| 1 | AR3T Regenerative Rehabilitation | Regenerative Medicine 101 | 1783 | 27 | | 50.6 | neutral | 27:29 | Welcome you to this pre-conference workshop, Regenerative Medicine 101. As the name implies, the idea of this short talk will be really to give kind of a foundational overview of common terminology, techniques, and approaches in regenerative medicine. We decided to start this pre-conference workshop, actually year two of our symposium. It was really in response to feedback after our first year where many of the individuals in the audience were clinicians, for example, and they said that they were really excited about the talks that they heard throughout the program agenda, but that they felt that they could really benefit from kind of this rapid, basics primer on, you know, to kind of overview some of the common terminology that's used in regenerative medicine. And so that's kind of the intention of this short talk. So I think many of you in the field are rehabilitation trainees or clinicians. And so you know well that in the field of rehabilitation, our primary goal is really restoration of function. And throughout our field's history, the rehabilitation has really come to be a very broad field with a range of expertise and specializations. And I certainly don't want to minimize those specializations or oversimplify. But I do highlight two, I think, important aspects of the rehab field. So in the first part, really a primary goal in rehabilitation is oftentimes to try and tap into the body's endogenous healing capacity to try and maximize functional recovery after an injury or a disease, for example. And so we do this using modalities, physical therapeutics, such as exercise or other mechanical loading paradigms. Another important aspect of our practice is that we've worked very closely with individuals from other disciplines where, for example, with the application of prosthetic devices, whether it be limb prostheses or joint prostheses, we work very carefully with, for example, prosthetics and orthopedic surgeons. And here the idea is to kind of compensate for the functional tissue loss or the disease tissue. And try and again maximize functional recovery. And so I think we, you know, throughout our history, we've really come to work closely with these individuals to develop very targeted and specific protocols. Well, likewise, the field of regenerative disease to restore functional recovery after injury and disease or with aging, for example. And it does so, it does so taking a little bit different approach. And with regenerative medicine, is there seeking to replace injured disease or missing body parts with functional and site-appropriate tissue? So the idea here is that at the end of the day, the injured tissue or limb, for example, looks almost identical to the limb, the way it did prior to injury or prior to disease. Well, there's actually a biological precedence for this kind of idea. And this is a video that you see oftentimes in various regenerative medicine or tissue engineering conferences. And it's a video of a salamander limb regenerating. And this is showing the regeneration of the limb over the course of, I believe it's something like 90 days. And you can see again, at the end of the day, the limb looks almost identical to the way that it did before imputation. And so regenerative medicine scientists are paying a lot of attention to trying to understand the biological processes underlying this remarkable capacity to regenerate. And so you can see at the amputated limb, you can see the wound closure. And what we're beginning to really invest are the activity and the mobilization of the cells at that injury site. And you can see that the cells are colored or have various colors. And the idea there is to represent the stem cells that will then go on to become differentiated tissue so that this regenerated limb then has a bony structure, a muscular structure, a neural structure and a vascular structure that again looks identical to the way that it did prior to the imputation. So again, the objectives of this short talk will be to one, define regenerative medicine and more specifically focusing on stem cell biology and some of the basics, including what exactly is a stem cell. And we heard a little bit about that from yesterday's talk with Max Gomez. And then I'd like to take just a few minutes to overview the state of the science in some of some, some I think exciting clinical progress that we've made. So when looking at regeneration, one distinction that I like to make is this distinction between repair and regeneration. And these are two words that are commonly used pretty interchangeably. But for the purposes of my talk, I'd like to distinguish between them, really kind of emphasizing, for example, in an adult wound healing process, what we see is oftentimes this default towards repair. And that is to say, you oftentimes get this scar tissue formation. The scar tissue formation is great because it of course helps to minimize further damage to that injury site, for example, and it helps to kind of contain injury and protect it. Of course, it will do this compromising the tissue's function. And we contrast that then with regeneration, with the idea of true regeneration, is to really recapitulate, at least in part, the original program that directed tissue formation. And so many investigators are very interested in understanding the developmental cascade, the way that our stem cells behaved and act and mobilized for that original tissue formation during development. And trying to understand is there a way to tap into that capacity throughout adulthood and throughout our life spans. So like rehabilitation, the field of regenerative medicine is also very broad. And there are a number of different approaches that are being pursued with this