Webinar

Joshua Snow
Welcome everybody to Trailhead’s third webinar of the year. My name is Josh Snow. I’m the Chief Commercial Officer here at Trailhead Biosystems. Before we begin, a few things to mention. I’d like to let everybody know that today’s webinar is being recorded and the replay will be available shortly on our website.

Also, all attendee microphones have been disabled for the presentation. We will have a short Q&A session at the end of today’s presentation. You can submit your questions to our presenter, Nooshin and our team through the chat.
You should be able to see a question submission button in your team’s navigation bar and we’ll try to get through as many questions as possible within our allotted time. If you have any questions after the webinar is over, you can also reach out directly to our team at support@trailbio.com.

So today I have the pleasure of introducing Dr. Nooshin Amini, one of our scientific directors at Trailhead Biosystems, and she’ll be presenting today’s topic, Precision in Neuronal Development Generation of iPSC-derived A9 Dopaminergic Neurons.

Doctor Amini is a stem cell biologist specializing in embryonic and induced pluripotent stem cells. Her expertise includes stem cell differentiation across all three germ layers, protocol adaptation for bioreactors in both 2D and 3D culture systems.
She’s worked extensively with stem cell applications across various platforms, including polymeric scaffolds, microfluidic chips and standard tissue culture systems. And currently Doctor Amini is focused on developing differentiation protocols for ectoderm-derived cell types, including glial cells and.
Multiple neuronal subtypes from the forebrain and midbrain regions. Nooshin, thank you for presenting today and I will pass it over to you now to get started.

Nooshin Amini
Thank you. Hi, everyone. I’m Nooshin. As Josh mentioned, the title of our talk today is Precision in Neuronal Development, Generation of iPSC-Derived A9 Dopaminergic Neurons. I’m going to cover an overview of Trailhead Biosystem, our approach in using.
HD-DoE, biology of dopaminergic neurons, limitations of published protocols, development of TrailBio A9 Dopaminergic Neurons, and the applications of the cells. And then we are going to have our Q&A session.

So the company was founded 10 years ago. The technology came out of Cleveland Clinic. Right now we have our own building in Beachwood, Oh. We have our own labs and manufacturing sites and all the work is being done on site in our building that you see in this photo.

The foundation of our worker stem cells. We use induced pluripotent stem cells. These cells are coming from adult human cells, from healthy donors. We don’t use embryonic stem cells, so there are no ethical concerns related to fetal tissue use.
And these cells, like all the other stem cells, have the potential to replace lost or dysfunctional cells in patients. The cells are reprogrammed using Yamanaka factors and they can be expanded indefinitely. However, these are stem cells, so they have three pluripotency markers.

And if you want to use them for specific applications, we should be able to differentiate them to specialized subtypes. For that we need to have access to robust protocols and if we have that, then that can open doors to scientists to work on a broader range of diseases.

As everyone knows in stem cell field, there are certain challenges using IPSC models. One of the challenges is that there is a very high demand for these cells, but the specification for differentiation of these cells are not fully defined yet.
The protocols that are being developed right now, it’s usually based on the published protocols and there’s just some variations or optimizations to those protocols. So basically these are just repetitive approaches and that’s leading to a heterogeneous culture that’s a mix of different cell types or just different subtypes of a cell type.

And that can affect the results of the experiments. Another problem is that most of these protocols are very lengthy and they rely on using high concentration of different cytokines and proteins and makes it difficult to scale up the products to meet the demand.

And lastly, all of the development and production processes are being done manually, which makes the processes slow and again very costly. So at Trailhead, we try to address each of these problems and find solutions for them.
The first thing that we do is we replace the manual discovery process through machine enablement. We can explore high-dimensional space of the design and identify regulatory inputs that are actually guiding the cells to the fate that we’re interested in and therefore removing those iterative approaches.

And that’s leading to an improved purity of the culture and we can focus on making a specialized subtypes. This automation also allows us to manufacture the cells at large scale and through doing all of this we have been able to discover novel biology and develop protocols from scratch or novel and.
Focus on new subtypes.

So how do we actually do this? I mentioned that we use HD-DoE high-dimensional design-of-experiments. So using this technology we can remove the trial and error methods and design the experiments systematically and all of these are data-driven.
We can explore the high dimensional space of the design and map the key regulators of the cell fate and that’s leading to a reproducible protocol because we’re able to identify critical process parameters and therefore we have improved purity, speed and efficiency.

