non-motor symptoms in my research.
Harry Powell, an amazing neuropathologist, also rocked my world during my clinical rotation. The first time I attended his post-mortem human brain cutting pathology clinic, I really grasped the magnitude of difference between a rodent and human brain. It suddenly made sense why my approach of placing neural stem cells into the midbrain wasn't going to be feasible in human patients. The sheer distance between the midbrain and striatum is orders of magnitude larger in the human versus the mouse. While that approach works in the rodent, it probably wouldn't work in the clinical setting.

As a result, we are now focusing our efforts on striatal transplantation.   

Of course my graduate advisor, Evan Snyder, was instrumental in my career path. In one of the first conversations I had with Evan, he said, "Stem cell biology only makes sense in the light of normal development."

He is absolutely right. All we are trying to do as stem cell investigators is recapitulate normal human development to the best of our ability. I think that's why a lot of the best investigators in our field are really great developmental biologists. Certainly in my field, people like Ron McKay and Lorenz Studer stand out as guys that really understand fundamentals of development and have applied them and made huge vertical advances in how we generate midbrain dopamine neurons from pluripotent stem cells.

Jeff Kordower, who I still work with side-by-side, was another immensely important mentor in my post-graduate career. My favorite quote from Jeff (and there are many) is, "There is no such thing as good data and bad data. Data is just that — data. It is your interpretation of the data that will determine your career and the impact you make on treating patients."

How simple and true is that statement. I really follow this everyday in the lab. It is important to be able to take a step back and look at your data in a completely unbiased manner. That's probably why I still like to have a second opinion of our results prior to publishing. I would estimate that Jeff has seen 95 to 99 percent of my data, and he has personally examined every brain section I have published since I joined his group.

It is important to have that check and balance with someone who has a proven track record and can give you that difficult unbiased feedback.

There are so many other people that have helped shape my career. I could go on forever, but there is one more person that I would not have had these opportunities without: Gene Redmond, who has been a great mentor and friend since my first year of graduate school. Gene understood the importance of this project that led to the award, and without his support in so many facets of life I don't know what I would be doing right now — maybe selling car insurance or something awful like that. (No offense to my State Farm representative. You are great.)      

What motivates me? That's easy: the future, developing new therapeutics and, of course, the patients. Nothing will motivate you like visiting patients in the clinic or at a support group. These people have your back more than anyone else ever will. They are your biggest advocates and talking with them will have a profound impact on your research and approach to science.

As Jeff regularly tells us, "Never forget why we do this — for the patients."

SCTM: Tell us a bit about your current position.

Dr. Wakeman: After graduate school I took an Instructor position in the Department of Neurology at Rush University Medical Center with Jeff Kordower. I am now an assistant professor, still working with Jeff as we build our stem cell program. Our main focus is still on Parkinson's disease, but we have a growing program in Huntington's disease and are actively pursuing other neurodegenerative conditions.

My primary research goals are directed at determining the long-term value of stem cell-based therapeutics in neurodevelopmental and neurodegenerative disease. I am primarily focused on pre-clinical testing of dopamine neurons derived from pluripotent stem cells, both human embryonic stem cells and induced pluripotent stem cells as a cell-based strategy for dopamine replacement using a rationale course of animal models to predict translational clinical outcome. I am also utilizing pluripotent stem cells to develop new strategies to model and treat disorders of the central nervous system. The goal is to use patient-derived iPSCs as an in vitro platform to model disease-specific phenotypes and develop new drugable targets, as well as in vivo to mimic human disease in the rodent and nonhuman primate brain.

SCTM: Is there anything else that you think is important to bring up about your paper, your work and what you think should happen next?

Dr. Wakeman: We are very excited about our current studies using cryopreserved, iPSC-derived midbrain dopamine neurons. We recently presented our functional data showing that these neurons survive, innervate the dopamine depleted striatum and rescue behavioral deficits in Parkinsonian rats up to six months post-transplantation. The grafts contain beautiful, healthy mature dopamine neurons without any proliferating cells in the grafted population of cells.

This is a huge advancement for the field as we push toward the clinic. Our initial functional and safety data is very strong, and we are very excited that these neurons can be efficiently cryopreserved, thawed and injected without any additional subculturing required. Generating a cryopreserved product like this that can be reliably and efficiently grown in large batches is a major breakthrough.

This was one of the major missing pieces of the puzzle that we needed to solve to make this a viable therapeutic option for a large cohort of patients. It immediately increased the range of eligible hospitals where the operation can take place and thus the number of patients that will have access to the therapy. Essentially, the requirements are the neurosurgeon and a standard clinical laboratory technician. It would not require special support staff or lengthy QA/QC processing steps to implement the procedure.

Accessibility is a major goal I wanted to achieve. You shouldn't have to live in a major metropolitan city to have access to state-of-the-art technologies and therapeutics. Ideally, every eligible patient that wants the procedure should have that option.

SCTM: Why did you select the journal STEM CELLS Translational Medicine to publish your paper?

Dr. Wakeman: We chose STEM CELLS Translational Medicine for this paper because of its readership and the scope of its mission. These experiments were very important for both translational stem cell researchers and clinicians, as they were informative for how to best move forward in the field. Namely, the use of human fetal neural stem cells was not going to provide the type of cell replacement needed for Parkinson's disease and that we should continue to pursue other cell sources that will differentiate into midbrain dopamine neurons.

As I mentioned, the paper also gives clinicians the hard data to discourage patients from considering an overseas operation that could be potentially dangerous and likely offer zero long-term benefit to them. I think STEM CELLS Translational Medicine is the exact type of journal that this work fits best in to reach our desired target audience.

SCTM: How do you think the Young Investigator Award might affect your career?

Dr. Wakeman: It is a great honor to receive this award, particularly because of the vision of STEM CELLS Translational Medicine and the scientists that I greatly admire and respect that sit on its editorial board.

I hope it brings awareness to our research and provides more exposure for our lab with other stem cell researchers. I would love to collaborate with other labs with expertise in biomedical engineering, protein chemistry and high throughput screening, to name just a few areas of interest.

I am also a big advocate of partnering with companies, large and small, to bridge the gap between academia and industry. These partnerships are critical to commercialize therapeutics and reach large patient populations.

Click here to read the best papers from our 2015 Young Investigators.