Webinar

<Rashmi Rajendra>
Hello everyone.
Thank you for joining today from all different time zones for Trailhead’s live webinar. We appreciate your participation. My name is Rashmi. I’m with Trailhead, and I will be your moderator for today.

Before we begin, I will quickly go through some housekeeping topics for today’s webinar. Your microphones have been muted and cameras turned off for the sake of a seamless webinar experience. You can enter your questions for the speaker at any time during the webinar via the Q&A feature of the webinar platform. We will ask as many questions as possible live, and in case any are missed, our scientific team will respond to you by e-mail.
Recording of the webinar will be made available to you as soon as it is processed and you will receive a notification link to view it.

With that, it’s my pleasure to introduce our speaker for today, Doctor Nooshin Amini.

Dr. Amini is a stem cell biologist with expertise in embryonic stem cells and induced pluripotent stem cell work, differentiation of stem cells to three germ layers, protocol transfers to bioreactors, 2D and 3D cell culture, differentiation of stem cells on various modalities such as polymeric scaffolds, microchips and conventional plates.

As a scientific director of the new ectoderm program, she and her R&D team are currently developing differentiation protocols for ectoderm derived cells, including glial cells and multiple neuronal subtypes of forebrain and midbrain regions.

Doctor Amini received her PhD in nanobiosciences from SUNY Polytechnic Institute, where she worked on diagnostic and therapeutic applications of human stem cells. In today’s webinar, Dr. Amini will present “Unlocking the Blood-Brain Barrier: A Novel Research Approach with iPSC Derived Vascular Leptomeningeal Cells (VLMCs)” and she will also be giving you a brief overview of the of Trailhead Biosystems.

So welcome everyone, and thank you for joining today.

<Nooshin Amini>
Hi everyone.
Thank you, Rashmi for the introduction.
Like Rashmi said, we are going to talk about Undocking the Blood-Brain Barrier: A Novel Research Approach with iPSC-Derived VLMCs.

Today I’m going to go over multiple topics, I’m going to introduce Trailhead Biosystems, I’m going to go over the components of the blood-brain barrier briefly, I’m going to tell you about the discovery of VLMCs, Trailhead’s approach to generate these cells, and some applications of these cells in vitro.

Trailhead was founded in 2015. The company is based in Cleveland, OH, and the focus of the company is to generate the specialized cells from ectoderm, mesoderm and endoderm from stem cells.
Now to understand the mission of the company, it’s important for us to understand the potentials and challenges of working with stem cells.
We work with induced pluripotent stem cells -these are reprogrammed cells that are coming from healthy donors. And this way we are eliminating the ethical issues that  are related to working with embryonic stem cells. But we’re able to take advantage of the power of these cells, which is that they can differentiate to all cell types and then replace the lost or dysfunctional cells in patients.
Therefore, it’s important to be able to differentiate these cells robustly and reproducibly to different cell types. If you can do that, then we are opening the doors to doing research for drug discovery, disease modeling and cell therapy for a lot of these diseases that don’t have any treatment right now.

Now there are a lot of problems in the stem cells field currently. There is the problem of the fact that there is a high demand for these cells, but not that many options are available. And out of the cell types that are available, the purity of these cells is low. And if you worked with any directed cells from iPSCs, you know that there is a batch-to-batch variation with these cells. There is also the problem of upscaling the protocols.
The cells cannot be generated at high quantities, and more importantly, a lot of the processes of the protocol development and also production are being done manually – that takes a lot of resources, takes time and it’s very expensive.

Now at Trailhead, we try to tackle these problems and find solutions for them. The first thing that we did, and that is the foundation of the company, is replacing all those manual discovery processes with machine enabled tools.
Using that, we can explore high dimensional space of regulatory inputs efficiently, meaning that we can look into effect of different factors that has an effect on the behavior of the cells. This way we can improve the purity of the cultures. We can differentiate different cell types and, more importantly, specialized subtypes of those cells.
We can define critical process parameters that can be used to manufacture these cells at larger scale, and we can implement all of these factors to our production platform for different cell types and generate the cells at a more cost effective manner.

