Q: What are some of the main challenges when working with and differentiating stem cells in the lab and then scaling them for manufacturing purposes?
A: I started stem cell work at the University of Wisconsin–Madison in the year 2000. At that time, the field of induced pluripotent stem cells (iPSC) was just developing, and since then, tremendous progress has been made. Initially, we had difficulty just growing the cells, and over the past decades, we have learned how to differentiate the iPSC into different cell types that have clinical potential. We now have the ability to make cells of the central nervous system (CNS), heart, pancreas, liver, and a number of other organs. Now that we know how to grow and differentiate them, we are trying to find out how to use them to cure diseases.
Some of the challenges unique to cell-based therapies, compared with molecular therapeutics, include dealing with an entity you need to keep alive after transplantation. There are immune system considerations, and there is also the environment. For instance, if you put cells into a part of the body that has lost its vascularization, the cells won’t receive the nutrients they need to survive. That is the medical side, but my lab focuses more on the challenges with the manufacturing side of things. It is hard to go from growing a million cells in a dish to growing the number of cells you might need to treat a disease. For example, people who have a heart attack can lose a quarter of the cells in their heart, which is about a billion cells. Going from making a million to a billion cells requires rethinking how to differentiate and grow the cells. Instead of plates, you need to think bioreactors. The processes that work on the research scale need some tweaking to get to the clinical scale.
There is also the question of cell quality, in terms of both efficacy and safety. A lot of the processes developed for research use animal serum and Matrigel. We would like to get rid of those animal-derived products from our manufacturing platforms. Animal products have problems with consistency due to batch-to-batch variability, and they also increase the chance of immune system rejection. Trying to replace those products with fully defined, xeno-free differentiation processes is important. Another aspect on the manufacturing side is, how do you predict which cells will work and which will not? With a small molecule or protein therapeutic, you know the chemical structure or sequence of the drug. With a cell, it’s a little more complicated than that. How do you define the critical quality attributes of the cell? How do you measure those during the manufacturing process? These are some issues that the field is dealing with.
Q: What are scientists and clinicians doing to get around some of these issues with translation from bench to clinic?
A: It’s a chicken-and-egg problem: How do you develop a manufacturing process unless you know what the cell-based therapeutic will be? On the other hand, how do you develop cell therapies unless you have the manufacturing issues addressed? So, the field is looking to first address some common problems involved in making many cell types before a mainstream stem cell therapeutic is developed. Each of the cell types, whether it’s for treating cardiovascular disease, diabetes, or blindness, will have other specific requirements, and those can be addressed as products move toward and through clinical trials.
Q: Are certain cell types more likely to work well in the clinic?
A: That’s a tough one to answer. There are exciting results related to treating blindness, because you don’t need vascularization for the retinal cells, and the delivery is easy. Another example is beta cells in the pancreas that are used to treat diabetes. They can be delivered in clusters and don’t need vascularization either. For cardiovascular and CNS disorders, the scope and impact of therapy are tremendous, but there are some complex challenges in terms of how the iPSC can integrate into the tissue. Hence, those therapies may be a little further away from the clinic. Eventually, there will be several cell therapies being developed, and some may definitely be closer to the clinic than others, but all these therapies have the potential to regenerate tissue, which current treatments cannot offer.
Q: Can you talk about some of the recent advances in tissue engineering and gene editing and how that is helping stem cell research?
A: Genome editing is a game changer and allows us to design certain properties into a cell. For instance, you can reduce immunogenicity or increase efficacy of the cell if you know which pathways to edit. We can make changes at the molecular level and create ‘designer’ cells. A number of other technologies are becoming important as well. Omics analysis, such as single-cell RNA sequencing, allows us to probe into the composition of a single cell. We can begin to identify populations of cells that are safer and more potent. With imaging, we can track cells and see what is happening to them inside the body. If we know where the cells go, how many survive, and what they turn into, then we don’t have to just sit around hoping for the best. So, there are a lot of technologies that the stem cell field is adopting, which is pushing us forward.
Q: Can you discuss some of the stem cell work your lab is doing and where you see its impact?
A: We are interested in studying how metabolism changes as cells differentiate. We use certain genetic or protein markers and functional changes to monitor how the cell is changing. As the cell goes from a stem cell into a differentiated state, its metabolic needs change. We are profiling the cells to see what these metabolic changes are so that in the manufacturing process we can sample the medium, see what the cells are secreting, and then determine what needs to be adjusted to get the process where it needs to be. This is a good way to monitor the cell differentiation process.
One of the challenges that we have with different cell lines or with different batches in the same cell line is that the differentiation process can move at different rates. Or the differentiation may fail, and you want to know that early. By monitoring the media to identify metabolites the cells are utilizing or secreting, or by sampling the cells and looking at the lysates, we can determine in real time the differentiation states of the cells and adjust our process using that information. Right now, most of our protocols read like cookbooks, where you add a certain factor after a certain time. Instead, we would like to have protocols based on certain milestones that the cells reach during differentiation. Having all this automated, and having the information collected and processed, is where we would like to be, and we are still in the early stages of having those close-looped systems developed.
Another project that we are working on is to find ways to build tissues. Stem cell manufacturing has generally focused on generating pure populations of a certain cell type. For instance, we grow cardiomyocytes and endothelial cells and then find a way to combine them. However, in the body, cells grow from their progenitor cells and organize themselves into tissues as they develop. So rather than growing individual cell types and then building tissues, we are starting to engineer a system where cells can differentiate and organize as they develop. For example, can we build a heart tissue where we have the cardiac cells, endothelial cells, and supporting fibroblasts all together? This requires us not only to provide the cues to make a certain cell type, but also to provide spatially organized signals. We can use materials, microfluidic delivery, and a number of engineering technologies to try and create these spatial gradients. The cells have the innate capacity to form complex tissues and organs in the body, and what we are trying to do is just support their ability to do that. There has been success in this field with developing organoids; however, we are in very early stages of this work with cell therapy.
Q: Can you comment on how the regulatory agencies are handling issues surrounding cell therapies?
A: The regulatory agencies are learning about what these cells can do and the unique challenges they bring along with them. At the same time, we are learning about how the manufacturing processes can be made better. With new cell therapies like Chimeric Antigen Receptor T-cell moving to the forefront, we need guidance from the regulatory agencies to ensure the safety of these cells. What assays need to be done to ensure safety? What needs to be tested to determine cell potency? Regulatory processes are evolving but moving in the right direction. If both sides, scientists and regulators, keep an open mind and stay open to discussions, the road to further success will be paved.
Sean Palecek, PhD, is the Milton J. and Maude Shoemaker Professor and a Vilas Distinguished Achievement Professor in the Department of Chemical & Biological Engineering at the University of Wisconsin–Madison. He received a bachelor’s degree in chemical engineering at the University of Delaware, an MS in chemical engineering from the University of Illinois at Urbana- Champaign, and a PhD in chemical engineering from MIT, and performed postdoctoral research in molecular genetics and cell biology at the University of Chicago. Sean’s lab engages in team-based research working with labs in biology, medicine, physical sciences, and engineering to tackle problems at the forefront of regenerative medicine.