And lastly, we are executing the experiments robotically and that also allows us to have unprecedented speed and precision in our development and also in scalability. Here’s an example of what I actually mean by improving the speed of the process.
So let’s say we wanted to screen 12 different factors and learn what’s their effect on the behavior of the cells. If you wanted to do it in a traditional way, we have to do more than 4000 experiments to understand the effect of each of the factors individually and also in the presence of the other factors.

But by using HD-DoE, we can actually compress that to only 96 conditions, which is just one plate of experiments, and that allows us to screen a large number of inputs, different proteins, small molecules, whatever that makes sense.
And find the find the correct inputs that are actually guiding the cells to whatever fate that you are interested in.
And we have been doing that and in the past five years we were able to develop a lot of new protocols from scratch. So of course we are looking at published protocols, we are learning from them, understanding what’s going on in the field.
But all these protocols are basically redeveloped. We have been able to make cells from the three germ layers. We have already launched some of these cells. We have our a9 dopaminergic neurons, hematopoietic progenitors, progenitor cells and vascular leptomeningeal cells that are available on our website right now.
And we’re also going to release other cells like different subtypes of interneurons, islet like aggregates, macrophages and all of these protocols are novel and we made them from stage one using our technology.

Now I’m going to focus on dopaminergic neurons. First, I’m going to talk about their biology and how the cells are actually developed in vivo. So these cells are coming from the midbrain region, specifically the ventral midbrain. The progenitors in that region are migrating to two different areas, substantia nigra and ventral tegmental area and as you can see in the figure we are showing them with two different colors, two with green and red and kind of highlighting the differences between these populations. The green cells in substantia nigra are expressed in SOX 6 and the cells in VTA are expressing OTX 2.
And that’s just one of the differences. Their function in the brain is also different. The cells in SNC are innervating dorsolateral striatum and the cells in VTA are innervating the cortical and limbic areas.

And the cells in SNC, which are A9 dopaminergic neurons, are involved with controlling voluntary movement and A10 dopaminergic neurons that are in VTA are involved with cognitive functions, reward pathway motivations and such.
And another important difference between the two is that the population that’s in SNC are a9 dopaminergic neurons are the ones that are actually lost in Parkinson’s disease and most mostly the cells in VTA are being preserved. So we want to understand why is that what is making the cells in SNC specifically more vulnerable in Parkinson’s disease. So we can start with by looking at their gene expression during the development. In the figure on the right side, I’m showing the timeline of the development of these cells in mouse spray. The figure is coming from a paper published in 2016.
So we can see that from the beginning we have expression of midbrain genes in all populations. We have flux A2, LMX 1A 1B, EN1 and two and this is as the cells are maturing, we are seeing expression of more mature markers NURR1 and PTX 3. And you see that we have these colored bubbles on there and that’s showing their involvement in mitochondrial dysfunction and oxidative stress that are some of the causes of Parkinson’s disease.

So one thing that we know is that these cells are behaving different, these genes are behaving differently, in different regions. And what I mean by that is when we have this regulation of the expression, it’s affecting the cells in each of those regions differently. So for example, if we have down regulation of expression of NURR1, what’s happening is that it is affecting the downstream genes that are mostly involved with mitochondrial functions. And we are also seeing that that’s leading to an increase in expression of alpha-synuclein. That’s another cause of Parkinson’s disease. Another example is PTX3. If we lose PTX3, it’s affecting a downstream gene of this, uh, I’ll predict three that’s called ALDH 1A1, and that’s only expressed in cells in substantia nigra. And it’s known that this gene is involved in production of retinoic acid and also it’s involved in oxidative stress and mitochondrial functions. So when we have this regulation of this gene, it’s affecting genes that are associated with that.

Another way that we can look at these genes is categorizing them into three groups. So some of these genes are only expressed in substantia nigra, some of them are expressed more broadly but are behaving differently, and some of them are only expressed in VTA. So the genes that are only expressed in VTA like Calbindin 1 and OTX 2 they are showing some kind of protective role in VTA and they’re saving those cells in Parkinson’s disease. The genes that are expressed only in substantial nigra, like ALDH 1A1 that I just mentioned, is making those cells more vulnerable.

And some of these other genes that are involved that are expressed in both regions, they have a different effect in each one and we are seeing the reflection of that in the way that the cells are lost in Parkinson disease patients as well. The cells that in VTA are lost only 0 to 40% but we are losing from 50 to 90% of the cells that are expressed in substantial nigra, and that is going to explain the differences between the cells in these two regions. But the differences are not just that we have more than genetic differences. One of the main differences is the way that the firing pattern of the cells. We have slow and broad firing pattern in A9 dopaminergic neurons and that’s causing a higher entry of calcium into the cells that’s leading to hyperpolarization of mitochondria.

