Alla Danilkovitch, RN, MS, PhD gives an in-depth look at mesenchymal stem cells and their role in wound healing. Dr Danilkovitch reviews the function of MSCs, where they are found in the human body and how they change during the human lifecycle.
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Alla Danilkovitch has disclosed that she is an employee of Osiris Therapeutics, Inc.
Male Speaker: Return engagement is Dr. Alla Danilkovitch who is one of the Chief Scientific Officers at Osiris Therapeutics. And who I often turn to when I need some detailed information on stem cell function or dressings, etcetera, etcetera. Since I have a hard time pronouncing her Russian name, I just call her Alla. She’s a good friend, a very, very knowledgeable person. Let’s welcome her back to tell her to – tell us about the biology of stem cells so that we can have a good understanding of this new and exciting wound technology. Alla.
Alla Danilkovitch: Thank you very much. I’m enjoying double dose of the podium so I hope that you will enjoy my talk also. So I have to disclose this is an educational lecture and the only disclosure I am making that I am a full-time paid employee of Osiris Therapeutics. But my presentation will be on promotional and nothing except of this pure science. I have only one objective for this lecture. To overview our current understanding of mesenchymal stem cells properties and they are all in wound healing. If I will be able to do that, I think that it’s a very good objective to reach. But before we will go to mesenchymal stem cells, I wanted to overview stem cells in general because there are a lot of misunderstanding what are stem cells, what are they doing, what type of stem cells exist. And then we’ll go to mesenchymal stem cells and to look on their history, identity, biological activities and particularly with accent on the MSC role in the wound healing. So stem cells, what are stem cells? By definition, there are two key criteria to identify stem cells and how you can distinguish stem cells from all other cells in the body. Number one property is cellular unit. You can see here that stem cell mother will divide and create two daughters. One daughter will be exactly the replica of the mother. That’s so called cellular renewing, to create the exact replica. The second one will be different from the mother and these cells will be committed to become some type of tissue or differentiated tissue and specialized cell. So renewing is exist to maintain pool of stem cells in the body. Differentiation, this is a mechanism of how our body repairing damaged and deceased tissues. In the beginning, we are thinking that stem cells, they only exist at the stage of their fetal development. But right now we know that stem cells consist in our body through the entire life. But stem cells undergo several changes during their development in our life. Step number one that you can see that, embryonic stem cells, they exist only for very short time in the development, and the stage of blastocyst, when the embryo is just a mass of 100 to 500 cells. And distinguished property of these embryonic stem cells, that they can create all types of tissues and organs in the body. So after this blastocyst stage, there are no anymore embryonic stem cells, they become fetal. There are also stem cells so they can be cell renewed and create differentiated cells. But there are differentiating potential is already bone marrow. So the quality of stem cell, you cannot recreate all tissues and organs. You can only recreate only some of them. So when we are here after birth, we’re still carry of what stem cells, they considered already adult stem cells. In the time period from one to four weeks after birth, these so called young adult or neonatal stem cells. And during the life we carry already so called classified adult stem cells. These stem cells called the multi potent because again they can be differentiated in different types of tissues, but usually they are more lineage restricted. So in general, there are two big classes; embryonic stem cells and adult stem cells and the difference between these two classes is that embryonic stem cells, you can develop all types of tissues and organs. Adult stem cells, they are only restricted. Here are two examples of adult stem cells found in bone marrow.
