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WSCS 2015: Restoring Vision - Discoveries And Clinical Trials

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WSCS 2015: Restoring Vision - Discoveries And Clinical Trials

This session will provide an overview of current efforts to apply stem cells toward the treatment of retinal degenerative disease using stem cell derived retinal pigment epithelium. Participants will address a number of key areas. I) strategies for the functional validation of stem cell derived retinal pigment epithelia (rpe), ii) design and planning for an ind to test autologous ips cell derived rpe in patients with geographic atrophy due to age-related macular degeneration, and iii) use of tissue derived stem cells and the level of differentiation required prior to transplantation for effective therapy.

Video Transcript

MODERATOR

► ALAN D. MARMORSTEIN, PHD, MAYO CLINIC



SPEAKERS

► KAPIL BHARTI, PHD, NATIONAL INSTITUTES OF HEALTH (NIH)

► SHELDON S. MILLER, PHD, NATIONAL INSTITUTES OF HEALTH (NIH)

► JEFFREY H. STERN, PHD, MD, NEURAL STEM CELL INSTITUTE



Start with this first slide is this schematic diagram in some very brief outline for the initial of nids your cell based clinical trials and for today. when i'm going to focus on is this as a starting point for this discussion is these highly purified differentiate rpe cells that we've been able to develop as they exist in intact i'm layers and what i want to consider in this presentation. how we authenticate those cells to determine if their functioning as normal epithelium what is your standard is a long effort in primary cultures of human she'll being a whole variety of other systems as well in the second to mark the heartaches gonna do the heavy lifting i think because he's going to discuss the transformation of these are the creation and gmp cells from me sells the use of these cells on i'm scaffolds and and their transplantation into an animal model of the disease from a readily generations. so that will be the second talk so the first thing i want to say is a few background things about the features of the retinal pigment epithelial cell itself and access at least as three different kinds of cells in the first place it's a it's a matter of hours and its legacy choosing the rides during the day to come tonight and maintain june 30 sep. thigh functioning over many decades of your life the second function has to do with visual cycle and then grow more vitamin e selenium plays a very critical role in the recycling of this vitamin a and the maintenance of this visual cycle and assertive role is just based on the fact that the run the ceiling of the photo receptors real relationship and by virtue of the relationship the red carpet manipulate controls the homeostasis of the cell itself and its wrongs with input from the red curry. so what i want to start is with the cell lines that we've created from four different donors is about 15 lines obviously an issue to talk we don't have time to go through all of them so i just want to focus on one pair one of the day one of her doing ourselves from is fetal i the second 1502 shoot second owner some adult adult don't inform each other stations we're going to compare and drive. I guess ok i.e from itself vs cornea and one of the conclusions is gonna come out of this is that the variance between donors in this case between the fetal and adult donor its larger significantly larger than the variance across different tissues from that same donor that's a real income come up again and again. at how do we are sending out of missus indication process consists of city obvious points four seasons structural analysis 2nd a similar analysis of gene expression incriminatory expression and it's a dysfunctional we don't have time to go through outings functional characteristics. so i'm just gonna focus on the road transport electrocardiography and the calcium input and just skip over the imaging in the diagnosis the point here is easier transmission electron micro graphs the study points to note a vehicle prophecies well described in human while developing huge deal rpi already sold sorry located and you can see space allowed only located. so that speaks to the boys nation the cells and as a very close similarity between these cells ips cells ipscs cells derived from the dog the other team the other point i want to make with regard to this scanning blocks. Em scanning what picture of the ip which is a very high resolution shows you again the nucleus but the important point is the elaboration of the able to see what that conveys in these cells gives these shows enormous surface to volume ratio and allows him to join the board also had some nutrients the red knight equity and the other direction as well as. A new set of chemistry and biochemistry top-line shares you need a means to chemistry for him in fear of being raped cool located and sweet basil ecology and it's time for individual visual cycle that's critical cycle and other one simple word melons insist so the main take-home point here is that the representation of these these critical proteins are very similar in all the lines gonna show and important point is just just that and not that you can differentiate from one room to another by this using this kind of data. so i can now i want to shift from a structural considerations considerations of expression and gene expression in this first slide is a plot of the expression of $165 be so-called are being seen as a function of developmental genes in the first point to note any sense and so the comparisons made across all 15 lines