And consequently higher production of reactive oxygen species and alpha synuclein and lower electron transfer chain. And all of this is happening in these cells that are very they have very long axons and very large arbors with a lot of neurotransmitter release sites and that’s making the cells even more vulnerable. So basically it’s providing a toxic loop in the cell that’s leading to loss of these cells in Parkinson disease patients. And it’s kind of makes it clear how why it’s important to have the correct subtype of the cells to study Parkinson’s disease.

Now I’m going to talk about the limitations of published protocols. So we looked at what’s available in the literature to differentiate stem cells to the polymeric neurons. The first thing that we learned is that there are so many different protocols out there. But most of them are basically different optimizations on one original protocol. So the main approach is to use the SMAD inhibition to start a neural induction and then we are using different Wnt agonist, Sonic Hedgehog agonist and FGF 8 to commit those cells to mid brain reject and then keeping the cells in the culture to just measure the homologic neurons. However, there is no mention of control over specific subtypes and over SNC versus VTA in these cultures.  And most of them are leading to a mixed population of A9 and A10 cells. And also they require a long maturation time for the cells.

There are different reports on the TH positivity in these cultures. So interestingly, in this one paper, Kirk’s 2011, they report that 80% positivity of TH. Of course it’s a mixed population and it’s coming up after 50 days. But surprisingly, all these other protocols that are coming out after that, they are achieving much less purity and we have reports of like 11% TH positivity, which was an improvement over another protocol and then up to like 50% maybe most of them around 30 to 50%. But again, all of these are mixed populations.

In our protocol, we don’t use dual smad inhibition, using HD-DoE, we realize that we don’t need that and we are able to control the subtype specificity of the cells. Our protocol is around 23 days and according to the full cytometry assays that we’ve done, we are getting around 70% positivity of TH in our population.
One thing that we did was we tested one of these protocols in our lab. This was a published protocol in 2021 from Kim. And what they’re doing is that they’re trying to optimize generation of dopaminergic neurons by changing the concentration and timing of.
Agonist called CHIR. We followed their protocol and stained the cells on day 17 and we checked the expression of tubulin 3, just a normal marker, more mature markers like PTX 3 and NURR1. And it’s too early, it’s only day 17, so we didn’t expect really to see NURR1. But the main market that we’re most interested was SOX6. As I mentioned, this is a market that is expressing SNC and we didn’t detect any SOX6 expression in these cells, whereas in our own protocol, on day 17, we do see expression of SOX6 at that point. And we also check their paper. They have their RNA sequencing report on there and there is no mention of them tracking expression of SOX6 in these cells. They do mention that if the cells are transplanted in vivo, they can differentiate to the both subtypes. And they’re showing that with expression of GIRK2 and Calbindin 1, GIRK2 is expressed in A9 population and Calbindin 1 is expressed in A10 population. So the cells have the potential to differentiate to both, but it’s a mixed culture.

Knowing all of that, we started developing our own protocol. So the first thing that we do is we look at the single cell RNA-seq data that is available. And one of the papers that we really like is this paper from 2017. They looked at all the other single cell RNA seq data that was available and they tried to cluster all of that and kind of define a lineage tree for the dopaminergic neurons. What they show is that there is a common progenitor for these cells called DA0 and it’s branching to two different clusters.

Cluster one that includes SNC population has expression of SOX6, ALDH1A1 and very low or no presence of Calbindin 1 and then that is branching out to two different subclusters. We have our cluster of SNC that as the cells fully mature they maintain that Calbindin 1 negative condition. However, the second group did start expressing Calbindin 1 when the cells mature, even though we have SOX6 and ALDH 1A1 in it as well. The other branch that goes to cluster two is just different subpopulations of VTA.

These cells are expressing OTX2 and Calbindin 1, but there are other differences in their highly expressed genes. BK3 has expression of CCK and BK4 has expression of SLC 32 A1.

