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MS Masterclass: Selected Short Talk: Modulation of neuro-inflammation in murine immune-competent brain organoids | Julia di Stefano, University of Antwerp, Belgium

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Summary

This educational session will explore brain organoid models, derived from induced pluripotent stem cells, embryonic stem cells and microglia progenitors. Experience the firsthand journey into stimulating organoid models with different stimulants and testing the anti-inflammatory effects of certain molecules. Get a comprehensive insight into the development of organoid models, microglial integration, testing viability, and increasing cytokine production in vitro. All medical professionals interested in learning more about organoid models and their application in medical research are welcome.

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Learning objectives

Learning Objectives:

  1. Understand the process of deriving brain organoids from somatic or embryonic stem cells
  2. Identify the unguided and guided protocols for obtaining brain organoid
  3. Appreciate the use of flow cytometry and ELISA for evaluating microglia activation and cytokine production
  4. Explain the potential of using different molecules to reduce induced inflammation in organoids
  5. Analyze the differences in viability of brain organoids with or without microglia.
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Computer generated transcript

Warning!
The following transcript was generated automatically from the content and has not been checked or corrected manually.

jury is from on. Where? In Belgium. Thank you. Good afternoon, everyone. So I will talk about, uh, brain organoid, and I don't know how to. Okay, So for those of you who are not familiar with this model, uh, brain organized can be derived from two different cell types from somatic cells that then reprogrammed into induced pluripotent stem cells or from blastocyst, from which we can derive embryonic stem cells independently from the source of cells. These are normally cultured into 96. Well, plates were employed by this form, and when they are too big, they are transferred to dishes plus or other plates so that they're going to grow further into brain organize. So there are two main protocols used to obtain brain organoid. One is the unguided protocol. So you can obtain kind of whole grains in vitro or the guided protocol to obtain a region specific brain organoid. Um, the goal of my project is to obtain urine, brain organoid that contain neurons and astrocytes on one side, or neurons, astrocytes and microglia on the other side. So I have these two models. The second step is to induce an inflammatory stress into these organize and I will use different stimulant for the two of them. So in the first one, we've only neurons and astrocytes. I'm using interleukin one beta the NFL find the interferon gamma. In the one where also microglia is present, I will use LPs and interferon gamma and finally I will try to test different molecules to verify their anti inflammatory effects. So when I want to obtain the first model without microglia, I start from my PC and a few days later I can already see the organize forming. When I want to obtain the one with microglia, I start from your own stem cells instead. Mixing them together with IPC derived microglia since the beginning of the experiment and the microglia I'm using is green fluorescent because of the e g f p protein linked to the 63 cr one, which is expressed only microglia. The techniques that I use are flow cytometry to assess the cellular viability and the presence of the e g f p positive microglia the Eliza to verify the microglia activation after the stimulation. So to test whether there are inflammatory cytokines in the medium and immunocytochemistry to show the presence of the cell types of interest. So the first model neurons and astrocytes I start from ABC. I wait around three weeks and this is an example of what I see here. You can see on the left in red better free tumbling, which stands for neurons and on the right G f a PPI for astrocytes and the blue You set up for the nuclear I. So we can say that both cell types are present and you can also appreciate the cellular structures of both of them. Then I try to induce an inflammation into these organoid using different cocktails of stimuli. And then I verify the presence of interlocking six in the medium. And as you can see in all the condition tested apart from ATP, there was an increase of interleukin six compared to the UN stimulated control. Then I did the same for six cl 10. And again, um, the amount of these proinflammatory cytokines was higher in all the conditions tested apart from 80 compared to the control. So we can say that it's possible to obtain urine brain organoid that contain major neurons and astrocytes, and it's also possible to stimulate them, causing an increase with the cytokine production for the organize with Michael. Yeah, I start from your own stem cells and microglia progenitors. I keep them in culture for four weeks and this is what I see. Uh, so again, we have read better free to bring on the left for neurons g f A PPI for astrocytes on the right. And in green, you see the microglia And there was a lot of variability in the amount of microglia present between one organized and the other. So to try to solve this issue, I did this other experiment where I tried 12 different conditions of neuro stemcell medium together with microglia medium. So I added different percentages of microglia medium, 50% in red, 20% in yellow. And I was adding this, uh, different time points and for different time spans. And I saw that the 12 condition for me was the one that works best because I obtained 5% microglia, which is very similar to the percentage that is found in the mouse cortex in vivo. So once I knew that all the free cell types of interest were present and that the microglia was present in the correct amount. I try to stimulate them with LPs and interferon gamma and again here. I did analyze a for interleukin six, and I saw that the presence of interleukin six was higher compared to the control. And furthermore, I also did a flow cytometry to verify the viability, and the percentage of viability was decreased