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AUGS/IUGA Scientific Meeting 2019
Basic Science Lecture - Stem Cells and Scaffolds f ...
Basic Science Lecture - Stem Cells and Scaffolds for POP: Designing the Future
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Thank you very much to the organisers for the invitation to speak to you today about our work on mesenchymal stem cells and scaffolds. And we're talking about prolapse today. These are my disclosures and some of the grants have funded the work I'm describing. I'm going to talk about mesenchymal stem cells. And they can be sourced from many different tissues, like bone marrow and adipose tissue. These are really highly invasive sources and require an anaesthetic for acquisition. They can also be acquired from waste tissues like umbilical cord and placenta. And we look at endometrium, which is minimally invasive source. We were very interested to use mesenchymal stem cells from endometrium because it's a highly regenerative tissue, one of the most regenerative tissues in the body. And so these cells are used to producing a vascularized stroma each month. And in postmenopausal women who have a thin, atrophic endometrium, it can regrow when they take estrogen replacement therapy for a fairly short time. We characterized these mesenchymal stem cells in the classic manner that the ISCT suggests, and also more of their stem cell properties as well. They're clonogenic cells and they're quite rare, only about one to 5% of the tissue. And then we went on to discover specific surface markers of these cells so that we could identify what their actual cell type was and their niche in the endometrium. And I'm just gonna focus on the SUS-D2 today because it's a single marker. But both sets of markers show that these cells are perivascular cells. They're around the blood vessels in basalis and in the functionalis of the endometrium. This is really important because it means we can acquire these cells by endometrial biopsy, which is relatively non-invasive. And I've already told you that we can get them from postmenopausal women because, remember, they're the main clientele of prolapse, or who have prolapse. We've spent a lot of time in developing culture expansion protocols so that the cells remain in their undifferentiated state. And we're also working under GMP-type conditions. And a lot of this work has been done by PhD student Shanti Gurung. We've got rid of serum. We're still working to get rid of all animal products. But the main thing that we've done, I think, is we used a small molecule screen to pick up a small molecule that would keep the cells undifferentiated. One of the problems with a lot of the clinical trials using MSC of various sources, that they've been rather underwhelming in their effect. And a lot of this is because of the heterogeneity of the cultures. And a lot of them are probably really senescent cells. But the A8301 is a TGF beta receptor inhibitor, and it maintains the cells in an undifferentiated state. And we can monitor this by the percentage of SUS-D2 cells. We get about 90 to 95% for our various patient samples. This is really important if you're looking at an autologous product, where you've got to have a release criteria that's relatively standardized and quite difficult between different patients. And we can generate large numbers of cells. We've done global gene expression profiling to see just exactly what happens to the cells so that they're well characterized before we try to put them into patients. And some of the things that we've found are that the very recently described mesenchymal stem cell potency genes, the twist genes, the twist one and two, and then we found a whole lot of up-regulated angiogenic genes, angiogenesis genes, anti-inflammatory genes, immunomodulatory genes, anti-apoptotic genes, and anti-fibrotic genes. And these are really a very desirable phenotype if you're wanting to repair tissue. So now we're just developing Xeno3 and our potency assays. At the same time, we've been developing various biomaterials for prolapses, starting from fresh, actually. And we did start with a non-degradable material, and this work's been done with CSIRO. You can see in the little blue logo. And they, material scientists and textile scientists, and they designed the material and chose polyamide out of a series of polymers that, because it matched the biomechanical properties of human vaginal tissue. And then we knitted the material in patterns that were around at the time we made these. To carry cells, we needed to, because the pores are quite large, they're about a millimeter in diameter, we dip-coated in gelatin, and then the cells are seeded onto that, and you can see that in the greeny color, the cells are growing on that gelatin. We tested these in small animal models, and here we have to do subcutaneous wound repair type model. And we found lots of things, but I think one of the most amazing findings, or probably the most important, some of the most important findings were how the collagen was laid down. And you can see where the, if you look at the scanning electron microscope, where the EMSC are, you can see that crimping, versus where there's no cells, more of that banding or scar-type collagen. That's physiological, the crimp in collagen fibrils. And when we looked at 90-day time point at the biomechanical properties, then, of