Another paper that we really like is this paper from 2016 from La Manno. What they did was also looking at single cell RNA seq from human, but they’re also comparing it to mouse and they came up with this timeline for expression of the genes during development and they’re showing differences between the two. So the first thing that we noticed is that there are differences between human and mouse data. Another thing is the differences between DA0, DA1 and DA2 populations. We want to be able to make cells that express DA0 profile and later mature them to DA2 profile. So we tried to find genes that are highly expressed in each of these and kind of differentiate the populations based on that. So we found this cluster that has very high expression of genes that are only present in DA2 and also and of course it includes genes like SOX6, ALDH1A1 and LMO 3 and there were other things that we noticed that were interesting too. For example, we have expression of Foxa2 in DA0 and DA1, but it’s much lower in DA2. We saw the kind of the same trend with PTX 3 as well. It is expressed in DA2, but it is much lower compared to the other groups. So there are subtle differences between the two and we try to be mindful of all of that when we are developing our protocol.

So to develop our protocol for stage one, the first thing we need to do is that to commit the cells to the mid-brain region. So we focus on expression of OTX2. It’s one of the first genes that’s coming up in that area. We also check expression of other mid-brain markers like LMX1A. But we also focused on GBX2, which is the competing fate that’s that can come up in the cells. GBX2 is expressed in hindbrain cells. As you can see, when we stain the cells at the end of stage one, majority of the cells express OTX2. We have some cells that are expressing GBX2 and very weak expression of LMX1A. So we had to track expression of GBX2 going into stage two to make sure that we are minimizing that expression. And we also wanted to bring up the other mid-brain markers like Foxa2 and LMX1A.
And as you can see with this ICC result at the stage two, the majority of the cells are expressing Foxa2 and LMX1A and OTX2 and we were able to bring down GBX2 as well. By the end of the stage two, we were confident that we made neural progenitor cells that are committed to the ventral Midbrain region. And from there we decided to focus on expression of SOX6 to make sure that we have cells that can become A9 to dopaminergic neurons. So we try to bring up SOX6 expression and then we measure that using flow cytometry. We can see that we have more than 70% expression of SOX6 in the population, which was exactly what we were trying to achieve. After that, we did bulk RNA sequencing on the cells to better understand their genetic profile. We took samples from stage one, stage two and stage three and check the expression of mid brain markers, some of more mature markers just to understand if we have any expression of those cells, panormal markers and also we checked the expression of pluripotency markers to see if there is any stem cells left in the culture.

As you can see, we have high expression of our mid-brain markers, FOXA2, LMX1A and LMX1B. We have expression of SOX6 that’s increasing over time. We also see expression of early mid-brain markers like CORIN, DDC, MSX2 and for the more mature markers, this one we decided to check AGTR1 because we signed a paper that between the different populations of A9 dopas, there is one specific population that expresses AGTR1 and those cells are more vulnerable in SNC. So we just were curious if we have expression of that gene in ourselves as well. And we see that expression and we see that the cells are starting to express some of the mature markers like tubulin 3 and we are losing the pluripotency markers NANOG, POU5F1 of one and the proliferated marker.
MKI67 is also going down as the cells are maturing more.

After that, we decided to develop our maturation protocol to get the cells to the mature state of dopaminergic neurons. You can see that when we do immunocytochemistry on these cells, we can detect expression of mature marker synapsin 1, neurofilament, the main dopaminergic marker tyrosine hydroxylase, KCNJ6 that’s presenting A9 dopaminergic neurons. And more importantly, we don’t have expression of calbindin 1 in these cells. As I said, calbindin 1 is only expressed in VTA population and it’s not presenting the cells even though the cells are fully mature at that point.

We also did a PCR on the cells to understand their gene expression and here I’m showing the data from stage one, stage three and stage four. And you can see that as the cells are maturing more, we have an increase in expression of the target markers.
We are seeing an increase in SOX6, a huge jump in expression of TH and expression of synapsin 1, two and three and GIRK2, NURR1. All the markers that we wanted to see is increasing over time. It was important for us to also track expression of GFAT. That’s a.
That’s a gene in astrocytes because we do know that in most of the development work for dopas, the glial population will be present. So we wanted to have some understanding of that as well. We also checked the ratio of the neurons and glial cells in the culture for that exact reason. We used flow cytometry, and based on the data that we got, we had more than 70% of the population expressing TH and only less than 10% of the population are expressing CD44, which is the glial marker that we chose.

We also checked the function of the cells. We wanted to see if the cell can release dopamine. We use that ELISA assay to measure that. We also purchased the commercially available vial of dopaminergic neurons just to compare it to our cells who have an understanding of where these cells stand functionally. As you can see, we took samples from day seven and day 14 from our culture and compared that to the competitor cells and we are getting from 4 to 12 nanogram per mil of dopamine from these cells.
And we were seeing much, much less dopamine release from the competitor. It was around like .01 nanogram per milliliter, much lower. And that kind of shows the function of the cells and how they do in the culture.