Now to give you a better idea about exactly what we do, I’m going to talk about high-dimensional design-of-experiment (HD-DoE^®). That’s the technology that we use. We have our proprietary, internally developed software.
We use these tools at the beginning of the process when we are designing the experiments. Therefore, experiments are data-driven, unbiased, and then we can execute these experiments robotically, and therefore we can achieve unprecedented speed, precision, and scalability.
And all this data that we are creating our own BIOS, we can always go back to them and look at different coordinates in that space and look for other cell types that are available there and were not our target initially.
Now to give you an example, this is speed that I’m talking about here uou can see that if we wanted to look at, for example, effect of 12 factors on the behavior of the cells, we have to do 4000 experiments to understand their individual and combinatorial effect on cells. Of course, that takes a lot of resources, but we can compress that 40 times and do 96 experiments instead and collect the same amount of data, model those, and predict the behavior of the cells when they are treated with those factors.
And that is why we can generate a lot of differentiation protocols and basically make a lot of different cells at a much shorter time than the conventional methods.

Now we’re going to talk about the blood-brain barrier.
There are two types of barriers in the brain. We have the blood-brain barrier, which is the barrier that’s made up of endothelial cells, and it makes a barrier between the components of the plot and the brain. We have the blood-CSF barrier that is made up of epithelial cells of choroid plexus, and it makes a barrier between the blood and cerebral spinal fluid. We also have the arachnoid barrier that encases the whole CNS, and basically it’s protecting the extracellular fluid of the brain from the rest of the body.
Development of the blood-brain barrier starts when the cells of the subventricular ectoderm, produce VGF and material cells are formed along the concentration gradient of VGF and as other cells in the environment are developing, other signaling pathways are involved and the BBB matures. Some of these factors are TGF beta, for example, it’s produced by both endothelial cells and pericytes, and they have receptors for the cells. And it helps with adhesion of pericytes to endothelial cells and also maturation of endothelial cells. When pathways also involved, it helps with expression of some of the B genes that are involved with nutrient transport like SLC 2A1.
Also, astrocyte precursors in the environment produce Sonic hedgehog that also helps with barrier function of blood-brain barrier. Now as the name suggests, the main function is forming a physical barrier that can protect the brain from the rest of the body. It can maintain the concentration of ions and neurons and maintain the homeostasis of the brain and while protecting it from the plasma components.
Now there are a lot of studies that show that in many diseases related to CNS, like MS, Parkinson’s disease, Alzheimer’s disease, brain tumors. In all of these diseases, we have disruption of the blood-brain barrier.
Therefore, it becomes really important for us to understand the components of blood-brain barrier and also, the mechanisms of its function to be able to find treatments for these diseases.

Now we are going to take a closer look at the blood-brain barrier components. There are cellular components and non-cellular components in blood-brain barrier.
Cellular components are brain endothelial cells. These are different endothelial cells in their body. They have continuous tight junctions and they lack venous pressure and they experience collagens, optical proteins, and form that barrier. We have the astrocytes in the environment. They are contacting the endothelial cells through their entry. They express pouring 4. They help with maintaining water homeostasis and ionic concentration in that environment.
We also have the pericytes. They wrap around the endothelial cells. As I mentioned, they play a role in development of the BBB, but also they are regulating the blood flow and they are involved in phagocytic activities because of their function in immune response.
We also have non cellular components in the environment. One is tight junctions, so they are actually the physical barriers. Basically, they are preventing the compounds from entering the brain. They prevent their paracellular movement and force them to use a transcellular movement to enter the brain.
We also have the basement membrane that’s made-up of collagens, laminin, different proteins, and it basically provides a bed for all these cells that exist in the environment so that they can communicate with each other. And it’s important for the signaling events to happen and basically helps with maintaining the barrier function.
Now I mentioned all of these and we haven’t talked about VLMCs yet. So where do they fall in all of this? And as you can see in the cartoon they are also in the BBB, in this space that you see here, they are contacting the material cells and astrocytes.

And with that, we are going to talk about the discovery of VLMCs.
So in 2016, a group led by Marques and Zeisel in Karolinska Institute, they were doing a research on understanding the differentiation of oligodendrocytes progenitor cells to mature oligodendrocytes in mouse brain. And they were doing single cell RNA sequencing. They found two clusters that their pdgfra positive and you can see on the figure with the blue and the red clusters – both of them were pdgfra positive, but they are two distinct clusters.
The second cluster was expressing some of the oligodendrocyte genes, but also some pericyte genes like tbx19 and vitronectin. And it was also expressing very highly some other genes, like Lumican, decorin, laminin, and different collagens. So they wanted to understand what the second cluster is. They did immunofluorescence staining and they used DAPI to stain the nucleus, they use collagen 1A1 for the second population, electing to visualize the blood vessel Pdgfra that was positive in this cluster. And they found that these cells exist on blood vessels and also on meninges, so they called them vascular leptomeningeal cells. And they also looked up the insituisation images on Allen Institute database and they saw that, imagine with what they saw. There is a population of cells that express the decorin and also vitronectin and they are on meninges and blood vessels.