One hematopoietic stem cell. Hematopoietic stem cell can recreate all type of blood cells but, can you develop brain or liver from hematopoietic stem cells? The answer is no. The same mesenchymal stem cells, they are also see in bone marrow and they can become any type of connective tissues but no brain or blood cells. So if you look again what is the difference between embryonic and adult stem cells is at the present time or current knowledge, we all know about the adult stem cells and just a little bit about embryonic. If you asked me how many patients treated right now with embryonic stem cells, the answer will be zero. How many patients treated with adult stem cells, millions. Because if you look on the hematopoietic stem cells, bone marrow transplantation, it’s treatment of patients with stem cells. We just used to it and not thinking that this is the actual treatment with stem cells. And they already celebrated to our 40-years since time of the – since bone marrow transplant took place. So out of patients we have treated. Mesenchymal stem cells, they also have a pretty long history and I will talk about it on this slide. It is a crowded slide and what is very important that the very first kind of thinking that there are specific cells in bone marrow that can become one here in the 1869. And then they took us hundred years to really to get prove that yes, bone marrow will have stem cells that can become one. So two key persons who actually discovers mesenchymal stem cells where a Russian scientist Alexander Friedenstein and he discovered the Maximow’s bone marrow. And then later, Arnold Caplan who was the professor of Case Western University, he described in 1991 similar cells found in human bone marrow. Then another interesting milestones that we started treatment with mesenchymal stem cells, the very first patients in 1998. And since that time, we continuously treating patients and we started like treating patients with genetic diseases from mesenchymal origin like OI and some other diseases where cells from mesenchymal origin, they are using defective collagen or other proteins. And we have observed some clinical benefits. So then we spent our knowledge about the stem cells and realized that they are actually have a broader therapeutic potential. Two other milestone where that in 2005, Osiris Therapeutics brought to the market Osteocel which was a tissue allograft containing bone viable cells and as you know bone will have MSCs in it. It was the very first MSCs stem cell based product in the market. And then in 2012, again the Osiris Therapeutics good approval of Prochymal which was very first in the world stem cell drug approved. This drug is based on the culture expanded bone marrow isolated donor derived MSC. So where we are today? If you look right now, more than 400 clinical trials ongoing and registered at clinicaltrials.gov. These clinical trials are for different indications with different MSC derived from different organs from placenta, from bone marrow, from adipose tissue and from other sources. And if you look in PubMed, you can find more than 3,000 publications so far dedicated and describing features and different characteristics of MSCs. So if you give me a cue with cells and ask, can you tell me whether I hear from the cue mesenchymal stem cells? Yes, definitely we can do that. So to identify cells that is MSCs, you will run all these tests described on this slide. Number one, you plate the cells and you should observe that these cells will appear to plastic and will have certain specific cell morphology. Then you can take the cells and using specific antibodies, recognizing different cell surface markers, you can say, “Okay, based on what these cells – expressed on the cell’s surface or do not express, you also can utilize this parameter to characterize cells. And what is more important is that you need to run trilineage differentiation.
So you put cells in the culture and create adipogenic, chondrogenic and osteogenic causative conditions and usually they absorb that these cells become adipocytes, chondrocytes, or osteoblast. So if all three parameters will meet certain criteria, you can say, “Yes. In this tube are really mesenchymal stem cells.” Where are MSCs are coming from? Right now there are three hypothesis. In all three they have some evidence that it may happen but the last one is getting more and more solid facts. So that it’s probably MSCs are coming as parasites. So hypothesis number one that MSCs are they resides in one home organ. And probably this home organ bone marrow. And there when you have injury or tissue damage or damage due to inflammation or disease, these MSCs will leave bone marrow and will come and rescue will help to repair damaged tissues. There are some evidences for that. For example, in a blood circulation you practically don’t have MSCs. However, if I push you to run a marathon after 26 miles if I collect you a blood and measure I can detect a lot of MSCs circulating. Why it’s happening? Because it’s like MSCs they know that you damage your beating your muscles to death, you need to repair it. And they are coming to help you to repair your muscle damage. So in another hypothesis is actually MSCs – if you can take any organ or a tissue, you can isolate MSCs practically from any tissues. So it means that probably MSCs during our development, they just spread and now they are sitting in all organs. So there are proof or some proof for this hypothesis. And the last one which Arnold Caplan is thinking that right now, this is the true origin of MSCs. That all MSCs are pericytes. So because majority of our organs and tissues they are vascularized. And all blood cells they have satellite cells called pericytes. If you isolate pericytes and will run MSC identity test as I described on the previous slide, you may say yes. This pericytes are truly MSCs. They have all characteristics. And the idea is that