first point to note is what is the definition of the army gene signature so that is a transcription analysis of fidelity one rbi double herein are keen along with native and primary cultures of p and and these are the least 763 genes expressed at a level that life after 10 exceeds expression in those jeans you know in a whole set of transcripts. those 80 or so from cells throughout the body so this the end the expression levels tenfold higher than all those other cells so it's kind of data allows you to distinguish the rpe from all of our cells in the body. that's a major statement what you see is an expression levels are at least as high density control are being in some even hot wire so that tells you these rhymes so potentially well-suited for august trial and and and some of them at the same time were clearly distinguish from all the cells in the body than those that this in a second. I'm comparison use a set of 13 differentiation associated my cronies there since my carney that are stem cell associated and what we find compared to the control micro gop is that they are closely culture clustered around so that we know that the micronesia. also in adult human are every similar to these if you love me so therefore all of these lines show an appropriate expression of my crime he's so they wouldn't be invaluable for you sooner targets child in need do in to see outlives that would be directed i'd like to shift to some functional assays as i can be for electrocardiography and future its word and once. I want i want to start this he said i need yes ok i remember hearing this ringing the point is that what this with this diagram shows you is a set of proteins function the self so for example membrane receptors alphanumerical receptors dopa mine receptors a whole set of others so the purpose in-this discussion. we just can't focus on these periodic receptors that can deactivated for example by atp and all the examples we in terms of that particular receptor by what you see from me that all of these receptors are coupled to consent approaching turns out to be very important to get across from the rain inside of the tissue to the corr ado side of the tissue into one of the points. I want to make with the slide is that these specifically cellular function shave read out in the intact i and so these are signals it come from taxing when i as a result of the activation of the of these the singles net showing slides over the next couple slides were going to activators when you see activation the light induced activation of activity in his cage and the activation of this receptor with atp so for example activation of this cage channel is what generates the same wave of electroencephalogram. which is commonly used to measure functional renal function chance of cftr which in cystic fibrosis trans membrane conductance regulator that that could channel instead jimmy so-called fast oscillation in the clinically observed a link to a kilogram in the third channel which is a calcium-activated shells which generates the so-called like peak. which are also be these are our measurements so i won't start to slide just by focusing on what happens with potassium in the intact i when i primates passing conservation and some space between the borders is about 5 you more when you flash a light that concentration drops of 5 to 12 million more in social purpose just need a step change in the s:p 500 million dollar and so i won't tell you what happens when we do that first so we need to dads driving force for potassium out of the cell in selling hyper polarizing by 2300 both know how do we make sure that there was your experience right herewith some liquor lectured in the cell recognition the apical membrane potential which is about 50 to 60 votes to get it the bases side is somewhat less but still inside negative and really sensitive measure is the trans epithelial potential because it's it's a difference between the small difference between these two large measurements and third national that's made reservation the alabaster membrane resistance and all of these resistances. can be eventually calculated and this is electrical characteristics of the tissue and so in his first experiment make the step change in potassium dark to light and the testing drops to space when you see a drop in the apical basal potential hyper polarization sorry of both of those membrane potentials end result is a very large increasing productivity potential and we'll talk more about that in the next like we were going to look at all the lines but the second thing that happens we did you activate is that when you do that with atp. in a case what happens here is a very different set of events activation of this receptor causes ipc to be generated which activates receptor in particular which increases calcium the cell which activates described blocks as potassium channel in both of these events are depolarizing events and you see that electrical response so that is something that comes up in going to be shown on this site. so now we making seen comparisons again one donor is it you don't don't don't don't we look at two kinds of dr tissues when he went from corneal we compare those responses and basically what comes out of this is the notion that the variance between landowners between two different donor sis slicing. The variance that you would get if he's different tissues from the same to her that's an important outcome this now i want to shift to a different kind of physiological measurements which is atp induced region support across. he may not be so jin we start with the sort of run diagrammed when you