They did another study 2 years later, in 2018, led by Vanlandewijk, and they focus on blood-brain barrier. First, they did a single cell sequencing to find the gene set that can identify each of the cell population that exists in blood-brain barrier. So they found genes that are highly expressed in endothelial cells in pericytes, astrocytes and also the VLMCs. And then they did immunofluorescence standing on the mouse brain to understand the location of each of these cells in relation to each other.
So in panel A they are showing the blood vessels with PECAM1 in white the red staining is Cspg 4 is showing the pericytes that are around endothelial cells and in green we have Pdgfrb and Pdgfra that are pericytes and the VLMCs.
Now you can see that the green cells on the vessel, those are them, those are the VLMCs and they are in contact with astrocytes, and that’s visible on panel B when they’re using aquaporin-4 to send the astrocyte. We see that VLMCs are on the vessel and on the other side we have the astrocytes touching them.

So they found these cells and they saw that they are loosely attached to endothelial cells. They wanted to make sure that they are not misidentifying these cells with the oligodendrocyte that are in the brain are also positive for Pdgfra.
So they did another set of straining and they looked at the location of these two cell types in relation to each other and they saw that the oligodendrocytes that were Pdgfra positive are on the outside of the astrocyte on the brain side and the VLMCs are on the inside in the PvP region in between the vessels and the astrocytes.
And shown here in green staining, you can see on the astrocytes are in red and the blood vessel is shown with CD31 in blue.

Now there is another study that the same group did in 2018 and they were looking at different progenitors that are in spinal cord and the brain of mouse, and they differentiate to mature oligodendrocytes and they found that the same early progenitors can either go to the oligodendrocyte fate or they can become VLMCs. And they saw that there are a lot of common genes as we knew between these cell types, the VLMCs, are also expressing Olig2, Sox10, Cspg4, Prdgfra, of course they’re missing the mature oligodendrocyte markers like Plp1, and of course they express the signature VLMC genes like the decorin and Lumican.

So that is what we knew so far in the literature about the VLMCs and now we can get into our approach to generate these cells.

Our work also started when we were doing research on developing a protocol for oligodendrocytes. We had an early glial progenitor and we were using our HD-DoE^® technology to guide the cells to mature oligodendrocytes. We’re looking at optimization of different genes and how we can guide these cells, and we found that there are two routes with these cells. And the second population that we are getting, they share a lot of the genes with the first population. When we did full cytometry on them, we had expression of A2B5 and CD9 in these cells, but they all have very high levels of PDGFRa and NG2 at a much later time point that is not necessary, that is not seen in mature oligodendrocytes. And they also had a very distinct morphology. They had this fibroblast like morphology that we don’t expect to see the with oligodendrocytes. So we decided to do a deeper characterization on these cells to understand what they are.

And this is the morphology that I was talking about that fibroblast-like culture homogeneously had that morphology.
So, we sent samples from these two populations for bulk RNA sequencing and when we compared the results, we saw that of course many genes are common between the two populations, but some genes are specifically expressed in the second population and they’re missing in the oligodendrocyte population. So, and those genes were Limican, decorin, and FBLN1, RGS4 all of which match the gene list that those grouping Karolinska Institute found that are expressed in VLMCs.
So at this point, looking at that flow cytometry data, our bulk RNA-seq data and the morphology of the cells, we’re fairly certain that we made VLMCs.

