0:00
I'd come up and introduce Malika and explain a bit about who I am, the company I work for, because I mean it's a sort of joint presentation.
0:07
So I work for, my name's Dominic Hussey.
0:09
I work for a company called BiP.bio.
0:11
So we make the cells that are used in this.
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We're a partner of Charles River.
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We've been very tightly integrated with them over the past few years.
0:17
We essentially make transcription factor mediated and forward reprogrammed iPSC derived cells.
0:24
That's quite a mouthful.
0:26
If anyone wants to talk to us, we have a booth.
0:28
I think it's booth 13.
0:30
We can explain the technology that goes into making these cells and our portfolio in more detail.
0:34
So I won't take any more time up and I'll hand it over to Malika and she can talk about the science with using the cells that we provide.
0:40
So thank you.
0:42
Good morning.
0:43
Thank you for the introduction and thank you giving me the opportunity to present some work done at Charles River.
0:52
First of all, I would like to give you an introduction to Charles River.
0:57
Charles River laboratories are a CRO founded in 1974.
1:15
So we currently operate in more than 100 facilities in more than 20 different countries.
1:22
So far, we supported development of 86% of the novel FDA approval drugs in 2021.
1:35
Our focus in timelines and currency in every stage of development.
1:40
We have a lot of multiple divisions with own knowledge and expertise.
1:46
So we offer our clients end to end programmes, meaning that we can guide our clients from target discovery to safety in vivo pharmacology.
1:58
Today I will focus more on target discovery.
2:02
I am based in Leiden, and we do a lot of complex cell biology assays.
2:07
So we use primary and iPSC cell lines to validate targets in cell based assays.
2:21
Also, I will present some data on the lead optimization and vitro tox.
2:27
The goal of this phase is to select drugs before going in vivo.
2:32
One of our goals is to reduce as much as possible animals in drug discovery.
2:39
So in vitro, I will focus more in vitro neuroinflammation assays to understand the role of microglia in neurodegeneration to support CNS drug discovery platforms.
2:54
So as all we know, microglia plays an important role in the innate immune system, in the in central nervous system, they called the first line of defence in the brains, they play a role in the most neurodegenerative disease.
3:11
It's just as Alzheimer's, Parkinson's and ALS.
3:15
A lot of studies shown that you have a lot of activated microglia in different diseases, just MS and Parkinson's.
3:23
The activation of microglia leads to the production of cytokines and chemokines and nitric oxides, leading to neuronal damage and finally to neurodegeneration.
3:34
The most of the of drugs targets microglia activation to inhibit cytokine release, but also to enhance phagocytosis.
3:45
So at Charles River we developed a couple of models which can be used in neuro inflammation.
3:52
I will tell more about the Dutch Brain bank.
3:54
We use primary cells isolated from human brains and we have a partnership BiT.Bio which provide us with different cell types, neural cell types.
4:05
They can also provide us neural cell types with certain mutations.
4:09
We do a lot of ALS work, Parkinson's work, but today I will more focus on microglia activation.
4:17
We use a lot of readouts, MSDs and a sequence in high content imaging.
4:26
We do also a lot of in vivo models, but today I will focus on in vitro models that we set up.
So,33
So as I already told you, we have the Netherlands Brain Bank, which provide us with human brains from controls, but also from patients who have different diseases.
4:44
And we developed isolation protocols for primary astrocytes and primary microglia.
4:50
And Dutch brain bank has also a large collection of fixed and frozen material, CSF, which can be used for the biomarker analysis.
5:00
As we heard today, biomarkers is important to for neurodegenerative drug discovery.
5:09
So we optimise an isolation protocol for human primary microglia from controls and diseased patients.
5:17
We optimise assays for the activation of NF, Kappa B and inflammasome pathways because we know that in the brains of diseased patients, you have cytokine release, you have damage of neurons and damage of oligodendrocytes.
5:32
As shown here, we are able to isolate human primary microglia from brain tissue with a high purity.
5:39
We use specific markers for microglia such as Iba1, TMEM, and CD11b, and we also take the GFAP and NeuN staining to be sure that we have a pure microglia cultures.
