Channel: Twist Bioscience clear
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| 1 | Twist Bioscience | Cellular reprogramming for the scalable production of human cells | 2485 | 50 | 53.4 | 36:32 | hello everyone my name is ernie guzman i'm the manager senior manager of technical support at twist bioscience and i'd like to welcome all of you to today's presentation entitled cellular reprogramming for the scalable production of human cells presented by dr michael d'angelo senior scientist at bid bio just a couple of quick housekeeping rules to cover first is that all lines will be muted during the webinar and this is to reduce any distractions during the presentation secondly we will have a q a session at the end please submit your questions in the q a box we will answer your questions after the presentation is complete and finally following the presentation there will be a short one-minute survey that we would really appreciate you taking your feedback will help us improve all future webinars and now i'd like to introduce our speaker today's speaker is dr michael d'angelo michael is a molecular biologist and genome engineer he leads the bit bio team conducting technology development and gene editing projects which are at the core of all of bitbio's cellular reprogramming activities and with that michael the floor is yours you so much for that introduction ernesto and i'd like to thank twist bioscience for the invitation to speak today on behalf of bit buyer today in this webinar i'd like to tell you about the need for better human cells and the promise of cellular reprogramming i'll tell you about the bios approach to making human cells and how twist gene synthesis has helped us and i'll finish off by showing you some data and showing you uh some of the products that we've made using our optix technology so why do researchers need human cells well human biology is often different from the animal models and tumor cell line models used in pre-clinical drug testing and as a result many drugs often fail in human clinical trials because of a lack of efficacy or toxicity that was not appreciated using these non-human or non-physiological human cell models so where research is going to get good human cell models for drug testing or you might think that the perfect human cell model would be primary cells from donor tissue however these have some problems they are of limited source because they need to come each time from tissue donations and they often display highly variable biology because they need to be isolated from complex tissues and they come from donors with different genetic backgrounds adding you know heterogeneity and noise to the system there's also classical tumor-derived cell line models these have advantages because they're easy and simple to grow however they don't they're transformed and they don't represent normal human biology and then you've got pluripotent stem cell derived models which in principle have the ability to provide an unlimited source for human cell types at the moment however their bottleneck is really realising their potential and at the moment all of these three sources suffer from a lack of consistency scalability maturity and purity so if i tell you a little bit more about ips cells so these are cells that have been reprogrammed from mature human cell types back into a pluripotent state so that they're very similar to embryonic stem cells in their ability to differentiate into all the three germ layers of the human body and in principle have the ability to become any cell type of the human body and so they have advantages in that because depending on where you get that starting cell type from they have the ability to um represent different human genetic diversity they are self-renewing and therefore they're inherently scalable and they are now have really robust cell culture procedures in place which means that they're amenable for genetic engineering and so what we really want to use however is these cells on the right over here so we've got a neuron or white blood cells and red blood cells for example we want to get to a mature cell type from these potent stem cells and the traditional protocols for doing these are called directed differentiation and really with these protocols what you're looking to do is mimic development in the dish so you're putting generally growth factor combinations on the cells to mimic what happens during a regular developmental pathways via various intermediaries and and precursor cell types to end up at the final mature cell type that you're interested in however these tend to suffer from having very complex protocols with very expensive and complex media that needs to be prepared and therefore they struggle with scalability also the maturity and the purity of the final cell type you end up with is not always particularly where you want it to be and so synthetic biology provides us with a new paradigm for how to make mature cell types from pluripotent stem cells and this is the idea of reprogramming so now instead of the cells having to interpret a complicated extrinsic signal we're now giving them a strong simple to interpret intrinsic signal by over expressing transcription factors and in this case we're imposing the cell identity and transcriptional network upon the cells and therefore speeding up the differentiation process however standard reprogramming methods lead to polyclonal heterogeneity and low scalability and this is where our opto x technology at bitbio comes in and we're able to now get cells at the end that have high purity maturity scalability and consistency so what are we all about at bitbio so bitbuyers mission is to code cells to advance the well-being of humanity and to do so we apply the principles of computation to biology our current focus is to develop a scalable technology platform capable of producing consistent batches of every human cell type and to enable research and drug discovery to move on from less efficient models and work with the cells that are actually affected by human disease and the scalable platform of consistent cells will also be the basis for a new generation of cell and tissue therapies so at bitbio we have two pillars on which we're based so in order to reprogram cells we