when you have tissue injury, so these pericytes will come to the tissue and will they do their job to help with tissue repair. And this is also why probably you can isolate MSCs practically from all organs and tissues. So if you look how many MSCs you can find in different organs, actually bone marrow is not very rich source of MSCs. It’s good enough too because bone marrow is not very difficult to obtain from donors. And it’s not very difficult to isolate MSCs from bone marrow and then grow them [indecipherable] [13:17]. So that’s why bone marrow is widely used for isolation and expansion of MSCs and Prochymal drug was based on bone marrow isolated MSCs. But if you – what if you use bone marrow as a source of stem cells as it is, you can see it’s not very rich. And then you can see scroll down that all other tissues, they have variety of different number of MSCs, I wanted to point out that MSCs are also found in skin. So in amniotic fluid, amniotic fluid is not very rich source of MSCs, just a few pericystic amniotic fluid. And surprisingly for us, stem cells was that amnio and chorion to placental membranes actually the richest source of mesenchymal stem cells. Almost every secondary for a cell in the membrane will be mesenchymal stem cell. In another very key feature of mesenchymal stem cell, they immune privilege. If you wanted to transplant tissues or organs for almost all of them, you need to match between donor and recipient. If you do not match what will happen that immune cells from the patient will recognize each molecules or human leukocyte antigens, this is the key molecules that you need to match between donor and the recipient. And then together with co-stimulatory molecules, they will become activated and reject transplanted cells, tissues or organs. In contrast, mesenchymal stem cells, they don’t have HLA class II. They have very low HLA class I and they don’t have a costolateral molecules.
In this case, immune cells from the patient will do not recognize allogeneic mesenchymal stem cells as foreign. And you will not observe any adverse reactions and rejection. That gives you serial advantages. Number one, you can create products or drugs based on knowledge in allogeneic of MSCs and such products will be valuable off shelf. And you can use it for acute conditions. Number two, very often, on patient the MSCs they are not so functional. So especially with underlying diseases especially with each patients. So it gives you a very nice model, a very nice opportunity to use highly potent functional allogeneic stem cells. So as for functionality, if I will give you this work 10 years ago, I will get MSCs is a bone cell. So if you take the cell, you can use it for your orthopedic indications and they will be differentiated. And osteoblast and will help to repair bone. If it’s still true that MSCs why differentiation can help you to rebuilt tissues of mesenchymal origin. However, right now we know that this is only the small part with MSCs can do. On the daily basis, sitting in different organs and tissues, they’re really regulating immune and inflammatory responses wide secretion of growth factors. And they also provide tissue protective growth factors to other specialized cells in heart, liver and other organs. That’s why you can find a lot of ongoing clinical trials. They are utilizing the MSCs to treat autoimmune diseases to treat inflammatory diseases. Or utilize MSCs to treat a myocardial infarction. It’s not because the cells will be differentiated into something, it’s because these cells are very powerful to produce the right growth factors and provide therapeutic benefits. So here’s a trophic effect of MSCs one of them endogenous. These cells can secrete high levels of vascular growth factors and on the left panel, you can see MSCs associated with new blood vessels form after myocardial infarction in animals. So in the right picture, the same on the bone reparative model in animal, so that these MSCs on one hand become osteoblast and participated directly in new bone formation. But also they have found associated with blood vessel formation. And it’s happening through secretion of growth factors. It’s very interesting when Osiris was doing a various studies using MSCs for myocardial infarction. Our thinking was that, “Okay, MSCs will become myocardiomyocytes and this will be the underlying mechanism how the cells can help.” No, it didn’t happen. The main mechanism is through secretion of growth factors to support a vascularization to prevent eschar formation. That the eschar after myocardial infarction will not be so scarry. Heart will be more functional. So another feature of MSCs, their true immunomodulatory activity. So you can see on this slide that practically MSCs can regulate a biological functionality and activity of all types of immune cells. What is very important that MSCs everyone probably hearing, MSCs are immunosuppressive. MSCs if no inflammation will be not immunosuppressive. They are immunosuppressive only if there is a very high level of inflammation. So they are responsive to local microenvironment. This is in contrast to classical pharmaceutical drugs. If you are dealing with immunosuppressive drug, whatever you put in patient, there is no down regulation. There is no upper regulation. So that’s why so easy to over suppress patient and then you will get negative side effects. With the cellular therapy like MSCs, it will not happen because you see how it works. On the 11th graph, you see while putting in a laboratory tube, MSCs with active inflammatory cells, active immune cells. These cells will produce inflammatory cytokines. This is red graph, you can see over time upper levels of TNF is going up. So with the small delay MSCs will sense rising levels of TNF and start production of PGII. PGII is direct inhibitor of TNF. So what happen after three days, levels of TNF going down and then MSCs stopped production of PGE.
