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We're developing highly innovative drugs and very specific for the disease we want to treat, but we are testing these in models that are a bit updated.
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So usually when we go into the clinic in the patients, there's always a gap in the response.
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And this is where our models come into play to really bridge this gap between the traditional models and the patients in the clinic.
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But first of all, what are HUB organoids?
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Because organoid is quite a broad term, it can be used to describe anything that grows in 3D.
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But in specific, our models are derived from adult stem cells, so they're derived from the progenitor cells that reside in the tissues of our body.
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And what we do, we start with the resection from a patient, we dissociate it into single cells and we grow these cells in a 3D matrix.
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And with the right combination of growth factors that really depends on the organ that you're growing, we can have overtime the outgrowth of these structures and they self-assemble they’re self-sufficient.
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We can derive them basically from any patient and from most epithelium organs.
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They can really capture very well the heterogeneity from one patient to the other.
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And also in terms of if we're looking at a tumour, they can really recapitulate the heterogeneity that you have in a tumour.
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We know that they are stable in culture.
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Over time, we can expand them almost like a satellite.
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And so far by now we have very good evidence that they really have high value and high clinical predictive value.
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First of all, before I go any further, I would like to give you a bit of information of our history.
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We celebrated last year our 10th anniversary.
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So we already exist for 10 years.
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We were funded in 2013 as a foundation and as a spin off from the lab that invented this technology and everything
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dates back to 2007 when Lgr5 was discovered as the marker of the intestinal stem cells at the bottom of the mouse crypt.
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2 years later, this knowledge was used to develop the first Organoid model from the mouse small intestine, derived from adult stem cells of the mouse small intestine.
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And in 2011, this was expanded also to human cultures and that's when HUB was born two years later to be able to really commercialise this technology.
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And since then there have been a number of really big milestones in organic research.
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Like for example, in 2015, we had the first cystic fibrosis patient was treated based on Organoid data.
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And also in 2018, we had the first oncological drug target entering the clinical trials based on drug screening data done on organoids.
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And this is basically what fuelled our growth.
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We expanded our facility in 2022.
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And yeah, last year we became from a foundation, actual commercial entity.
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So we have come a long way, but there's a lot more than we can do.
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And here I have just a brief recap of the key features of our models.
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As I said, they're derived from adult stem cells.
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They are physiologically relevant.
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So in this system we have not only the progenitor cells that are the ones that make us expand this model, but we also have most of the differentiated cell types that are present in the tissue of origin.
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They're stable in culture and they are predictive of the patient response and can be derived from many different patients.
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We can expand them so we can have a high throughput for our assays.
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So normally we use 96 or 384 well plates for our essays and we can genetically manipulate them.
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And we have high establishment efficiency.
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Of course, that changes from organ to organ, but in general, it's quite high.
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And this is a bit of overview of our biobank.
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The size of the cartoon correlates with the size of the biobank.
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So colon and breast are among our largest biobanks.
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And we can derive these models from normal tissue, healthiest tissue, but also from diseased tissue, from patients with cystic fibrosis, cancer, IBD as I will talk about later, genetic disorders, COPDS and so forth.
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And we can characterise them in a number of ways.
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This is just an example with DNA RNA sequencing, but all our models go through our QC checks, which is checking their growth, their identity with snip typing and also of course mycoplasma.
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So how do we use these models that we establish - mainly in three ways.
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So there's three ways in which we work.
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We can have really customised assay development type of project and this is really tailored to specific research question and that can really differ a lot.
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Otherwise we have a more standardised type of assay, which is a drug screening that we normally use for testing the efficacy of compounds in our organoids.
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And we can also do this in parallel with a clinical trial.
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So we have co-clinical drug development and as you can see here, our therapeutic areas are quite broad, immune oncology, cystic fibrosis, COPD, but also genetic and infectious diseases and IBD, which I will talk a bit more about.
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Yeah, in a few minutes.
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So IBD, inflammatory bowel disease and I chose this example as an example of our capabilities and what these models can be really useful for.
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And IBD is this chronic inflammation and damage of the epithelial cell compartment of the intestine.
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We do have therapies for example, trying to block the inflammation anti-TNF of therapies, but 40% of the patients are resistant and not responsive to these treatments.
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So there is really a need to identify new therapeutic targets for IBD.
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What we know so far, so the aetiology is unknown which makes it more difficult.
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But if we compare a healthy situation that you have on the right with the homeostatic condition of the of the intestine with the stroma underneath and epithelium.
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We know that in a disease condition we have this cross stalk between immune cells from inflammatory fibroblasts, and this leads to an overproduction of pre-inflammatory cytokines that ultimately damage the integrity of the epithelium and there is also a shift in the gut microbiota.
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So in this sense, yeah, there's many players in this disease and the therapies that are being developed at the moment are targeting either the chronic inflammation that we have in the gut, trying to restore the integrity of the barrier or targeting the pro inflammatory fibroblasts.
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And what we have in our biobank is at the moment 30 models that were established from 15 patients.
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They are from the large intestine, small intestine, from both Crohn's disease or ulcerative colitis, which are the 2 main types of IBD.
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And yeah, we have characterised them with RNA sequencing or risk loci profile.
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So what do we do with these models?
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We've seen that the disease is quite complex.
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There's many players involved.
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And what we would like to propose and what we follow is a modular approach.
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So based on how complex your research question is or how complex you want it to be, we can decide to look either at really the integrity of the barriers.
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So really focusing on epithelium itself.
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And we have this PDO monolayer system, PDO patient direct organoids where we grow our organoids in 2D on a transferal membrane.