ultimate goal of functional tissue and organ recovery after, after nutrient disease. And so here you see some examples, organ tissue engineering where there's organs and processing organs within the laboratory, IO inductive scaffolds, and probably the area of regenerative medicine that has most captured the public attention is that of cellular therapeutics. And so stem cells, as you know, are thrown around a lot. I know in the grocery store, you can see, even find shampoo that says there's a stem cell infusion. So I thought it would be worthwhile to, when we throw out this term stem cell, what exactly do we mean? Because there are some very distinct features that are required to, in fact, determine that a cell is a stem cell. So one of the first ones, and again, we heard a little bit about this yesterday, is this concept of multi and pluripotency. And the idea here is that an undifferentiated cell has the capacity to differentiate into multiple different tissue lineages. And so you can see here examples of blood, scar tissue formation is a possibility, and in many ways it's believed that, throughout adulthood, our stem cells have an increased predisposition to differentiate towards that fibroidic pathway, and that that might be contributing to some of the fibrosis formation that we see so often into adulthood, and then even more so with increasing age. Of course, we know that embryonic stem cells are probably the ones that have, that we have access to, that have the most potency and are able to develop into the most different tissue lineages. These embryonic stem cells are found within the inner cell mass of the blastocyst, and again, when given the right extrinsic signals, then these cells have the capacity to form a number of different tissue types. We know now, and we're continuing to learn more and more about it, that there are adult stem cells as well that are found throughout our body. And I'll apologize because I don't have the beautiful videos that Max Gomez showed yesterday, but this is just kind of intended to show that we now understand that stem cells are found in multiple tissues throughout the body. And so bone marrow, bone marrow, is something that we've been interested in in stem cell grafting for decades now. Dental pulp is an area where we're starting to see a lot of new research coming out. One area of highly abundant stem cell presence is the fact, and of course that's something that's getting a lot of interest because nobody has any qualms with parting with their fact, and kind of making a donation to isolate stem cells and expand them and then use them for subsequent therapeutics. Skeletal muscle is one tissue that really has a remarkable capacity to regenerate in cases of relatively minor injuries. And again, in the field of rehabilitation, that's something that's that remarkable capacity that we really try and tap into and promote with a lot of our interventions. So I mentioned that we have the characteristic of stem cells, and that is potency, that it must be able to differentiate into more than one tissue lineage. So how do we test that then? And so one of the very common ways of, if you have a stem cell or a population in question, and you'd like to know whether it has stem cell capacities, we can test this in vitro. And what we do is we have our stem cell population for evaluation, and then again, we can expose the cells to various inducing agents or different types of media and microenvironmental factors that would then drive them to a various tissue lineage. And so you can add a progenic media, and then you can, of those cells in the culture dish to actually form myotubes in vitro. You can do this with different conger sites, bone. And then those that do not respond to the different type of inducing agents, and you might be able to make a statement that they don't have, for example, osteogenic capacity. And so many times when individuals identify a new stem cell population, a part of the paper will be exposing them to various different factors and seeing what kind of lineage capacity the cells have. We can also look at this in vivo, and so one example is that you can take your labeled cells, or the cells in question, and you can actually inject them into a host embryo. And so then you create a chimeric embryo with your labeled cells inside. You'll implant it into the female, and then you evaluate the offspring. And so you can make some statements there again. If within the offspring you can see muscle tissue or bone or whiskers, and if you see that that's of a donor of a labeled cell origin, then you can again make some statements about the potency of those particular cells. Another important characteristic of stem cells is this idea of a cell for a new one. As you can imagine, stem cells then are the primary cell population that are responsible for dictating the regenerative cascade. Upon activation, stem cells go into action, and they begin to rapidly divide and differentiate into the various different tissue types of the tissue of interest. What's going to be important, however, is that somehow you have a mechanism for being able to maintain that stem cell reservoir such that you still have stem cells available for future rounds of regeneration should you need it. And so the way that biology has handled this is this capacity of stem cells to sell for new. And that is at the end of the day, you'll have a cell that looks identical to the mother stem cell, not differentiated in possessing these characteristics. And so this is kind of what a cell lineage tree will look like. Again, you have the stem cell in question. It becomes increasingly committed and then can differentiate in response to specific cues. It can differentiate into various differentiated cells. And so really you're kind of talking about this interplay and this