But we wanted to look at the protein expression of the cells as well.
We decided to do immunocytochemistry on these cells. We looked at expression of collagen 1A1, deconrin, Lumican, PDGFRa, and NG2 as we expected to see those because we had the gene expressions. We also included PMP22 that’s a neural crest marker and TUBB3 that’s a neural marker. As you can see, the majority of the cells express all the VLMC proteins that we expected to see, and the neural marker and the neural crest marker are missing in these cells.
At this point, we knew that we made VLMCs, but we wanted to check the robustness of our protocols. So we differentiated another iPSC line from Reprocell. We use our protocol on them and then we did flow cytometry and immunocytochemistry to characterize those cells. In our flow cytometry, we looked at expression of NG2, CD146, CD44 and Pdgfra as our surface markers. And you can see that more than 80% of the population express all those markers. And we also did immunocytochemistry on the cells we looked at expression of collagen 1A1, decorin, Lumican, SOX10, PDGFRa and again included PMP22.
This time we included SOX10 because based on the signals that RNA sequence that we had from those papers, the cells express are the markers. So we want to see if we see that.
Now you can see that the majority of the cells express the proteins, we can visualize expression of collagen 1A1, decorin, Lumican, all these VLMC genes and also PMP22 the neural crest marker is missing in the population. That confirms the origin of the cells.

Next, we wanted to see if we can cryopreserve these cells and thaw them successfully, and will they become mature VLMCs at that point?
So we froze down immature VLMCs. We thawed them, cultured them for seven days in our differentiation media. Then we use RT-PCR immunocytochemistry and bulk RNA sequencing to characterize those cells.
With PCR, we looked at expression of VLMC genes, decorin, SPP1 and collagen 1A1. And those three were experienced at high levels. And of course, we don’t have any expression of neural marker TUBB3.

In our immunocytochemistry we looked at expression of GFAP as an astrocyte marker, because those early glial progenitors that we’re using have the potential to also become astrocyte. So we wanted to make sure that we don’t have any contamination of that fate in our culture.
We send the cells with collagen 1A1, lumican and vimentin. And as you can see, the VLMC markers are present and we don’t have expression of GFAP in these cells. To analyze our bulk RNA-seq data, we made a gene list based on the single cell RNA-seq data that we saw in those papers and we tried to see if other cells that are present in the brain, such as the PB cells, pericyte, muscle cells and astrocytes and also like microglia and oligs, if we have any expression of those genes in our cells. As you can see, we don’t have any expression of genes of microglia or oligodendrocyte. We also don’t have expression of mesodermal markers in the culture or astrocytes.
We have some of the genes of pericytes, but muscle cells present in the cells, but that aligns with what we know that these cells share a lot of the genes with each other, but we are missing the signature pericyte markers KCNJ8 and ABCC9. So not all of them are present. And of course the VLMC genes are expressed very highly. Different collagen’s MMP2, SPP1, Lumican, decorin, they’re all present in the cells upon time and also seven days after culturing them.

And with that, I’m going to talk about some of the in vitro application of these cells. But I should say that this work is ongoing, so I’m only going to show some of the data that we collected so far.
So first assay that comes to mind is making a blood-brain barrier model in vitro, with these cells as we know they are present in that region. We have our own astrocytes and of course these VLMCs that we make at Trailhead, but for making the brain endothelial cells and pericytes, we used published protocols. We needed to define a set of markers to identify each of these cell types so we can confirm their presence in our co-culture.
For the astrocytes, we are going to check CD44, ACSA-1, ACSA-2, GFAP and CD38. For brain endothelial cells we check CD31, Occludin, VE-Cadherin and CLDN5. For pericytes we check expression of CD44, CD146, CD13, PDGFRB, CD90 and NG2. And for VLMCs we are checking CD44, CD146, PDGFRA, A2B5, CD90 and we expect to see some NG2 expression.

As you can see a lot of the markers between the cells are common. So, it’s really important to do a comprehensive study and understand exactly which cell types are present in the culture.

To make the BBB model refer we paired two cell types at a time with each other to understand the interactions with each other. Because there is no data on culture VLMCs with any cell type.
So we paired them with astrocytes, we paired them with pericytes, we paired astrocytes and pericytes, we paired them with brain endothelial cells, and then astrocytes, pericytes, VLMCs together, and at the end 4 cell types.

We made the model on a mono layer so we can use an MEA plate to get their barrier function. And also on a transwell having the brain endothelial cells on the apical side and the other cell types on the basal side, as direct transwells are commonly used for blood-brain barrier work in vitro.