So that feature dynamic reaction and the response of MSCs to local microenvironment, will help you to reach good therapeutic effect without negative side effects. On the right graph similar situation but we put the cells on hypoxic condition. Again hypoxic will drive production of VGF because these cells will hear that no oxygen, no nutrients what we need to do, we need to simulate new blood vessel formation and bring to the area oxygen and nutrients. So VGF is one of the key factors to promote vascularization. As you know the healing happening in three key phases. And MSCs very important to support all three phases particularly they are very good to shutdown inflammation and transit wounds from inflammatory phase to proliferative phase. And this is very important particularly for chronic wounds that stopped in the inflammatory phase. So here is an example how MSC can assist with their epidermis formation. What was done for the study? Dermal equivalent was created. Fibroblast were seated on collagen and then epithelial cells replaced, this is figure A, and you can see a reconstruction of epidermis on top of the cytodermal equivalent. However, if you add 10% of MSCs and this is very relevant amount that found in the kids to cytodermal equivalent. You can see the thickness of epidermis significant the better. So truly demonstrates that MSCs in these are very important to – in participate in variety of different processes ongoing in the skin wound healing. So if you read 300 different papers dedicated to understand MSC role in wound repair, you can summarize this in one slide like that. So MSCs number one can inhibit inflammation through secretion of anti-inflammatory factors and can control wound by a burden because they are capable to secrete antimicrobial factors. They also will support all other cells essential for wound healing like epithelial cells, endothelial cells, fibroblast and even they secrete some of cytokines that will attract circulating in the blood different variety of stem cells. And that they will come to the area of the wound if no cyto cells and will become endothelial cells or nerve cells and will help with tissue repair. MSCs are very anti-fibrotic. And how they do it? Again, by regulating MMPs sometimes [phonetic] and also by secretion of anti-fibrotic growth factors like hepatocyte growth factors, VGF and very important they balance TGF. Very often people talking about TGF as fibrotic factor. But reality, there are three different types of TGF. TGF beta 1, beta 2 and beta 3. All three required for wound healing but what you need, you need the right ratio between TGF beta 1 and beta 3. When we are young, we have more MSCs and we have more TGF beta 3 in the wound, that’s why kids heal without scar formation. With aging, we are losing MSCs, we’re losing TGF beta 3, the ratio will be shifted toward TGF beta 1, that’s why adults they form scars. And right now some evidences that MSCs can promote regeneration of skin glands. It’s very important for skins functionality and also for cosmetic effect because you wanted to restore pigmentation. You wanted to restore everything what was in the skin. So as I mentioned already, unfortunately with age we are losing cells. Their numbers are declining and here what is going on in bone marrow, but again also MSC number and functionality dramatically change with diseases. Here is what is going on in type II diabetic animals. But also some other conditions will dramatically decrease potency of the cells. Here’s interesting, MSCs are isolated from bone marrow of patients with chronic wounds. Non-diabetic patients but wounds persisted for longer than two years. And then researchers wanted to understand how these MSCs can attract fibroblast because migration of fibroblast very important for wound healing. So they use the so called Transwell assay where you can put fibroblast on the top of the porous membrane.