need to identify the optimal transcription factor code that can reprogram cells from the pluripotent state into the mature cell type of interest and to do that we've built our bit bio discovery platform and this is a platform that allows high throughput experimentation coupled with machine learning to determine the optimal transcription factor code of interest that we can then use to program a mature human cell type of interest and then once we've identified the optimal transcription factors they are then ready to be put into our optix platform which is the way by which we over express the transcription factors needed for cellular reprogramming into the new cell type of interest and i'll go into detail about how the optix platform works on the next few slides what this allows us is to have cells that now have very high consistency very quick uh speed to maturity high scalability they're very easy to use and cost effective so optics stands for optimized over expression and so older methods for example using lentivirus or so non-specific integration methods lead to high variability in reprogramming and this is because they each gene expression cassette will land in a different genome location and therefore leading to differences in expression level of the transcription factors and therefore differences in the outcome of the reprogramming so initially experiments were done using a targeted system so into a genome safe harbour site using an inducible system so in this case the doxycycline inducible system uh however when this was at first uh tested what was seen was low expression and silencing of the transgene in this case gfp so you can see patchy and low expression in this ips colony here and so from this is where optix was developed and so in a seminal paper from our founder dr mark cotter's academic lab what they did was a simple trick what they did was to split the two components of the doxycycline inducible system so we have a constitutive expression of the rtta transactivator protein from one genome safe harbour site and then add a second genome safe harbour site we now have the uh tetracycline response element uh inducible promoter driving expression of the transgene of interest and now what you see is strong stable expression every cell in this ips colony is now expressing gfp and so optox is able to overcome these limitations of silencing so this is great because with the inducible system what we have is in the absence of doxycycline pleuric potency is sustained and so these cells are now self-renewing and inherently scalable and now when we add doxycycline we have deterministic induction of a new cellular identity and so these are examples from this paper showing reprogramming into these three cell types of interest in very short time frames and so this is what it looks like in action this is a time-lapse image of cells differentiating into glutamatergic neurons what you can see is that within just three or four days they're already displaying neurite outgrowth and then they've become post-mitotic and they form a dense neural network within a week so what's really exciting about this is the speed at which they're able to form such a dense neural network this normally takes weeks with classic directed differentiation cultures and also the homogeneity that you can see really every cell in that culture is reprogramming they're changing morphology at the same time and the resulting cell population is extremely pure so how has twisted synthesis helped us in our mission so in order for us to get access to different transcription factors that we want to test with the op2x platform we use twist gene synthesis and so what we used to do was when we had a new transcription factor we would get it synthesized in the twist cloning vector then they would send it to bitbio where me and my team would then perform sub cloning into our updx vectors however now it's even better we've completed custom vector onboarding so they now have our targeting vectors there and what we can do is whenever we want new transcription factors to be tested we can order gene synthesis and twist will clone them directly into our into our optics vectors and ship them back to us ready to use so this is fantastic because it's fast and it frees up the time that my team would have spent at the bench doing the doing the cloning it's also inherently scalable because if my if we were to order a large number of transcription factors well the amount of time that we would take to sub-clone them uh would scale somewhat linearly with the number of factors that we ordered whereas at twist it takes roughly the same amount of time to get one transcription factor as it takes to get 50 because of their high throughput platform and we're also really excited to take advantage of their new higher yield dna prep offering which will hopefully get us access to transfection-ready dna so we'll spend even less time at the molecular biology bench and more time doing exciting things uh plugging these into ips cells and making exciting new human cell models so bit bias cellular reprogramming is really making a new benchmark for human cell models due to the high purity the speed of differentiation the consistency not only batch to batch but really cell to cell in each vial and also the scale at which we're able to do production and this enables a number of applications for basic research both normal and disease models and we're really building up a great capacity for genetic engineering of disease models and this is enabling for developmental and cell biology experiments it's also enabling for drug discovery so both normal and disease models can be used for high throughput drug screening target discovery and high throughput toxicology screening an interesting application is in bioproduction and actually our founder mark cotter is also a co-founder of a clean meat company using the oc2x technology in cells derived from livestock animals to make a scalable source of lab-grown meat and of course a really exciting application for this would be cell therapy whether it be immune cell therapy or other regenerative medicine applications for different cell types so now i'd like to show you some data from some of our human cell models and so these will be our