5:52
At day zero, we use FACS staining to see if we have indeed CD11b positive cells to start with the experiments.
5:59
We also optimised a couple of assays where we measure the cytokine release you will see just some representative data.
6:13
We optimise the LPS.
6:15
LPS is classic trigger for the inflammation, and we use dexamethasone as an inhibitor for the release of anti-inflammatory cytokines.
6:26
As shown here, we see a nice induction of cytokines in an in concentration dependent manner, but also in time dependent manner, which can be inhibited by dexamethasone.
6:36
And we also developed assays for to study the inflammasome pathway where we first prime with LPS and then we add the second trigger such as BzATP or Nigericin to assess that the release for IL-1 beta and IL-18.
6:52
It's known that in the brains of for instance, for Alzheimer's and in Parkinson's, you have a lot of IL-1beta and IL-18 which leads to neuronal damage.
7:01
And we used relevant trigger which we know that it's upregulated in for instance, in Parkinson brains is we treated microglia with alpha synuclein, and we test, and we measured the release of cytokines.
7:16
Here you see just an example of IL-1beta and yes, alpha synuclein fibrous leads to the release of cytokines in human primary microglia.
7:31
So far, we have a success protocol.
7:36
We isolate pure microglia assays.
7:44
Second, we I'll go back to the this slide I showed you that isolated microglia are functional.
7:52
They respond to LPS.
7:54
It triggers different triggers.
7:56
And the second question, are they, if they have phagocytosis capacity?
8:02
Yes, we isolated myelin from human.
8:12
I don't know if you can see the video, it's not working, but we isolated myelin from human brain tissue from controls and Alzheimer's and we fed microglia with labelled myelin.
8:27
Maybe I can go to the to show you the video.
8:31
Hopefully, yes, it will work.
8:33
So what we have done, we isolated myelin from human brains and we labelled myelin with pHrodo which gives intensity when it's inside the cells.
8:44
So we, the isolated microglia are able, they have capacity for phagocytosis which can be used for the target validation for drug development.
9:00
I'll go to the present presentation mode.
9:05
So I showed you that we are able to isolate primary microglia from human brains.
9:11
Yes, they are functional.
9:12
They express that microglia markers, they have phagocytosis capacity, but we have limitations.
9:20
The isolate, the yield of the isolate microglia is very low to perform any large screenings.
9:28
So we can use primary microglia at the final stage of the hit validation where we can test the up to 12 compounds.
9:39
So the induced the iPSC derived microglia that can be a tool to perform large scale compound validation.
9:50
And therefore we use iPSC derived microglia.
9:53
BiT.bio uses optic technology where they use transcription factors to differentiate iPSCs toward a certain phenotype.
10:03
And we use ioMicroglia, we characterise them using ICC and we use the same markers that we use for the for human primary microglia.
10:12
And we can, and the ioMicroglia expresses the specific microglia markers, they are also functional.
10:22
We treat them, we use the same treatments as we use for primary microglia.
10:27
And indeed they respond to LPS by the release of pro inflammatory cytokines which can be inhibited by dexamethasone.
10:35
And we also optimise an inflammasome pathway where we prime the cells with LPS and we treat them with nigericin leading to the IL-1beta release which can be inhibited by MCC950, which is a known inhibitor of inflammation pathway.
10:51
And we also treated the primary the iPSC derived microglia with beta amyloid fibrils.
10:58
And we assessed the release of pro inflammatory cytokines and the treatment of ioMicroglia with different concentrations of beta amyloid leads to the release of the cytokines IL-6 and TNF alpha.
11:17
And next we optimised, we use the same assay that we use for the for primary microglia where we treated the ioMicroglia with pHrodo particles.
11:31
And we use Cytochalasin as a as an inhibitor for the phagocytosis.
11:36
And yes, we've shown that ioMicroglia also able to phagocytose the beads, which can be inhibited by Cytochalasin D. We also want to compare the gene expression profile of primary microglia and ioMicroglia.