balance between the potency of the stem cell and the capacity to differentiate into multiple lineages versus commitment towards a specific tissue type. So let me give you just one example. And in my laboratory, we're very interested in skeletal muscle regeneration and understanding some of the declines that occur with the healing capacity of skeletal muscle over time. And in fact, the muscle stem cell was, you can see here by Mato et al, was a first to identify the muscle stem cell. That was in 1961. And so we've really come a long way in terms of our basic biological understanding of how stem cells work. What we know now is that a muscle stem cell resides in a normally quiescent state at the periphery of, I'm gonna give you one. It resides that the periphery of your muscle fiber between the basal lamina is well membrane. And so I've highlighted the muscle stem cells here in red. And in fact, now we've come to learn what are specific transcription factors that we can say definitively, this is indeed a muscle stem cell. But again, most characteristics, I would say, that it's really that anatomically location in a quiescent state at the periphery of the muscle fiber. Now, these muscle stem cells make up a very small percentage of the total myonuclear number. And I think it's somewhere between two to three percent. In the case of an active muscle injury, however, these, this small population of stem cells at the injury site will then be evaluated and will begin a phase of proliferative expansion. And so they'll start to rapidly divide. Again, they need to amplify their cell numbers in order to have enough stem cells available to do the job necessary for the injury. Again, this arrow going to be presenting self renewal again to reinstate that quiescent stem cell reservoir. Proliferating cells slowly begin to fuse together. And in muscle, we have this great advantage of knowing an actively regenerated muscle fiber by this very characteristic central nucleation where the nuclei line up in the middle of the muscle fiber. And we can go in and look at that with various approaches and kind of have a great indication of how well a tissue is regenerating in response to an acute injury event, for example. And then eventually these centrally nucleated muscle fibers will migrate out toward the periphery. And again, at the end of the day, you see a structure of the myofiber that looks pretty much identical to the way that it did prior to injury. So I mentioned PAC-7 is a canonical muscle stem cell marker that we use to definitely say, for example, when we're isolating muscle stem cells, this is a good muscle stem cell population. However, because PAC-7 is within the nucleus in order to be able to assess it, then we compromise the viability of our stem cells. So one way around that, what we want to do is be able to isolate a very pure population of stem cells. But then we want to be able to go on and further evaluate it, challenge it, manipulate it so that we can really better understand some of the characteristics of the stem cell population themselves. So what we do is we take advantage of the presence of cell surface markers. And these are specific proteins that are found. And again, in the case of muscle stem cells, for example, we have a very good understanding of what cell, cell surface markers stem cell, muscle stem cells possess and which ones they do not. We can then use a flow cytometry assay in which we can take our cell population and then label the cells for the specific surface markers of interest. And then with flow sorting, we can actually isolate a highly purified population of just those muscle stem cells. And so this is a really powerful tool for us, for again, being able to extract out only those cells of interest. So I mentioned that cellulotherapeutics then are probably one of the most widespread and most widely discussed aspect of the protective medicine. But in fact, there are a number of really beautiful studies going on that are looking at combination approaches. And so investigations that are looking at not only using the isolated and expanded cell vinculture, but in combining this with other approaches, such as scaffold approaches. And the idea here with tissue engineering is to kind of provide the cells with the microenvironment that they need to be more effectively incorporated into the host tissue. And so these scaffolds can be synthetic. And so there's really some great advances going on with biomaterials and the development of scaffolds that will really promote specific aspects of the stem cells that will make them more amenable to the targeted regeneration of interest. There's a lot of interest in native scaffolds and isolating native scaffold materials and kind of combining that with a cellular therapeutic approach. And in many cases, then the scaffold after implanting the cells and the scaffold material into the host, the scaffold many times will degrade away. So again, after longer periods of time, you really won't see that presence of the scaffold material. And I think another important aspect of tissue and organ engineering is that there's a lot of attention paid again to this microenvironment. And so it's not only scaffold materials and growth factors, but it's also a lot of attention to physical and mechanical forces that will then give the stem cells the information that they need to behave in the way that we intended them to even following implantation into the host. So now I'd like to move on and just quickly kind of talk about the state of science and translation of some of these regenerative medicine technologies. And even though we oftentimes think about regenerative medicine as being very modern day science and the truth of the matter is we've actually been doing regenerative medicine and stem cell transplantation for decades