see the new kaskade cassette approaches at the apical and alabaster membrane that drive through it across the apical to alabaster side the tissue to the blood and all of these proteins are involved in all the pathways that are connected to them and technically in a kind of measurement. what happens is the fluid as he leaves the apical side level drops in his capacity proposed can measure that drop and gets to the base aside the level rises and tsunami in the nearly two per unit range the outcome of this nation is shown here being too fluid from the debate a side. its mission to make leading scorer senator hour and positive values for movement in that direction and what you see is a significant increase fluid transport across the epithelium the activation of the receptor on the apical membrane and that in this event is reversible and that's all of that is as again covered in this comparison between fetal donors and an adult donors for the same exact measurements. so i won't take obviously this is a kind of work that goes across all the donors in all the clones about this he was representation of the data so all you have to do understand what's going on is first of all understand did you read the red color are those clones those clone scan use to resemble pilar pia pilar pia phenotype and the blue nose at least that phenotype and you when you get an overall view you just go into this plan summary run and those assertions that are red indicate lines would be potentially useful for autonomous trials. because they have a very close similarity to allow don't harpy another point to note the sparks of physiological jan supporters of physiological events here is very is it can distinguish very well ryan said. I would be appropriate at all of this used versus those that are not in the way to notice that is just look at this set of responses here and see how much blue you find going down you're always places that you see a roof lines that you wouldn't change as a first choice for transplantation contrasting red lines which would which would be so stop with lap and just pick me make the conclusions that the show the as i said that the present experiments provide a kind of structure electrocardiography criteria for determining the ipsw suitability for a target target such transplants and provide some limits. in terms of actual responses in the second point is for these 15 ipsw lines you know orientation doing this is is much larger than the donor or so this suggests that the geneticist differences between different tissues is is is not it's not a problem for is not recognized by these measurements. is not a problem is that decries deviations in these measurements third point is a general rule that has to do with all of the mountains they'reconnected to these signaling pathways and and and that are activated and sent a letter and all of this program together is what maintains homeostasis of the rpe length input from the court. the renault and in this is stations is made and maintained to alterations in darkness and light so this goes to the question functionality of these measurements and finally the last point is what i just showed you in that heat map and that the set of physiological assets are particularly important they provide a necessary measure of rpi fantasy for thrones that were that came out in the scale of it and i've read in the last 142012 obviously not used to i'm gonna stop there and just people that did the experiments which sharma have led kristopher and she and shake her haha john did all the use of chemistry kinds of the expression data change. in iran did the electrocardiography stopped working and i just john joshua joshua mediation did the calcium work why did all the cell culture he does did this game like you and i metro lines and they were made in selling stop there i'll be talking to them about how we are approaching towards developing an tautologous cell tower before amd using ips cells derived army patch. so i have a nice introduction by andy just to briefly betrayed that this is a disease that affects patients and regions or whatever they're looking in the center part of which they are pretty much blind to victor large population of about 30 million people worldwide suffering from amd and its a huge unmet medical need billion dollars per year taking care of these patients there's no real treatment for this disease and of course there's no cure the idea here is that these people are are blind in the central part of the region and that's because if were captor cells that's the light-sensitive cells. which had an indisputable and these cells die for receptor cells die because the rpe leathers layer of this pigmented epithelium cells situation in the previous talk about his dad and in the center part of the ice and and the reason is that the death of our pieces for us because our be spends all its life taking care of receptor function and held and is like kind of nicely illustrates what really happens in mt patient's. you can see this is what we called how these on where the rpe and the poor receptors are still alive so that towards the periphery of the eye as we move towards the center as we move towards the center you see this area what is called a transition zone we are b cells are dead but before receptors are still alive. but probably not fully functional and they're about to die and that's because i support functions support layer is missing and enter the ip layer is gone and of course as long gone and for receptor cells so the idea is that people have called for a long time and jeff mentioned that you put in a layer of rpe cell. here