ips derived neurons and myocytes so first up is our fully launched product which is the human io neurons glute product these are ready for experiments as early as two days post revival and they are a majority are glutamatergic neurons a something like 88 and a smaller subpopulation of cholinergic neurons so what you can see here is some immunofluorescence staining for pan neuronal markers beta-3 tubulin and map2 and then in orange here are the two glutamate transporters which are specific for glutamatergic neurons v-glute 1 and v-glut2 bulk rna-seq analysis has shown that the neurons have a rostral cns identity they exhibit robust neurone outgrowth and form functional neural networks in as little as 17 days that demonstrate drug response so what do our customers get so at bitbio the first phase here is performed which is induction so for three days the cells are primed towards the neural fate and then their cryopreserved and then you will receive one of these cryovials over here with cells that are ready for ready for plating and rapidly mature upon revival and then it's a very simple process in the customers hands they go through a very they go through a four day stabilization phase during which doxycycline is kept in the media and then a maintenance phase for however long you want to culture them where doxycycline is now removed and during that whole time they've just got a simple glutamatergic neuron media which is sim is a fully disclosed formulation and it this is enabling for our customers to customize it for their particular experiments so we've done some deep characterization on these cells so we've done single cell rna sequencing so what you can see here is a time course of reprogramming so following anti-clockwise from the ips state through some early time points here 12 24 48 and then 72 and 96 hours and then also some very mature cells over here two and two and three weeks and what you can see is the pan neuronal marker map t starts to come on around that day three to four mark and then he's really expressed in every cell at the two to three week mark and most of the cells are expressing this v-glute glutamatergic marker with a smaller population that express this this cholinergic neuron marker and so our partners charles river have used these cells in various different experiments you can email us for a copy of this poster if you're interested at info at bit.bio so what they've performed are some functional characterization using high resolution multi-electrode arrays so that is showing that the cells demonstrate electrical electrophysiological activity and so um both at two weeks and then increasing at three weeks in culture the firing rate percentage of active electrodes and the spike amplitude all increase and also they have validated these for high throughput assays in 384 well plates and so if you're interested please email us and we can give you a copy of these this poster and now i'd like to talk to you about another cell model that we've created which is the skeletal myocyte so iomiocytes skeletal this is in beta testing phase and will be hopefully launched soon but if you're interested in being on the beta testing being one of our beta testers please get in touch these are also ready for experiments as soon as two days post revival and contractility can be assessed as early as three days post revival they exhibit visible striated fibers and multi-nucleated myotube formation by day 10 in culture and uh what you can see here is immunofluorescent staining demonstrating robust expression of components of the contractile apparatus including desmond dystrophin troponin and titan and also we validated by qpcr the expression of key markers including various myosin heavy chain isoforms as well as the transcription factor myogenic and so here we have a video showing spontaneous contraction of these cells so this is in the absence of any stimulation 15 days post revival there they go always freaks me out when i see that down the microscope and importantly also these cells if you add a stimulus such as acetylcholine these cells contract very strongly then so what i've shown you is data from our i o neurons glute fully launched product and coming soon our imi site skeletal and also before the end of the year we expect to launch our io neurons gaba which is a gabaergic neuron product as well as our io glio ioglia oligo product which is a human oligodendrocyte cell model and if you're interested in those please get in touch and we can let you know when they're going to launch so at big bio we're developing the next generation of human cell models and providing the best cells for research and drug discovery and our cells are used in research and disease modeling they've also seen applications in in 3d printing into 3d scaffolds and so there was a recent paper out of oxford university using our cell using our neuron cells in a 3d printed cell scaffolds and also in high throughput screening and at this point i'd like to thank our partners charles river who are using ourselves in high throughput in their high throughput screening applications and abcam who where ourselves are available for purchase if you want to go to the ad cam website you can find the cells available there and if you are an academic researcher or working in a not-for-profit organization please make sure you take advantage of the discount code there so that you can get access to our sales for a really great price so bitbio allows for the scalable and consistent generation of defined human cells and the development of the bit bio lineage portfolio portfolio is driven by our discovery platform for the identification of novel and optimized transcription factors for new cell type reprogramming and our optio x technology for the controllable expression of transcription factor combinations and all of this results in cells that have high consistency high speed to maturity scalability ease of use they're cost effective and they show really high purity with that all that's left is to thank the team at bitbio that really make it a joy to come to work every day um the one problem with fun jumping pictures is that i think all you can see of me is maybe my shoe right there behind my boss who's blocking me but it's a fun picture