11:59
And we in this graph we compare the top 50 express genes in human primer Microglia with ioMicroglia and in black dots you see the high expressed genes in both primary and iPSC derived microglia.
12:14
In green you see the high expressed genes in primary microglia and not in iPSC microglia.
12:24
We see a huge similarity within primary microglia with some differences for instance in toll like receptor repertoire.
12:32
As we know toll like receptors are the innate immune receptors and microglia.
12:38
Primary microglia expresses the whole repertoire of toll like receptors and ioMicroglia have limited expression of toll like receptors.
12:49
So I showed you that both primary and iPSC derived microglia show a strong expression of specific microglia markers are functional in response to LPS.
13:01
They are able to phagocytose beads and myelin.
13:05
And the top 50 expressed genes in primary microglia receptors have similar levels in ioMicroglia with some differences.
13:14
So both cell types can be used as a tool for to evaluate the potency and efficacy of prospective drugs for multiple neurological diseases associated with macroglia activation, such as Alzheimer's, Parkinson's and in monocultures or co-cultures.
13:34
The next part of my presentation, as I told you at the beginning, Charles River aims to develop complex cell models to be used to select the drugs before going to animals with the goal to reduce the amount of animals using in vivo pharmacology.
13:55
So we used BiT.bio iPSC derived cells and we developed a couple of models.
14:02
The first one is the coculture of astrocytes and cortical neurons.
14:07
The second one is where we have oligodendrocytes, macroglia and cortical neurons in one dish.
14:14
And we have also developed the complex culture model where we have the three different glial cells, astrocytes, macroglia and oligodendrocytes together with cortical like neurons.
14:28
So we in this slide to see a case study we used in the cocultures of neurons and astrocytes in an in vitro neurotox for one of our clients where we assess the effect of ASOs owned neurotoxicity.
14:44
We are not allowed to show you the data on ASOs because they are own data for the clients.
14:50
But we I can show you the assay that we used.
14:53
We use a high content imaging to quantify the neuronal growth, and we use nigericin.
14:58
Nigericin is known to be toxic for cells where we quantify the length of neurites and we've shown that indeed the treatment with nigericin leads to the death of neurons, which can be distributed by we can use ASOs or small molecules to protect neurons from neurite degeneration.
15:19
And one of the known markers used in the field is NFL release.
15:24
So we measured the NFL release in treatments with vehicle, LPS, nigericin.
15:32
We used also a condition made in from microglia treated with LPS and nigericin and with nigericin alone and we show that we have enhancement for NFL release in neuronal cocultures treated with nigericin. And in this slide we optimise in vitro oligodendrocyte maturation assay where we use IO oligodendrocyte like cells from BiT.bio and neurons.
15:59
We cultured them in a dish, and we quantified the MBP production using high content imaging.
16:07
And we also developed an algorithm where we can quantify the number of MBP positive cells and the intensity of MBP in inside the oligodendrocytes.
16:18
As shown here, we the addition of astrocytes leads to the production of MBP positive cells compared where when we culture neurons and oligodendrocytes, we see nice maturation and enhancement of MBP production and both day 7 and day 14 proceeding.
16:41
Finally, we case study #3 where we developed an in vitro neuroinflammation assay where we have the whole glial cells, astrocytes, microglia and oligodendrocytes together with neurons in the well.
16:55
And we treated them with the beta amyloid and measured the NFL release and the this experiment is still ongoing.
17:07
We will use high content imaging to quantify the dendrite outgrowth.
17:12
And as shown here, we show that the treatment with beta amyloid fibrils leads to the NFL release, meaning that we have no reiteration, which hopefully we can measure with high content imaging where we will quantify the neurotic growth and the health of neurons.
17:32
So finally we developed a couple of complex cell models which can be used in the target discovery, we can select targets before going to in vivo to reduce the amount of the animals.
17:50
So the complex cell models can be used to predict neurotoxicity and neuroinflammation of hits and select drugs before going to the animals.
18:00
And finally, I would like to thank my colleagues at Charles River and BiT.bio.
18:05
Thank you for your time and attention.
18:12
Thank you for that great presentation.