and you hope that would protect these receptor sin the transition zone from from dying and you would protect at least region from declining in this area and as again jeff mentioned that people have tried this approach cleaning so you can leave for a long time. they basically transplanting and tautologous patch from the penny from from from this house apart into the market and have seen that in cases where the sou successful they could protect vision loss in those vision and for us that's the guiding principle for the approach we're taking which is now looking at all of this patch for amputations. so the idea is you start with a named patient somatic tissue blood or fiberglass you reprogram those using these new programming factors into ips cells differentiated ips cells into rbw patch and treat those patients back into the same patients back within our own selves and i want to mention that it's an important point as you saw the rpe cells work as a polarized moralistic is important that we transplant them as apple as morally to get most african. see all of them so there will be a patch is critical and again back to have mentioned you know and that also allows you to make mature cells that can right away stop functioning was transplanted in the eye and i shoes completely filled out that we have been able to demonstrate that in fig on a pig model set matches actually function so just the take home message here is that we actually have deliberate streamline manufacturing process you'll be in ga and manufacturing process we're basically starting from day 0 all the way when we draw blood from amd patients to making clinical product from them and transplanting it back into them it takes 142 days so little later on for a half months will optimize everything those step of the process starting from making cd for positive cells to prospecting and making many banks of ips cells to setting up the position and getting pure rpe cells putting them on a scaffold for making the clinical patch again so you can imagine a process that is such long we need to have in process quality controls and of course we have been able to set up all the politicking externally to measuring still in the process of checking the quality of ips cells in the asses and of course. I don't be off the scaffolds and at the same time if you have such a long cross as you don't want you to fail and if it does fail you don't want to go back to the patient ought to the beginning of the process of we have been able to institute crowd preservation steps to all the process the patient material intermediate stages and my favorite is the last page which is a clinical product we have been able to preserve intact rpg patch. you can imagine the land a job having a patch that means we just age when we want to centralize manufacturing and be able to ship the patch anywhere in the world we should be able to do that is to just use some background process runs so it's a very good mentally guided process the differentiation of rpe cells from ips cells follows different stages of differentiation. as to normal cells do in an embryo cells go from ips cells tonier acted on them to argue progenitor all the way to maturity stage and we we do that by just coaxing them gently by user previous growth factors at different stages. the cells and are being majority in you saw some data in the peace talks from sheldon allergies are more data off rrp patches going on scaffolds and how we characterize their functionality so needless to say that we do get this pure ip cultures at a very very high efficiency we can easily get 98% yourselves as an an again an important feature off making these cells from ips cells is that the efficiency is too high we don't need to passage of ourselves too much since. its apical cells they do not like to be passage sortie of passage you know we call we can put them on scaffolds and we get the best originating marseilles mentally laid down ourselves being characterized as fully rpe cells so we can make yourselves we put them on scaffolds this is an example of rpe cells going on a scaffold which as you can see the scaffold by percy its appeal gb's hold its biodegradable and what time it degrades away and the whole tissue integrates into the eye of animals and of course. will do for patients and you can see in this tms the cells make this a beagle process like chill in talk about to have a typically located between neighboring cells and a very interesting and important feature of rpe cells is busily unfolding scenario. where he sells which you never see when you grow cells on plastic dishes on paula story but when we go this cells on plga patches we can see they sell stock to make me as a simple things so they have much more native like structural properties as compared to cells grown up on a plastic dish. I won't go into the details of this food transport a substantial and spend some time explaining how disaster works so the bottom line is that i ps3 barbie grown on this on our own patches on scaffold touches are electrically tight and are able to put forward as they should in a native are free so and then i'm gonna shoot data from two different animal models and compare the two animal models. because i think this is an important come back as soon as we're moving forward to the first animal model is like these rats have a magician and in one gene called motivate its receptor which is responsible for figures they told us of our assignments so member from interdiction children talk with these two segments are segments and that's part of their function so since these rats the are being these rats