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And this really allow us to test therapies that can strengthen the barrier.
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But we can also decide to look more at the interaction between organoids, so epithelial cells and fibre blasts.
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So we can do Co-cultures of our organoids with fibroblasts and we can do this in 3D like we normally grow our organoids, but we can also study the interactions between microbes and epithelial cells again in the Co-culture.
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And we believe that ultimately this all together will really drive and speed up the development of new therapies in IBD.
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So I will start by talking about the PDO monolayer system.
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This is how it looks like.
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We have this transfer membrane with our monolayers from organoids and we can of course have access to the apical side.
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So we can apply our stimuli there.
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It can look like different types of stimuli and we can also tweak the composition of the culture medium.
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We can have them in a bit more expansion state with an expansion medium where you have a high presence of proliferative cells, as you can see from the Histology with Ki67 positive cells and also from the RNA sequencing, our expansion medium.
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But we can also decide to differentiate these models towards, for example, enterocytes if we're mostly interested in the cell types or what we normally do.
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What we prefer to do is to have a combination medium that you see here in the middle and also in the Histology.
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And this combination medium allows us to have both progenitor cells that proliferate but also differentiated cell types.
10:48
And here you have an example of gold blood cells that are present in in this model.
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And yeah, in terms of stimulated, we can apply.
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Of course, inflammatory cytokines are one obvious candidate for when we look at IBD.
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What we do is seed our organoids, we let them differentiate for three days and then we apply cytokines, for example, TNF alpha or interferon gamma and we do the treatment for 24 hours.
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And in terms of readout during the whole experiment we always measure the tier, which is the trans epithelial electrical resistance across the monolayer.
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That tells us about the integrity of the barrier.
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And as endpoint assays, we also look at the permeability of the system with Lucifer yellow assay and the viability.
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And as you can see on the right side the plot, when we apply increasing concentrations of cytokines, we really observe decrease in the tier values until we have a drop at 24 hours with the highest cytokine concentrations.
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And also from the pictures we see that the monologues don't look very healthy and very happy.
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Good thing is that we can treat them with for example, in this case have we used Tofacitinib which is a (JAK) inhibitor.
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So it really interferes with the signalling pathway of the inflammatory cytokines.
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And when we apply Tofacitinib in cytokine treated monolayers, we can have improvement of the inflammatory and the integrity of the barrier.
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And this is reflected both in the tier data where we look at the barrier integrity, but also in viability.
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So with the highest concentration of cytokines, we see a reduction in both the integrity and the viability with Tofacitinib.
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This can be nicely rescued, and the permeability is the third readout that we do and it works the other way around.
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So when epithelium is damaged then it's more highly permeable and when we apply to Tofacitinib together then we restore the integrity so the permeability goes down.
13:05
Just very quick information.
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We recently launched a package that is called IntegriGut Screen if you're interested in testing drugs that's really restore the integrity of the epithelial barrier.
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So if you want to know more, you can stop by our booth, which is number 10 and you can ask more questions.
13:26
But now I would like to continue with the Co-cultures between organoids and macrophages.
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So we said that this trauma is really a major player in the development and also maintenance of IBD.
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We know that there's an imbalance between myofibroblasts, inflammatory fibroblasts and this is what keeps the also chronicity of the disease and also the resistance to the anti TNF treatment that we have at the moment.
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So what we did was culturing together our organoids with fibroblasts and you see them in red in the picture and we can see how close closely to one another they come.
14:08
So they fibroblasts really gets in direct contact with the organoids and in the culture medium from this co-culture we can quantify the release of different cytokines that are produced either by the fibroblasts but also by the organoids.
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So we can measure what extents the epithelial cells response to the presence of these inflammatory fibroblasts and the plot in the middle you can see that with inflammatory fibroblasts we have high responsive in the release by the organoids.
14:44
We can also do damage induction in the system and our readout in this case is imaging.
14:52
We quantify the caspase signal and in the last plot on the right-hand side you see with increasing concentrations of cytokines we have increased in apoptosis, so increased caspase signal and when we Co-culture organise with normal fibroblast we observe a protection.
15:14
So we have lower levels of cell death and this is completely lost when we use pro inflammatory fibroblasts.
15:21
And what we're working on at the moment is to further increase the complexity of the system because we know immune cells also play a really big role into this.
15:30
So our goal now is to set up typical cultures where we have fibroblasts organised and immune cells, for example, T cells.
15:40
And last, I would like to show you what we did to mimic host microbe interactions.
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We wanted to have a proof of constant study in which we wanted to know if we were able to infect organoids.
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So we chose an E. coli strain that produces genotoxin, colibactin that is also found in up to 40% of the IBD patients and ingrained.
16:07
You can see again that the bacteria is strictly associated with the with the organoids here.
16:13
There's a little movie, but no, don't think it's going to play, never mind you just see it in 3D.
16:19
And as a readout we measured the gamma H2X.
16:25
Yeah, thank you very much.
16:28
Quantification for DNA damage and we saw that the bacteria are producing the gelatoxin had high levels of DNA damage compared to bacteria without.
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So with this, I will get to the last slide of my talk.
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I showed you how we can follow modular approach to look at and really dissect the IBD complex scenario by looking at either the integrity of the barrier interactions between fibroblasts and epithelial cells or interactions with microbes.
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And as I said at the beginning, this was meant to be brilliant as an example, because we believe that this approach can be applied to any other disease, any other condition.
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And we know that organoids can be very valuable and really contribute to any aspect of the drug development pipeline from target discovery to late optimisation and also in the clinic.
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And with this, I would like to thank you for your attention and happy to take any questions.
17:33
Thank you.