Another thing that we did, we cryo preserved the cells and then after thawing the cells we took samples from day one and day 14 and did bulk RNA sequencing on them and then again checked the same markers that we checked before those of a mid brain markers and then more mature markers.
And as expected, we are seeing expression of midbrain markers. We have expression of SOX6 that’s increasing as the cells are maturing. We have expression of TH and the mature marker KCNJ 6 and NR4A2. Also, pan neuron markers mapped to TUBB3 and no presence of pluripotency markers in the cells. And that kind of confirms the identity of the cells that we are able to make dopaminergic neurons that are mature in in the culture.

Another thing we wanted to know was the robustness of the protocol. So when we moved to scaling up the protocol, we took samples from 8 bioreactors at stage one, stage two and stage three, and I’m showing that on the X-axis. And we measured expression of some the markers that we were interested in. Here I’m showing expression of FOXA2, LMX1A, Nestin, OTX2, SOX6 and TUBB3 and we are seeing the range of the expression of these genes throughout the differentiation from stage one to stage three in eight bioreactors and we are seeing that as the cells are differentiating more, we are getting a better control over their differentiation, and the range of expression is actually getting tighter for most of these genes.

Another thing that we needed to know was batch to batch variation in the in the bioreactors. So we sent samples of two bioreactors from the mature assay and the progenitors. We did block RNA sequencing on that and then we compared their expression on a PCA plot. And you can see that this is our starting point, that is our starting material iPSCs. And as the cells are differentiating to progenitors, the clusters of two different batches are very close to each other. And then again when the cells are fully mature also we are seeing there, we are seeing that the gene profile is very close to each other and they’re clustering closely. And that kind of shows that we have a low variation between our batches when we are using our protocol in bioreactors.

Now I’m going to talk about their applications.
So iPSCs based on the diseases that they are involved in, they are using different models. So for the dopaminergic neurons that is the Parkinson disease and there are different models for that. We have the patient derived iPSCs that are differentiating to DOPAS. We also have the genetically modified iPSC lines that are differentiated to DOPAS and those cells are usually used to study the genetic diversity in Parkinson’s disease or if you want to understand the disease pathway. Or if you want to focus on one specific gene mutation and understand how that is affecting the cellular function. Or if you want to develop a drug that’s focuses specifically on bringing down one of these gene mutations, then these cells are used for that. But the healthy iPSC lines are also used.

So one thing that they’re used for is without any prior treatment to them. They are used for a small and high throughput screening of compound libraries and people are checking the effects of different environmental toxins on the cells and they’re measuring the viability, the neurite outgrowth and neurotoxicity of these compounds on the cells. Another way that they’re used is that the cells are exposed to some kind of toxin like MPP or rotenone.
To mimic the conditions of PD and it’s being done in gradient concentration and then we can look at the effect of each of these compounds and then the effect of neuroprotective compounds and see how the cells can be saved actually. In that case, we can look at the how the mitochondria is functioning. We can look at the progression of the disease. We can look at different calcium channel blockers and understand the behavior of the cells in that way.

There are also models that are co-cultured or organoids. So I saw that there are some tanswell-based models that people are co-culturing BBB cells with dopaminergic neurons to understand drug transport through BBB. There are also co-cultures of dopaminergic neurons with astrocytes to either understand the cell-cell interaction between the two population or just understand the role of the astrocytes in Parkinson disease. And these are just some of the ways that the cells can be used. There are other applications as well.

One thing that we did ourselves was that we purchased the PD line that was available commercially. This was a patient derived line. And we tested our own differentiation protocol on these cells and what we found was very interesting. So the cells can differentiate to dopaminergic neurons. We see expression of tyrosine hydroxylates, PTX3, KCNJ6, neurofilament, TUBB 3. However, we are not, we weren’t able to detect any SOX6 expression in these cells. So basically these PD lines weren’t able to generate A9 subtype. Of course this was only one line that we tested, so we don’t know how the other lines would behave, but we just thought it was very interesting that our protocol was affecting the cells in this specific way.

To summarize, the dopaminergic neurons that we are generating express dopaminergic markers, TH, SOX6, DAT and KCNJ6. They’re provided as cryo preserved vials with a full media kit, we have lack of calbindin 1 in these cells to the and as I said, calbindin 1 is expressing A10 dopaminergic neurons. The cells are validated by RNA sequencing, flow cytometry and immunocytochemistry. And we are seeing dopamine release from the cells doing ELISA on that.