was not able to. because i told all the segments therefore this episode 2 degenerated and what time they die and this can be measured by the optimistically tracking so basically put the ride on a platform and we will still end up with black and white stripes on it and if the rad can see. it will track the stripes and plaid cans can see that it randomly move ahead on elections of course we can see this is in line for that are seen as rad as redness out to be generated slowly gets planning you can see the region is declining we inject ourselves in this case. we injecting cell suspension or transplanting the patch we just injecting cells at day 28 postnatal we look at them they 16 d90 and as you can see by day ninety these cells have been able to protect almost as close to all-time region in his words if you look closely into the into the eye of his residence as a histology off the whole i have staining you can clearly tell with his injection sites and this is where the injection was done. because the right here you see the right now has two different nuclear layer and hear both nuclei intact with us on this side with injection was and then there's only one nuclear they left the other has already generated so this site has been has been protected by the injection of ips are be a few min to that i wanna play now this very important feature so. you can see these are my ip is our b cells labeled with human marker and an rbi marker these cells do not form a mono layer these our self suspensions are they just going and aggregate but despite that they're able to protect. this will receptors right on top and you can see this protection is almost around the area where themselves are surviving if you move further away from the site of injections the production decreases so there's a temporary protection provided by these cells as sitting in the sub retinal space and other labs have shown that this injecting the sub retinal space actually do not survive long-term. because as a clam immune cells invade and the eatables cells and transplant them as a mono layer they would survive much more long term and and be able to predict which models and going back to that approach is again for amd patients with advanced planning erp package and be able to protect his voters so far that. we developed and other animal model which is a larger than a pig obedience wheeze and one is at the peak i size and anatomy is what is similar to mine i size to me so we wanted to double up surgical procedure test or transplantation to and the patch that. we're gonna transplanting human guinea pigs and be able to transfer all of that procedure from pig to human and have over so jones 39 pig so that they are ready for transplantation but since in pics we do not have a genetically defect genetic model of emdr rrp originally generation we developed a laser induce dare p injury models are basically the idea is we shine micro pulse laser in the back of the eye and that kills on the rpe cells. because the laser is absorbed by the pigment in rpe cells and you can see that by this window the fact that is seen here by incorporating againig. if you look at that by histology i cells clump and they die off and as a result for receptor started off to the goal is here now that we put our fb page right here and that should protect these were separated on top of the patch and this is the movie transplanting. the ip patching the pig model so generous preppy i was making their decision bigger so he can put the candle ah read the army patch in the eye and you can see his a candlelight tape this is erp patch like appalachia patches. but two-by-four millimeter patch this is a cut and the retina and this is a blog under the red now where he's going to go and place and come with a candlelight that has the irb patch on a scaffold as a gop great ip s rbe on the scuffle incision is gonna go underneath the right now and transplant the patch and their egos and sometimes it takes to the kind of comes out. but it has enough physical standards under the right now and in the redness flattened and the pats days and then the good thing with the eye is that we can follow this patch for weeks and is to follow and this is done by a multiple techniques i'm showing. you one example of which is based imaging of the back of the eye you can see the patch on fossil you and then in a cut we hear from oct is sitting right in the need the right now flat patch and the right now on top of it is protected you can tell that by this method which is called multi focal basically. the measuring electrical signals coming from the back of the eye but by shining multiple light pulses to the back of the eye so depending on the light is gonna shine the light shines on the healthy part of the eye when you get-this nice robust response. now if the light shines on the laser injured part of the eye you got no response and when the light shines on the transplanted part in the class landed part has protected the injured by you should get a response in between and that's exactly what we get to you. so this is a positive control in a negative control all within the same i response no response and transponders so that we can tell that these patches able to protect the right now from dying and last thing i want to show you is jeff talk badly about how do these cells that she would survive in a patient and nobody knows what will happen in a patient. but we're able to do some experiments to see how to be sexy survival of patients books memory so we are able to work with a collaborator towork to get any patients books membrane and and see themselves on that on