See the MSCs in the bottom chamber. MSCs are supposed to secrete growth factors. Even if there are sufficient growth factors, fibroblasts will migrate to the bottom. And you can visualize it. So what they found that statistical significant difference between functionality of MSCs derived from normal donors versus MSCs derived from patients with chronic wounds. Clearly, you can see that they are not very functional. However, there is a hope. So if patients don’t have of all functional MSCs, can we use potent healthy MSCs and to restore and help these patients to heal? Obviously data from this study indicates yes we can. So this was done in diabetic animals. The diabetic mice like humans if you compare wound healing versus a normal mice, it is delayed and the healing is not so good. So here you can see horizontal line representing closure in normal mice, day 15 approximately wounds will be shrinking by 50%. But only around less than 30% shrinking of the wound sites happen in diabetic animals. If you have healthy, normal fibroblast to such wounds, you can see the fibroblast didn’t work in the way we wanted them to work. However if you add healthy potent MSCs you can see that this MSCs will assist and bring chilling in the diabetic animals to the level like normal animals here. Really, there is a hope and this study indicates that the MSCs might be very beneficial for wound healing. Especially with patients, all the patients with underlying diseases. But right now, we know about use of MSCs for humans. Unfortunately, not a lot. It’s a crowded slide, but reality is just a few publication case study mostly whereas some kind of MSCs autologous bone marrow derived were used for wound treatment. And still even in these cases, there are some positive results. There are several ongoing clinical trials with variety of MSCs but these clinical trials unfortunately still and face one or two. If you ask me, when do we expect MSC drug for wound healing will be on the market? My answer will be probably we need to wait 10-15 years before such products will come to the market. But unfortunately, you need it right now something to help for your patients. And what is available right now containing MSCs for your patients. Number one you still can use autologous tissues. So you know very popular one bone marrow or I think it was tissue. In this case, you will rely on the patient own MSC number and quality but if you ask me, it’s still better than nothing. So in allogeneic products available right now, I can mention majority of them for bone repair. Osteocel Trinity and the [indecipherable] [00:28:22], they’re all viable bone allograft and they're designed to stimulate bone formation. They contain bone resident MSCs. All three products they are coming from Osiris, so Osteocel was invented in 2005. Now, we sold to NuVasive and NuVasive continue selling this product. Trinity is another version of Osteocel and [indecipherable] [28:45] is the current Osiris products that are available for bone repair. For wounds it’s Grafix because Grafix is a cryopreserved placental membrane as I showed you on the previous slide that placental membranes have very rich source of young, potent MSCs. So there are some other products based on amniotic fluid unfortunately I cannot include them as products containing MSCs because per CC they will heal in every three MSC. And we don’t know exactly how many MSCs are we need to reach therapeutic efficacy but definitely three MSC per CC is not enough. An example of such product is full graft and several others via the – they’re all containing like one CC of amniotic fluid. So in conclusion, MSCs found in skin and like all MSCs are playing a very important role in cutaneous wound healing. So majority, how MSCs the mechanism of fraction is not through MSC differentiation but through secretion of growth factors so called the atrophic and immune modulative effects.
So MSC over many times with good aging and diseases dramatical decrease as well as functionality decreased that would leading to impart wound healing. So healthy potent MSCs are capable to correct defects in patient own MSCs. So in MSC autoimmunogenecity, allows you to use allogeneic MSCs without matching between donor and the recipient. And right now just a few tissue allografts are readily available that you can use for your patients. And let me stop here and if you have any questions.