nonetheless and yeah thank you again to twist for inviting me to give this webinar today and with that i think we'll take some questions uh thank you michael for uh your presentation really appreciate it and now we'll take some questions from the attendees um as a reminder if you have any questions please make sure to ask them in the q a box um so a couple of questions here how does the opti-ox improve reprogramming so reprogramming the cells is by directly activating a program that defines particular cell identity by over expressing a transcription factor from within however typical methods for over expressing transcription factors leads to heterogeneous expression and therefore variability in the final cell outcome optics gives us source where every cell is expressing the transcription factors at the same time and at the same level and therefore what you end up with is a highly consistent reprogramming it is a highly consistent reprogramming event okay another question so you've shown the power of the optio x generate mature glutamatergic neurons and skeletal myocytes uh what are the applications of your human cell models and is bit biodeveloping any other cell types yeah so defined and consistent ips derived cells provide an excellent alternative to animal models and immortalized cell lines which diversify during long-term culture our human cell models are used for research for example the study of neurological activity in the case of the iron neurons glute they're also used in disease modeling and 3d printing because bit buyer cells are consistent and scalable they provide a good model for high throughputs screening and a great example of this is our partner charles river who have adopted our ips drive cells into their high throughput screening workflows for use in target discovery validation and screening services and our aim is to democratize access to high quality human cell models and provide researchers across academia and industry with reliable models for research and drug development and so we're working on a wide variety of different cell types so we will be releasing as i mentioned earlier some cell types in the central nervous system space and if you're interested in trying them sooner as a beta tester then please get in touch with us another question is how are you choosing which transcription factors to overexpress yeah sure so that really comes down to the power of our uh of our discovery platform and so that allows us to really do experiments in cells at the bench to screen high numbers and combinations of transcription factors and to determine the ideal transcription factor code that can make the final cell type of interest that we're interested in and this is really powerful and then the data coming out of that is coupled with machine learning methods which will allow us to predict these um in the future um okay another question is um are all the cells you provide diploid the cells that we provide the cells that go into our manufacturing processes are diploid so ips cells are diploid cells um with a normal carrier type we do that's part of our quality controls um that we have at bid bio and um i believe we also check ourselves at the end but we'll need to double check with our with our product specialists okay how many transcription factors can you overexpress at the same time so that's a good question and and it also i guess comes back to how many transcription factors do you really need to make a particular lineage um at the moment with relative ease we can express up to probably five or six transcription factors of course there are lots of ways that we can expand that out and we're looking into that at the moment um you you sort of brought this up earlier but uh another question is how do you qc that you get the right program cells of course yeah so there there's lots of different ways that you can that you can look at and benchmark the cells that you're making um one of the best methods which i showed some data from is single cell rna-seq analysis because that allows you to look not just at a bulk measurement of expression of a particular marker but it really lets you look is every single cell in that population expressing that marker and to the same level and it really gives you high high order information about really what is the transcriptome of each individual cell and how well does that match the cell type that you're trying to model of course the questions of what's the best benchmark is a little bit of a difficult one because even primary cells freshly isolated versus primary cells kept in culture will will display transcriptional differences so that can that can make it a little bit difficult to say uh exactly what um you know what's your what the goal you're trying to hit is but um in general that's a really good way of qc and then once once you know you know classical markers that they should be expressing then they can of course be tested either at the protein level by immunofluorescence staining or at the transcriptional level by quantitative reverse transcription pcr okay um another question do you try to engineer the expression level of the transcription factors or is it more of an overexpression versus not sorry could you repeat that i just missed that one sorry so do you try to engineer the expression level of the transcription factors or is it more just sort of an overexpression versus not expressed so i mean the beauty of the doxycycline inducible system is that it's um it's inherently titratable so if um like all of our products that we're releasing we have established the correct concentration that is required to reach the reprogramming that we need so that's in built into the optics system because of the dox inducible system so we have the ability to vary the expression level um have you ever made direct comparisons between the ips cells produced and versus native cells so did you say versus native cells yes uh i i believe so you don't mean ips sells you mean the um the final um the final reprogrammed cell product and and um yeah i believe that there's been some there was some slides in a recent webinar from our ceo that that looked at ways of comparing um the identity of the cells that we make in a reprogramming versus the mature human cell