that membrane and see over the course of three weeks all these cells are like oh this is a comparison of ips are be from our lab human dna previously published srp and you can see neither of these have actually survived on this book's main the cells have died and clamped over three weeks. whereas i piss rpe cells are able to remodel patients brooks membrane and and be able to nicely survived an attachment and go on this so that one out thank the entire team this is a huge undertaking there's a lot of people in walled shelling targeted many of these people who have worked in establishing the protocol and now there's a big team working to transfer all of this into gmp facility. we teamed up with the company's cdi in a lot of people from the company were involved in doing the gop optimization we have a number of surgeons and indeed experts working on the right patients and nutrition analysis and surgeries and then of course you saw a lot of people working on rats and pigs so that elected bank and taking questions i'll be talking about a primary rpe stem cell derived proxy for transplantation regeneration. wonderful introduction video and sheldon so i'm going to try to go quickly through this we've long known that our pe removed from human donor globe can proliferate what we found more recently is that operation occurs in clothes as a sub population of human rpe cells that sell for new which is the defining feature of a stem cell rpgs. them so we demonstrated this in various ways rpsc proliferate but also differentiate and they're poised to produce rpe progeny that have the characteristics of needed for p that sheldon and capri liberty described indeed. they've helped us identify these bone file or genuine or peace so most of my talks about transplanting these 4 a.m. the two that an important characteristic for durable and effective or pc replacement therapy is that the transplanted cells into parade and survive we've been looking at cfb labeled rpe drive i'm sorry rpsc droid arpey sprinkled onto existing human rpe model heirs and find local images good integration into the orp layer that is stable over time this allows us to study the factors that influence integration itself and stability figure psychosis is some major function of the rpd particularly. the rcs rat model used by the fda for demonstrating efficacy here rpsc dr dr peter shown taking up the screen labeled rod outer segment for a glimpse at a high rate of these ips arrived are p from the scleroses of the same patients that the rpsc were taken also figure 2 cyto sport to it to lesser degree our transplant studies are based on on this principle that we differentiate the cells for two weeks for weeks or eight weeks when mature cobblestone epithelium begins to appear and transplant those various stage sunder the right now of the rcs 400 dose of 50.000 selves we see integration here human nuclear marker expressed in the rat retina seventy days after transplant integrated apparently this is so tx rpe marker that's human biased and mitf and best one are also human specific rpe cell markers demonstrating survival and the integration of the rpi into the red or later we do see occasional clumps of cells as well. but these are rare most of the runners covered by these integrated so and as okapi so nicely demonstrated the outer nuclear layer representing the photo receptor cells remains near the clubs but also away from the club's this is slower and choroid with erp. this is a detached retina from another area sometimes the right now attaches. when we make these sections in a journey of a normal round you can see the photo receptor layer over nuclear layer degrade after two months-to a reason were only fragments of the photo receptor cells or or preserve after transplant here again illustrated with the outer nuclear layer survives nicely even out to work i think 180 days will be on 280 days before week a week or seven weeks sorry indication here are the is the time. these cells were differentiated in the rpd line a how much are they were both mature and intermediates of differentiation rescue photo receptor nuclear power circles form orphologic rescues stage independent. we're also measuring the outer nuclear layer with oct just to show that this injection site a large number of retinais rescued by a single injection there will be a few clumps scattered here but mostly these are normal appearing rpe mono layers in these high-level vision rescue is dependent. we use ok she showing bar spinning that the rapids reflexively following and the boys get thinner and thinner. we describe that's partial frequency angle as the cutie that the rat sees early all on at 30 in the rcs rat they see quite well near wild-type ip ninety two months later they're cuties dropped markedly the blue line and the red line represents this if we interact with selling no change for a little change if we do it with two week differentiated rpsc dr rpe very little change three weeks. we start seeing effect for weeks and five weeks back to optimal and then it drops off again so highly mature rpg modelers dissociated injected or less significantly less effective and again as i mentioned earlier this is in retrospect expected from a long history of studies of other cell types or less differentiated developmentally younger tissue integrates and repairs were more mature oligonucleotide and neurons. when transplanted into the brain do not i think this is a key results when considering which cell transplant. what is its developmental age because efficacy and vision rescue car leads with stage of differentiation the seller transplanting in most