First, we needed to confirm that we made the correct cell types using the published protocols to characterize the brain endothelial cells, we use immunocytochemistry. We looked at the expression of those tight junctions that I mentioned, Claudin-5, Occludin, and VE-Cadherin, and you can see the majority of the cells express those tight junction proteins. For pericytes we use flow cytometry, and you can see that CD13 that is the signature pericyte marker is present in 66% of the population. We don’t have any expression of A2B5 that these present in our VLMCs and we don’t have CD31, that’s an airline mesoderm marker and rest of the markers are expressed in almost 100% of the population.

When comparing that data to the VLMCs, because a lot of these markers are common, we can see that we have comparable levels of CD44 and CD146 as we expect higher levels of NG2 in pericytes and higher levels of A2B5 on VLMC.

After confirming that we have each of the cell types, we started making the co-culture. Here I’m showing just some of the data that we have. On the left panel we have the co-culture of brain endothelial cells and VLMCs. One interesting thing that we learned was that when we are adding the VLMCs to the brain endothelial cells, the VLMCs come together and form this little spheres in the culture, they’re still attached to the to the monolayer of brain endothelial cells, but that is the form they take.

They are loosely attached, so when we did immunocytochemistry and did all the washing steps, some of them came off, but there still some stayed in the culture. We use Occludin to stain the brain endothelial cells and collagen 1A1 to stain the VLMCs.
And you can see that we have those spheres in green in that co-culture. In the co-culture of the astrocytes and VLMCs that I’m showing you the middle panel, we learned that these two cell co-culture with each other really well. They adhere to each other and they interact with each other really well. I’ve used TAGLN to stain  the astrocytes and alpha-SMA to stain the VLMCs and you can clearly see the two population in the culture.
Then we added the pericytes to the culture, so we had astrocytes, pericytes and VLMCs. Again, we are using alpha-SMA to show the VLMCs in the culture and there it is showing the astrocytes. We have some low levels of TAGLN also expressed in pericytes, so that’s also what we are seeing here.
And on the right side, we have the images from the transwell that we are working on. As I mentioned, we have the brain endothelial cells on the apical side and we are sending those with Occludin. You can see that we have those tight junctions on that side of the transwell and use collagen 1A1 for the VLMCs and on the basal side, we have VLMCs and the astrocytes.

So you can see that we have the nucleus sustained DAPI in all the cells, but collagen 1A1 only in a subset of those cells. That shows that we have the co-culture on that side.

Of course, this work is not done, more work is needed. We need to have 4 subtypes together to be able to measure the tier value that shows the value function of these cells.

One thing that we learned that was very interesting was in the co-culture of astrocytes and VLMCs. While we’re doing that, we did flowcytometry on those and we learned that when we are culturing the astrocytes and VLMCs together, it has an effect on maturation of astrocytes. We cultured the two cell types together and we saw that when we were straining the cells with ACSA-2, that is the astrocytes at a specific marker. It’s used for isolation of astrocytes from mixed viral populations and also CD38, that’s another astrocyte marker, that when we are staining the cells with those two, more than, 63% of the culture express those. So meaning that out of that 100% mixed population, 64% of those cells are becoming mature astrocytes in presence of VLMCs. We wanted to see if the effect is coming from the VLMCs or our co-culture media.

So we did another experiment and we saw that when the astrocytes alone are cultured with that media, we only have around maximum 17% maturation of the cells. We have 17% expression of ACSA-2 and only 11% CD38 in the culture.
So this kind of confirms that the maturation was coming from the interaction between the VLMCs and astrocytes that was happening in the culture.
We also did immunocytochemistry on the cells. We used GFAP to stain the astrocytes and collagen 1A1 to stain VLMCs.

Now you can see we have the initial expression of GFAP in the subset of those cells, not in all of them, but it was important because we didn’t detect any GFAP in the culture of astrocytes when it was cultured alone without the VLMCs. So we thought that was very interesting.

Another assay that we did on this co-culture was using was culturing the cells on a NEA plate and then measuring the impedance. Impedance is the resistance of the cells to the current, and it’s an indirect way of measuring the barrier function of the cells.
We had the VLMCs alone in our column eight on the plate and the VLMCs and astrocytes together on column nine. We measured the impedance every 24 hours for four days.
And when you look at the bottom map that I have here, you see that after four days the impedance doesn’t really go up in the VLMCs when they’re alone, so they don’t show a barrier function. However, in the co-culture of VLMC and astrocytes, we have an increase of impedance in those four days. And that’s shown with the red dots in our column nine.
So we thought that was interesting. Of course, it’s very cool if we can culture all four cell type on the in NEA plate and then look at the barrier function, especially in presence of brain endothelial cells and see what is the effect of VLMCs on those.