type that we're trying to model so we have done benchmarking experiments like that i don't have the data in this slide deck though if you're interested again you can get in contact or you can check out that webinar which i think is accessible on our website okay um does your platform alleviate the need for growth factors during the differentiation process yeah that's a really good question um what i would say is that it it certainly allows the simplification of the um of the extrinsic signals so so the media components um however at the moment certainly there is still a need for some extrinsic cues there still needs to be media in which the final cell types are are happy and and performing their functions and so there are still um depending on the cell lineage some um growth factors or various additives in the media but typically they're simpler than the media required for direct differentiation protocols um the question is the optix integrating or non-integrating yes so as i mentioned it uh involves targeted integration into genome safe harbor sites so these are sites in the genome that have been widely used they have the advantages that it's established that they are transcribed in the majority of different human cell lineages and therefore they should remain open no matter which cell type you try to make and ese's targeted integration are at two different genome safe harbor sites for optiox okay another question that comes in have you considered making disease models using these cell lines for neurogenetic neuro neurodegenerative diseases i the question is i do see a need for this in our work at our research foundation and it is very hard to come by good human cell models yeah it definitely this is something that we are pursuing at the moment something that we're looking to expand and if you're interested in a particular model i urge you to get in touch with us and we can start a conversation that is something that i think is a really exciting application of of our cellular models there's a lot that can be done with kind of wild type human cell models that are reproducible and consistent however um it is um it's certainly also very powerful to be able to do disease models with these so something that we are actively exploring at the moment so please get in touch if you're and we can we can discuss what you're interested in exactly what one um okay um uh have you discovered many novel dna sequences that can cause a differentiation in ips cells i'm not quite sure i'm not quite sure what that question is asking in terms of novel dna sequences i mean if if we assume we're talking about novel transcription factor codes um i would say that our discovery platform is um is capable and is we've shown that it's we've demonstrated that it's able to generate new transcription factor codes that that haven't been published before so we are definitely generating and discovering new transcription factor [Music] codes okay um can you comment on the difference between your system and using an episomal plasmid to avoid any genetic changes so and an episomal plasmid um so the the main differences are going to be in the scalability and the homogeneity when you introduce an episomal plasmid you need to transfect the cells which means that inherently that's difficult to scale there are high throughput platforms for that now but they're not widely available or you know even those are somewhat limited in their scale so you you need to transfect the cells then you've got issues where maybe there's transfection efficiency differences so already you might have some cells that don't receive the episomal plasmid and don't therefore don't reprogram or you need to use some sort of drug selection to enrich and then you've also got the issue where you can't easily control the copy number of that that episomal vector and therefore you can lead to differences in the reprogramming outcome both sell to sell in that batch that you've just tried to create and also experiment to experiment because every time you do that transfection it's going to be a slightly different outcome and therefore for you know for some applications maybe that's okay but for things where you really need consistency and purity what you want to have is you know every cell in the dish doing the same thing at the same time and for things like high throughput drug screening this is imperative because if you don't have if you have any extra sources of noise in your system they can destroy your signal and therefore suddenly um you know what was a hit is due to noise or you miss out on hits or on things that could have been hits because um your system is not showing the sort of um signal definition that you need so um really the op dx platform allows for the scalable and reproducible production of large batches of these cells that all do the same thing um as well as the fact that they have the advantages that that they're all the cells are doing the same thing at the same time okay i think we're coming very close to our end so i just want to ask one last question the question is i would be keen on using these cells what do i get in the tube do i get the cell or the cell lines current cells available take weeks to differentiate neurons yeah sure so um our cells that uh we've produced so they will have undergone an induction phase so that they are primed to already reprogram to the lineage that we're making and then they're cryopreserved and so when they're shipped to you these cells rapidly mature and uh and become post-mitotic and they are so the speed to reprogramming and differentiation is much faster for the end user you also never have to know how to culture an ips cell they're ready to use um and really that's another great advantage of all the cells that that you get in that tube okay so i think we're just about out of time and we want to be respectful of everyone's schedule um as a quick reminder after you leave the webinar will be redirected to a brief survey if you have a moment to spare we would really appreciate your feedback thanks again to dr michael d'angelo of bitbio and thanks to all of you for joining us here today we really do appreciate it until next time remember science doesn't stop and neither will we stay safe and have a good day | ↗ |