in the stimulated condition compared to the UN stimulated one. So it's possible to obtain organized that contain all the free cell types, neurons, astrocytes and microglia. And when stimulated with LPs and interferon gamma, uh, this leads to an increased cytokine production and also to cell death, the next steps and also what I'm doing at the moment with the ongoing experiments, is to test different molecules and inflammatory molecules that should reduce or solve the induced inflammation. I would like to thank my professors and my colleagues for the help and support and all of you for listening to me. So any questions, I'm just wondering. In general speaking, what I'm seeing in Organoid is that they never develop muscular elements. How big a problem in, in your view is is this lack of muscular? Yeah, the This is one of the biggest problem with with organize. And there are different types of, uh ways that this can be solved. Um uh, in my case, I think it's a little bit less important because my organoid since they come from urine I pc, Uh, there are a lot smaller than human organized, so normally they reach one millimeter, which is a lot less compared to human organized that are up to five millimeters. But I think it's still an issue, and there is probably a necrotic or in the middle. Okay, Super. That actually nicely leads onto to what I think about their just at the end. So when you show the viability the control actually had quite low viability, it's less than 50%. So my question was, whether that's typical of any type of tissue organoid or is that something specific about the C. N s? Um, I am not sure about that because I always work with brain organized. So always, uh, brain, not other types. Um, I think, um, it could be because of the necrotic core, but it could also be because of the way I was obtaining the single cell suspension, which is quite harsh so I really have to type it a lot. So it's possible that during the process some cells die because of that. An opportunity. Maybe, you know, if it is because of the chronic core. You have a model of how healthy cells are reacting to dying cells, which is a really interesting aspect of pathology to think about as well. So what can sometimes look like a problem could be an opportunity. Thank you. Very nice. Thank you. Any further questions? Yeah. Um um I have a question about, uh, optimized it by changing the media concentration was like, uh, was there any indication of that brain or you just kind of just, um I think neurons and astrocytes were more or less more or less the same. It was just the microglia. That was a bit random. So before I did that, that experiment, it was really changing a lot from one organized to the other. What? Julian thanks very much. Um, did you get to a stage where you could see any kind of organization of the neurons or the sites in relation to each other or within the three d structure, where the lining in any particular way? Not really. Not really. Do you think it's a time question or what? What's missing? Uh, probably I never went. I never kept them in culture for more than five weeks, so I don't know if maybe keeping them for a longer time. They, uh the thing is that all the works I know, uh, with brain organized are all human cells. And in with human cells, yes. They also see some more organized structure. But not many labs are working with a million cells and proportionate cells to each other. A representative of what is a 5% a good representation. Microglia? Yes. The other cell types. I never quantified it. I think there are more neurons than all the other cells. Yes. Okay, so maybe you get up to the astrocyte. Seen right? I never I never tried to quantify exercise. Yeah, that's sort of have you done any single cell work on this after the organized, be informed to see what is the proportion of cells. The type of cells that they develop apart from what you expect? No. No. Yeah, because that's a potentially a good opportunity to see what extraterritoriality you might have in the tissue. All right. Uh, I just wonder because your system is so beautiful when you add the PS. The CEO uh, you doing okay? Mystery to observe changes in the configuration of my career? Um, no, No, because I think, um, no, I don't know, like doing the immunocytochemistry. Uh, I didn't see any difference in the shape or the organization. No, I don't know if it was because it wasn't strong enough or I don't know. Yeah, I don't know why, you know. Thank you. You know, I was actually we're doing similarly. So you had looked at I'll six, production. Um, in both the organized models that you've been developing and you were the levels are very different from the addition of microcephaly in your in your second organoid metal. And, uh, it says that you're expecting to be produced. You expect? You mean, if I use the same concentration to stimulate both organize? Thing is, I also read out. Yes. Your yes. Are you? So? Are you achieving similar concentrations Final six in your uh yes. No, it was similar. So the addition, So microwave included in the organ? I didn't change that. Maybe. Yeah. Maybe we should try something else. Also apart from from interleukin six and six and then because we also tried to test for a TNF alpha. But there wasn't really a difference compared to the control. Um, so we should maybe I'll look for something else apart from this super Thank you very much dot So thank you for staying with us and hopefully the people in, uh, hopefully the people on the, uh, the internet was able to hear the questions, answers and the talks and enjoy those Anyone has any questions? Remember, you have plenty of time to type that in to the, uh, to the online portal. So, Denise, is there any anything else too close because tomorrow to see See you back tomorrow at seven o'clock in the morning, because seven o'clock, Because you need a lot of time to look at the posters. And there is still time today. If if you are still hungry for more science to go up, look at the posters again. You can see that on the Internet, you can ask questions through the Internet, so don't be shy. And even if you are, you are able to do that in a much more impersonal way. And thank you very much for medals to putting on this show. It works very well. So thankful for for, uh, supporting this meeting. Thank you. And see you tomorrow.