Another application of these cells that we looked into was the information response, because the VLMCs share a lot of genes with pericytes and we know that pericytes have a role in immune response. We wanted to see if we see anything from the VLMCs. So we treated these cells with LPS for 24 hours. We collected samples at different time points during that time, and we also took phase images of the cells.
Now we didn’t see a big morphological change in the cells after 24 hours, but we were able to look at the cytokine concentration and learn some interesting things there.
So we use the Multiplex detection kit for the cytokines and looked at concentration of 13 different cytokines. 9 out of those 13 cytokines had elevated levels on hour 24 compared to hour zero. And three of those had a very high jump, so IL-6, IL-8, and IFN-a2 had more than 10,000 fold increase in their mean fluorescent intensity at 24 hours compared to untreated culture.
Now, these are pro-inflammatory cytokines. So this kind of points to the fact that these cells play a role inflammation. But what that is, how it’s going to change if they are in contact with other cells, these are the things that we still don’t know.

So, what we’ve learned so far: we know that VLMCs are part of the blood-brain barrier environment. They possibly play a role in the development barrier function and the neural information response in the brain. But we need to get a better understanding on some topics like the functional role of VLMCs in immune response in comparison with other immune cells, like is there a difference in their response, compared to the pericytes. What is the cell-to-cell interaction between VLMCs and astrocytes that cause the maturation of astrocyte? What is the interactions between the VLMC and pericytes?
We need to know more about this cytokine release of other components of the blood-brain barrier in presence of astrocytes, but in also VLMCs. And also we need to know more about the role of VLMCs in development, maturation and function of blood-brain barrier, especially because there are some papers that are showing VLMCs have an effect on development of pericytes early on in the development. So that would be really interesting to learn as well.

There are more information on our website about the product. We have our data sheets there. We have blogs and frequently asked questions. You can also contact us at support@trailbio.com if you have any questions.

Thank you for your attention. And I think now we are going to go into our Q&A section.

<Rashmi Rajendra>
Thank you so much, Doctor Amini for this lovely webinar. It was really good.
Please, I encourage everyone to please go ahead and input your questions into the Q&A. In the meantime, we do have a couple questions for you.
So how are these cells sold? Are they just cells or is it a kit?

<Nooshin Amini>
So it’s a kit, they come with the media that needed for seven days after thawing.

<Rashmi Rajendra>
OK. So do these cells need to be differentiated in culture? How long does it take to make these cells?

<Nooshin Amini>
Yeah. So, when you thaw them, there’s the immature VLMCs. So, with the media that we provide, culture them for seven days and at that point they should become mature VLMCs. They should have that fibroblast morphology that they showed and it should be a homogeneous culture.

<Rashmi Rajendra>
Very cool. Thanks. So for how long can these cells be maintained in culture for cell-based assays?

<Nooshin Amini>
We have kept them for a couple of weeks. They look fine. You can passage them every week to control the density. But they can be kept for a couple of weeks at least.

<Rashmi Rajendra>
Very cool. So that gives, a scientist some time for multiple co-culture assays and for the impedance assays that you showed us.

So what are the key markers that you use for QC? For analyzing these cells before you send them out?

<Nooshin Amini>
So, we look at some surface markers like CD146, CD44, Pdgfra and NG2.
But also there are those specific VLMC genes, like the decorin and Lumican and different collagens.
We do RNA sequencing on them to check the level of those, make sure that we have a good amount of expression of those compared to the starting material.
We also do immunocytochemistry and look at the protein expression of them as well.

<Rashmi Rajendra>
So does the cell type have any endogenously expressed fluorescent markers at all?

<Nooshin Amini>

No.

And one of the last questions I have for you is, can these cells be maintained in regular DMEM medium? Or is it something special?

<Nooshin Amini>
No, we need the differentiation media that we provide with the cells.

<Rashmi Rajendra>
Awesome. Cool. If there are no more questions from the audience, I just want to say thank you so much for this wonderful webinar and to the audience, thank you so much for attending and you know, please feel free to reach out to us with any questions you might have.
As you can see, it’s up on the screen: support@trailbio.com
You can also go to our website and follow us on LinkedIn.
Thank you so much for attending. Have a lovely rest of the day.