the mesh tissue complex, you can see in the red graph that it's much less stiff when we had the cells present. And also, there's that toe region just down between 0 and 2 on the x-axis. That's longer, and that's really important. And this is because of the crimping of the collagen, and this has give, okay? And this is really important in a vaginal area. Now, before we started to test in a large animal model, using the vaginal approach, you really need to have some outcome measures, more than just histology. And so, we looked at adapting the POPQ, and we did this with Anna Rosamilia and her team, and Nathania Young headed this work. And we looked at POPQ points that are shown there, and measured them. And when we looked at porous sheep, we actually found we kind of had like two populations. And there were those that seemed to have AA and AP or BA measures that were relatively abnormal in the human comparison, so greater than equal to naught. And we called them significant POP. And then we had another group that were relatively normal. And we looked at the demographics of these sheep. And when you looked at the age, this was quite significantly different, and they were older sheep. With parity, it was very significant. Look at the odds ratio. So, increased number of deliveries that those sheep had, and then also the number of lambs. We've also gone on to develop another device with John Arkwright, shown there, who's a biomedical engineer at Flinders University. And he put these fiber optic little pressure sensors on the blades of a speculum, both blades. He adjusted this, adapted this speculum so you could open it in a parallel manner. And we used these in sheep. And you can see the position of the device in that diagram. And we used those two groups of sheep, and we looked at the pressure profiles. Each of the numbers is one of the sensors. And you can see with the dotted line, I've got a, maybe, yes, with the dotted line down here, these are the ones that had that significant POP. And they had much weaker vaginal walls up at the cervix end. So, we were ready then to test our mesh with the cells on it, our polyamide mesh with the cells in the sheep with transvaginal surgery. And Stuart Emerson just recently graduated, did a lot of the analysis of this work. What we did here was, firstly, we selected the sheep from a flock. Parash sheep had these POPQ values. Okay, we selected the worst ones, and the average was minus one, or the median, at AP, and zero for AA. And we matched them across our three experimental groups. We also did fiber optic, but we haven't really analyzed it in that way. Now, when we began, when we put our cells in on the mesh, we were very disappointed transvaginally to find a lot of exposures. So, we changed our methodology. And what we did was we put the cells, we put the mesh in first. The polyamide mesh in this knit is very drapeable, and it just lays beautifully on the tissue. And then we had the cells in the gelatin, and in liquid phase, it was dripped over the mesh, and then cross-linked in situ with blue light, didn't take very long, and then stitched up. These mesh, in this two-step procedure, which I can tell you is far more surgeon-friendly than the single-step procedure, which is quite difficult, we found we had no exposures as opposed to five when we had PAG together in that way. We've been using autologous cells because this is the model that we're exploring, and so we had to get the cells from the sheep, and we want to track what they're doing when we've administered them. We had to label them with these paramagnetic nanoparticles. They also have a fluorescent dye attached to them, and you can just see the fluorescence shown here. After 30 days, we still had autologous cells there, and that's why they were there for that length of time. We found less myofibroblasts, so less fibrotic response, and less inflammatory macrophages. So the PA mesh with biomechanical properties that match human vaginal tissue has superior drapeability to the PA gelatin-type mesh, and we had no exposures in a vaginal surgery model using a vaginal wall weakness, and our autologous EMSC survived 30 days. I want to go back to that pressure sensor device because we've been using this and testing this in women, and John Arkwright has made a better prototype than the ones on the speculum, and you can see that there, and you can see the little pressure sensors on the one that's on at an angle, and then at the bottom one that's white, you can see that's open or dilated, and this is done now automatically without any human touching, and you can see just the diagram of it in situ. Now, this is the data we obtained, and our first ones, we also inserted a rectal balloon, and so we got intra-abdominal pressure measurements as well, and you can see that the nine, this one had nine sensors on both the anterior and posterior wall, and you can see that it could easily pick up a valsalva maneuver or cough in both the posterior and anterior, but when we did the dilations, the automatic dilations, we could see some changes in pressure in some of the sensors in both the anterior and posterior, but independently of the intra-abdominal pressure, but we still have to really analyze this and try and understand what it fully means, so this is quite a work in progress that I'm telling you about. We've also been given issues with the degradable