now. And again, the bone marrow transplant was in the 1960s. Now we know that actually bone marrow transplantation and blood stem cells are actually the standard of care for a number of blood diseases, including leukemia, lymphoma, and several inverted blood disorders. There are a number of ongoing stem cell studies. And by one reference, it's been suggested that there are over 2,500 active regenerative medicine clinical trials. If you do an NIH reporter query and you look up the just the terms stem cell transplantation, what you'll see is that there are 630 active studies and maybe even more remarkably, over 400 of those are clinical trials. And so even though there's a lot of enthusiasm and excitement about the field, I think we're starting to see more and more that there are more clinical trials being tested as well. So here is just one example of a clinical translation. I think this is a really exciting study that was published. I believe in 2010. And it was looking at a stem cell therapy for coronal regeneration. And so in the case of an acute injury to the coronia, what we see like in so many other tissues is this deposition of fibrosis. Well, just very briefly, what the investigators did here is they recruited individuals with a chronic coronal injury. And so you can see kind of what it looked like at the time of inclusion in the study. Most of these individuals had unilateral injuries. And so then they isolated the stem cells from the opposite un-injured eye. And they expanded the stem cells in culture. And then they injected it back into the same individual. So what is called an autologous transplantation where you get cells from the patient, expand it, and then put it back into the patient. And this is of course desirable because you don't have to worry about rejection issues. Well, they found, and they actually ended up following these individuals up to 10 years after stem cell transplantation. What they found was already after one year, a majority of the patients had seen, or had had what they would call a successful implantation where they saw kind of the restoration of that coronal clarity and structure following stem cell transplantation. And then again, they followed these individuals for up to 10 years later and found that of the 78% of the individuals who had successful engraphments, most of those, that most of that improvement was sustained as long as 10 years. And so when I mentioned that these are chronic injuries, I think the average injury, or time since injury to the stem cell transplantation was something like 10 years. So this was really chronic. And that fibrosis had been in the area for quite a long time. So I think this is one area where we're really starting to see some great progress. There are of course many challenges that we face when considering regenerative medicine technologies, when considering cellular therapies, for example, we really need to think about cell source. And so we have an abundance of potential sources available to us, whereas we can extract stem cells. As I mentioned, fat is also usually one where there's a lot of interest. But, you know, and kind of weighing the benefits and kind of the invasiveness of being able to expand the cells or isolate the cells and expand them. We need to think about donor matching. So if you do a transplant from a donor into a host, really trying to consider what type of rejection and how to handle that rejection. Of course, ethical considerations has always been something that's really been of great importance to the field of regenerative medicine. How do we deliver the cells? What is the best route to administer the cells? Is it a local delivery? Is it an intravenous delivery? These are all things that are very carefully considered and are really being tested in these preclinical trials. And I think it's also important to consider the over-hype. And so as a field, I think there's a lot of potential. I think we're making some really great progress. But we need to balance that with, you know, just being very careful in terms of our interpretation. And I really appreciated last night's keynote talk because he really spoke to this in terms of really needing to do our due diligence preclinically and not being over-enthusiastic in terms of translating these therapies to the clinic. And so I think another great advancement in the field of regenerative medicine is this idea of induced pluripotent stem cells. And this was a paper that came out in 2006 and cell with a senior author, Yamannaka. And here what they demonstrated is that you can take a skin cell or a blood cell from an adult. And with the simple application of just a few genes, you can actually induce pluripotency in these adult stem cells or in these adult cells. And this is really exciting because I think this has a lot of potential in terms of self-source and really opening up a wide range of possibilities. And this also kind of, it's amazing to see how you can kind of revert and induce this pluripotent state in what would be considered an already differentiated cell. And so this is a technology that is being investigated for a wide number of diseases, heart, liver. And it's actually even being investigated for cancer. And so I think this has a lot of promise. And I think there's a lot of considerations that need to be made as we look at the potential of these IPSEs. And so just kind of coming back to this, I think there's a lot of great advances that are being made in the field. And for example, these IPSEs and this capacity to kind of tap into some of the innate properties of the cells and kind of manipulate them with this ultimate goal of really enhancing tissue or restoration and ultimately functional recovery after injury and disease. So with that, I want to thank you and I'm willing to take a question. Zoom 2003 | ↗ |