tissues in many tissues that's true and then we we know. it to be true in the rpe certainly go back the difference between the two week and the four-week cell is really very minimal genetics through the same for nearly the same except for two weeks of differentiation and defined media so it's easy to compare the two week in four weeks lol echo early to find out what the differences are that correlate with the casino business rescue. we started doing that by obtaining are they seek from our cells which segregates into three groups early not efficacious cell sat the case result of cases of vision rescue and the later cells that are also not early dad a single single samples single time points for each. but there is one very interesting molecule that segregated with efficacy that we pursued and confirmed using pcr and we call it transient rescue expression factor t-rex correlates with applications rpe two weeks earlier where it's not application expression of the small occurs down and later its expression also declines this is a long non-coding rna that we are exploring the mechanism how this is involved in integration and vision rescue perhaps and development began early early experiences that they would find phone but i digress and we'll get back to the transport discussion. we are very grateful to the newyork stem cell or for funding ind enabling work and we are in the process of gmp manufacture animal advocacy experiments definitive once similar to the ones that were described farm talks is just starting a pilot is complete and vehicle design for an ind because we see more efficacy were applying to the fda to target in earlier-stage very eloquently described the zone where r p soldier lost reporters abuse have not died yet we're going earlier stage of intermediate back to you where there isn't lost their atrophy geographic a truce not progressed and this will be enough phase. one to a single site study in collaboration with kellogg they were planning for 2017 so are our role and create your different than other studies were looking vision loss modeling of rpe changes in the rpe layer preservation of macular thickness on clinical oct or excited to get to the store so in summary for the transplant comparison of our will group embryonic stem cells ips together concern for tumor formation forces extensive differentiation for safety reasons ourselves or less the rpc themselves less allowing transportation of younger stages of differentiation which have more efficacy of immune suppression ips couple is meant to do. what i guess there's also some potential or so so much less expensive only about five billion after two passages which were using for 500 pages from a single preparations singalong but the obvious difference between the rpg stem cells and the other sort is that the erp systems already present in the army so we take them out or remove them spend them back and and i'm already a surgeon have the time and just talk for a minute. about the possibility that we could activate these cells without removing them or putting them back so brutal surgery i think you've identified the surgeon who can do them all day and there are few but not many takes a lot of skill pieces go bad operating rooms have to be booked with teams with intracranial injection is much easier chiffer in-office procedure which we are targeting. so we're talking talk to three brief about transportation vs endogenous activation scoring a factor into the eye to very gently tickle these cells into proliferating 12 percent of them dividing once or twice would counteract that decade of war peace a loss about 0.5% of our pieces per year in typical triangulation. we work with fcf to stem leaves his small plga controlled-release growth factor be there were originally developed for spinal cord injury. I see some people interesting optic nerve some advocates here for spinal cord and we don't the masonic a drug release over a two-week period but now are putting various growth factors into these same controlled-release speeds and injecting them into the i this is our home sales of male side that have been injected with the fgf to be its forces not staying for ky 67 a marker of proliferation. you can see that some of the knee injury true or peculiar is activated to pull up for it but not without the beads in the control unattractive animal very little proliferation occurs in the harpy layers rather. yes from six weeks of gestation with the beach we can stimulate 5% up to 5% division now people have known this for a long time scientists like will ferrell many others have injected growth factors to stimulate our people information but i think that the new part is that these growth factors are very labile i'm back in the day large amounts had to be injected in order to stimulate proliferation massive quantities really and very high levels of these cytokines have off-target effects such as angiogenesis effort to stem beads. allow tight control of levels inside the debris gently tickle rpg with stimulating other pathways and others finish on this section of a male side that was injected with stem bees and other than very mild changes in our pe layer we see no into genesis from the choroid iris is free of angiogenesis. I will say my thanks to the group of neural stem cells to 230 scientists in the small independent institute led by sally temple again to new york state for funding the retinal stem cell consortium in rochester for cornell campus key maker measurements. Sylvia phenomenon expert in phagocytes thank you get out hope for organizing this.

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