mesh. We've been developing non-degradable mesh using electrospun nanofibers, and what we've been doing, what electrospinning really is is making a solution of your polymer, and extruding it through a narrow nozzle in a high-voltage electric field, and collecting it, and it deposits these nanofibers in quite a random arrangement, and this recapitulates the arrangement of fibers at the nanoscale in connective tissue, actually, and it provides a high volume-to-surface ratio and allows cells to attach. One of the issues, though, with a lot of these polymers are that they're hydrophobic, and cells don't really like to bind to them terribly much, although you can see that pink cell there. It has bound to the PLACL. So what Chianti Mukherjee, who's the post-doc who's driving this work, she blended the PLACL with gelatin, so during the electrospinning process, and you can see, if you compare between B and D, that many more cells, which are the dark shadows or dark areas, they're the cells that have penetrated through those two-micron pores because they've got binding sites provided by the gelatin. And also, the young modulus of these scaffolds is in the range of human vagina. Now, we've also looked at them in mouse models and shown that, we've looked at the macrophage response, but one of the issues with having a blend of a polymer and gelatin is that the gelatin degrades, the gelatin in the fibre degrades, and you can see that on the lower image there. But when you have the EMSC present, this is mitigated, and in fact, if you look at the bottom right-hand corner, you can see that there's the full width of that scaffold, but also, I want to show you that those images there, the green are the macrophages, where we didn't have the EMSC, there's a lot of macrophages within that scaffold, but where we had the EMSC, the macrophages are around the edge a little bit, but it's full of cells, because you can see lots of blue nuclei, but they're not the macrophages, so this is tissue cells infiltrating that scaffold. This is exactly what you need if you're using a degradable material. So, that blending of a polymer with a gelatin produced a scaffold of similar stiffness to human vagina, increased cell-binding sites, increased the degradation rate, but promoted cellular infiltration, but this was mitigated by the presence of the EMSC. Now, finally, we've also been doing some 3D printing, and bioprinting of the cells, and this work's been done by Kalian Paul, who's a PhD student in our team, and for this unit, he did melt electrospinning, so this is really, you have to have a polymer that you can melt, and he chose PCL, which is a very slowly degrading polymer, and then he designed the CAD so that he could print a design that would form like a mesh, layer by layer by layer. And this is just some of the data he's generated, but you can see that it is like a porous scaffold that will allow cells in, and it indeed does. The biomechanical properties are not terribly good. They're not, couldn't use them for prolapse. It's too weak at this point in time. So, but this is the start, okay? But what he also did is he took our sustitu positive EMSC, they were labelled genetically with a transgene that carries a fluorescent dye, so you can find these cells when you implant them. He put them in a non-animal gel, aloe vera and alginate, and then he used the 3D printer to print on top of the 3D scaffold, actually, and this is what it looks like. There's a scanning electron micrograph, and also the cells, you can see there, the little pink things in amongst and attached to those fibres. He looked at, in the mouse model, only one week. They looked at the inflammatory response, and really what that shows, if you look at B and D, is there's a lot less M1 inflammatory macrophages and a lot more M2 wound healing type macrophages. This is because of the EMSC, and this electron micrograph, which is just part of what he has in his paper, and the red areas are showing the increasing magnifications, but if you look at the one where the mess hide an EMSC, in comparison to the next door one, you can hardly see the fibrils of the printed scaffold because there's been a lot of cellular infiltration and collagen deposition in this mesh. So the EMSC, again, typically alter the immune response, the macrophage response, and they promoted collagen deposition and cellular ingrowth. So in summary, we're using autologous EMSC and degradable or non-degradable scaffolds, really focusing now on the degradable ones. We get the cells, the plan is to get the cells from the endometrium. We purify them with SSD2 magnetic beads, easy for cell production. We're working on our protocols to keep the cells in the undifferentiated state, so they will survive longer in viva and produce a lot more cytokines, et cetera, and perhaps even incorporate than they would if we just transplant fibroblasts. We've developed three different types of material at this stage. We've put cells with all of them. We've tested them in small animal models. We are developing the pressure sensor device and our sheep model, transvaginal surgery model, where we use sheep, harvest sheep, with weakened vaginal walls, and we match them in the groups. And we're now working on with the newer version of the device in women. And there's many people to thank. This is really a big multidisciplinary, multi-institutional project. I've just listed people in my group who've contributed to the work I've shown you today, and also from CSIRO, Sharon Edwards who did the non-degradable polyamide work for us, John Arkwright at Flinders University, and Monash, Anna Rosamilia, and her wonderful team of fellows who do all the surgery for us, and a lot of the pop cues and pressure sensor, and our funding bodies. And I'm just gonna leave you with a message. Thank you for your time and attention. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Mimi Lucas, San Diego. Wow, that's really awesome. Thank you. And gives us a lot of hope, especially with all the mesh problems that we have around the world. Can I guess that only women who have a uterus, potentially in the future, would be eligible for this kind of therapy? Or is it possible to get endometrial cells from somebody else? Look, you could. And a lot of the other MSC is used in the clinical trials, and I think there's over 500 for many different conditions. They're about half autologous and half allogeneic. And so they possibly could. I just don't think they'll last very long. When we put them xenogeneically, like human into animal, they don't survive as long. And while they're meant to be lowly immunogenic, I think they will be immunogenic. And we've got other ideas where we would like to perhaps use them more than once. So maybe a plug for uterine-sparing prolapse repairs until we get this technology up and running. That's right. I think that is a very encouraging thing to hear that that's what's going on now. Hey, I'm a second-year resident, so my knowledge about this is pretty limited. First of all, it was a great talk. Thank you. I wonder how you'll do pop cues in mices. That is impressive. Coming to my question, I see that you've used endometrial-derived stem cells. And in the presentation, you mentioned that you do an endometrial biopsy to get these cells. I was wondering if you or your group thought about using menstrual blood-derived stem cells for a similar purpose, as it's more non-invasive. We do. We work a lot with menstrual fluid as well, and more in our endometriosis work. But they're definitely there because they're present in the functionalis that's shed. And we've looked at, we've cultured them too with the A8301, and it's quite similar. Same with the, not quite as good. I do think the endometrial ones are the best and most proliferative, but they are okay. When we've done a comparison with bone marrow and fat, MSC, they are all better. The post-menopausal, the pre-menopausal are the best. Menstrual fluid as well. Thank you. And placental too, decidua basalis. Kathy Connell, University of Colorado. Carolyn, your work is amazing. It's been amazing to see how much you've come throughout the years, and I think you've done the most in terms of showing how important that matrix is, of the extracellular matrix, and how everything communicates. Now that I think, I've watched you grow this over the years, literally, I love that you have the pressure sensors to see where the biomechanics are coming into play. Do you think you could take this technology too to the uterus sacral ligaments or the paravaginal attachments? Because I think without reconstructing the anatomy, like if there's still some defects, you might have some pressure on those tissues. Because I think you could, no? Look, I think that's probably the most challenging because it's more deep within, and then to get an animal model that we could use. Really, we started with the vaginal wall because it seemed to be the easiest thing, and that's what most of the measures were going, and so that's where we thought we would commence. And I think there's also the pelvic floor muscles too, but I think you need to use the muscle-derived satellite cells. But I think it would be really good if we could do something in that line of things as well, but I think it'll be quite challenging. Cathy, I think you'll need to come and work with us. Okay, I'll go, won't you? To develop that along. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you.
Video Summary
The speaker discusses their work on mesenchymal stem cells (MSCs) and scaffolds in the context of prolapse. They explain that MSCs can be sourced from various tissues, including bone marrow, adipose tissue, umbilical cord, placenta, and endometrium. The focus is on using MSCs from the endometrium, as this tissue is highly regenerative. The speaker describes the characterization of these MSCs and the identification of specific surface markers. They also discuss the development of culture expansion protocols to keep the cells undifferentiated and the use of a small molecule screen to maintain their undifferentiated state. The speaker highlights the importance of standardized release criteria when using MSCs for autologous treatments. They then discuss the development of different biomaterials for prolapse repair, including a non-degradable polyamide mesh and degradable electrospun nanofibers. The speaker presents data from animal models to demonstrate the efficacy of these materials in promoting tissue repair. They also mention ongoing work on 3D printing and bioprinting of MSCs. The video concludes with acknowledgments and a Q&A session.
Asset Caption
Caroline Gargett, PhD
Keywords
mesenchymal stem cells
prolapse
endometrium
biomaterials
tissue repair
3D printing
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