We have more information on our website. We have our blog. We have a FAQ section. You can go to the website, learn more. You can also reach out to our technical account managers, Kritika and our senior business development manager Peter to learn more about the cells.

Thank you. And I think now we’re going to start our Q&A section.

Joshua Snow
Sorry, I was muted there, Nooshin. So thank you so much, Nooshin. That was a, that was a really great talk. So that brings us to the end of the presentation portion. We’re now going to shift gears and jump into the Q&A session.

So thank you to everyone who’s already submitted a question. If you have a question in mind and haven’t submitted that yet, now is the time, so please don’t be shy. Again, just use the the Q&A feature that’s at the top of your screen to drop your questions in and we will start answering those now.

So first question for you, Nooshin, is about the maturation step.
The question is, what’s the typical total time required from thawing the progenitor cells to having a fully mature assay ready A9 dopaminergic neuron and culture?

Nooshin Amini
So it takes around 14 days for the cells to fully mature and that is the amount of media that you are providing with the cells too. But they can be used for downstream applications from day 10 after thawing the cells.

Joshua Snow
OK, great. So the next one is can you provide more detail on the measured dopamine synthesis and the release compared to other sources of dopaminergic neurons?

Nooshin Amini
Yeah. So we did ELISA on the cells and our samples are just from untreated cells. So we didn’t do the stimulation on them, but I know that people can do KCRS stimulation on the cells and check the dopamine release in that as well. I’ve seen some people use HPLC too. But we only did ELISA on the cells and we we did detect dopamine release from the cells.

Joshua Snow
OK.
All right. Another one is if you add L DOPA to the cultures, do the A9 neurons take it up and then convert it to dopamine? This is a two-part. And why do you think the PD derived line does not generate A9 neurons effectively.

Nooshin Amini
Right. So for the first part, we do want to test that. Actually we haven’t tested it yet, but we are planning to add LDOPA to the cells and then do calcium imaging and NEA and and on NEA plate as well to understand how it’s affecting the cells, so that is that is coming. And the second question was why didn’t we are not sure we didn’t look into it closer, but it can be just because of the how the genetic mutations that the line had and that they are just not able to become A9 dopaminergic neurons at that point.

Joshua Snow
Great. We’ll keep going. So the next question is, have you done or do you plan to do any additional studies on the electrophysiological properties of these cells, for example, comparing to in vivo or primary midbrain A9 neurons?

Nooshin Amini
Yeah. So that’s one of the things that we we are really excited to do. We are planning to do some electrophysiology assays on the cells, yeah.

Joshua Snow
OK. Similarly here, what additional co-culture or organoid experiments have you performed or do you plan to perform?

Nooshin Amini
We haven’t done any co-culture on the sales yet. We are thinking about maybe doing a co-culture with astrocytes, but it also comes down to what kind of feedback we get from customers and if they’re interested in seeing that then then we can work on that.

Joshua Snow
OK. And then we’ve, it looks like we’ve got two more. One is you mentioned that A9 are more vulnerable in PD, but what are your thoughts on how relevant these cells are as a model for sporadic versus genetic PD.

Nooshin Amini
So I think for genetic PD there has to be some gene editing on the cells for the relevant gene that people want to study, but for sporadic I think they can be applicable.

Joshua Snow
Alright. Another is what is the expected viability of your frozen neurons after thaw? Is it possible to dissociate and replate these neurons after they’re seated?

Nooshin Amin
So we are seeing viability from 50% up to 60-70 that is that is the range we are seeing at least 50% and I don’t but we haven’t replayed it themselves.

I would say if that’s going to happen, it should be sooner in the culture rather than later. If we do that at the time that the cells are fully mature, there is a possibility that we’re going to damage the neurons and then the recovery after that won’t be that great.

Joshua Snow
OK. And then our last question is how long can you maintain the cells and culture for assays?

Nooshin Amini
So beyond that 14 days, we we tested keeping them for another two weeks in the same media and we could do that. We haven’t done longer time studies, so I don’t know how what will happen after that extra 2 weeks.

Joshua Snow
All right, great. Well, that’s all I see in the Q&A, but those were some good questions. So that’s where we will end today. So thank you Nooshin again for the great presentation.

Thank you very much to our attendees for your questions and engagement.

If there was anything that wasn’t answered for you today, again, please feel free to reach out to us at support@trailbio.com.

And then as a final reminder, the entire session has been recorded and will be hosted on the Trailhead website later this week.

So we hope you found today’s session valuable. Have a wonderful rest of your day, and we hope to see you again soon.