Channel: Case Western Reserve University clear
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| 1 | Case Western Reserve University | Anthony Wynshaw-Boris: Chromosome Therapy - Rescue of Ring Chromosomes... | 676 | 4 | 44.3 | 25:05 | first of all I'd like to thank Alan for organizing this wonderful Symposium uh and for our guests for coming all this way to give wonderful talks thanks so I'm going to talk today about a um novel system uh for abnormal chromosome correction that we uncovered uh in human cells and that is ring chromosome correction during induced furry potent stem cell reprogramming and as you can imagine this wasn't something that we set out to investigate although I have't studied uh genome instability in the past but we stumbled upon it when we were studying this disorder this human Disorder so now we're going to get away from yeast a little bit I think Stan's probably going to talk a little about human as well next we're studying this disorder we're studying for many years listen which is uh called which is refers to the smooth brain as you can imagine although the brains are very similar in organization brains miss the the normal gy and susai of primate development and so the children that have this have severe mental retardation seizures and early death there are many forms of lenuo but one of the most severe is Miller deaker syndrome and that results in a completely ayic brain here shown in this MRI and some recognizable cranofacial dysmorphisms at a medical geneticist of which G one could easily recognize all children with Miller deaker Syndrome have about at least a one megabase deletion on the tip of chromosome 17p that includes more than 20 genes including a gene called Lis one uh and although I'm not going to talk much about Evolution since evolution is the title of the seminar as well I just want to point out that we knew nothing about what list one did until its function was uncovered in the bread mold Aspirus nigin and was found that it interacts with dining and important for dining regul and function so that's my little uh forent Evolution for my talk so uh what we wanted to do although we found a lot out about the phenotypes and the mechanism of action of Liss one using Mouse models we were interested in trying to make human cellular models of this disorder and so in this case we're taking advantage of the recently described and now ubiquitous method of induced furry poent stem cell Generation by cellular reprogramming and as you all know this was developed by shamanaka who won the Nobel Prize for this several years ago and the the concept is you take a skin cell or any other cell from a patient and by uh transfecting uh transiently cells with four to six transcription and other factors those skin fiber blast will be reprogrammed into Pur poent stem cells similar to es cells and these cells can then be studied differentiated in vitro to any sort of cell type that one wants to study let's say again the disease is listen then one want would want to make brain cells study those cells and see what can go wrong for cellular disease model in addition if the genetic defect is corrected it could also be used for cell therapy in the patient if there is a Cell Therapy modality for the disorder so Marina burin a postto in my lab when I was at UCSF decided that she wanted to make these models and so what Marina did was to take three different patient cell lines with Miller deer syndrome and being interested in the history of Miller deer syndrome we wanted to reprogram this patient's line this patient was uh uh was uh born in the 1980s and a kype analysis indicated that the child had ring chromosome 17 and this is the first indication that any could be wrong on 17 for Miller deaker syndrome and so uh a number of other investigators then looked to see in uh other patients with Miller deer syndrome whether there was something wrong on chromosome 17 and in fact all patients had a deletion of the pr arm of chromosome 17 so this patient LED then the to the identification of the genetic cause of Miller deer syndrome these heterozygous deletions of 17p so Marina reprogrammed fber blast with a ring chromosome patient and two typically deleted patients first of all let me tell you a little about Ring chromosomes they form when um the PIR that normally protect the ends of the chromosome are lost or activated allowing a chromosome to form uh a ring and this could then result in both genetic and structural problems structural problems because ring chromosomes are diff difficult to uh uh undergo Division and uh genetic problems if there's any loss at either end as there is in Miller deer syndrome resulting in hin sufficiency in those problems so the Inus is quite rare but it can occur in every chromosome and there are human beings that have ring chromosomes in all of the autosomes as well as the X chromosome that have been described so these are known now one of the things that happens is that uh there's unstable behavior of ring chromosomes during mitosis there's a failure to pair with homologous chromosomes frequent wiing loss leading to monosomy there can be the formation of dcentric rings secondary ring derivatives and the formation of anaphase Bridges leading to genomic instability and because of that there's an increased rate of anupy and high cellular death rate in cells and culture in the patients themselves in fact patients that have no deletion resulting in any other abnormal phenotype almost always have small size so growth rate is reduced in patients to have ring chromosomes and that's almost Universal so we were able to collaborate in San Francisco with the the yamanaka lab and Marina was able to reprogram the patient fiber Blast from each of the three lines one with the ring and two typically deleted lines into uh IPS cells and those were proven to be I IPS cells because the cells were all able to make uh the endogenous factors that indicate that their IPs cells listed here also by inv vitro and inv Vivo methods were able to show that all the cells could uh contribute to all three germ layers maderm endoderm and ectoderm so in fact these cells were Pur potent the ring chromosome line as well as the two typically deleted lines all reprogrammed at about the same frequency and so when Marina first uh examined the fiber blast of course the ring chromosome as well as the typically deleted lines all had a reduction in both Lis one mRNA the gene that's at the edge of the deletion as well as another Gene coding for 1433 Epsilon ywh Hae in 50% levels and the fiberblast also had 50% levels of DNA however after reprogramming although the typically deleted lines had 50% reduction in list one genomic DNA the ring chromosome lines now had a normal level of Lis one genomic DNA they had the normal of list one mRNA and they also had uh Rescue of the to to normal levels of production of list one and 1433 Epsilon so in the fiber blast here's the level of wild type list one and 1433 Epsilon the typically the leaded lines and the ring chromosome line had 50% reduction in those proteins however after uh IPS formation that the typically deleted lines had 50% reduction those proteins but the ring chromosome line now had again correction to normal levels of these proteins and so the reason that that occurred this is published so I'm just going to summarize some of the data really quickly is that the ring chromosome that was in the fiber blast after reprogramming in several different clones the ring chromosome was lost and replaced by a completely normal chromosome 17 so that there seem to be two identical copies there and this uh occurred sometime around pass D this would just show the frequency of the Ring chromosome kot type in in doing kyp analysis in the fibr blast at two different passages and you can see that almost all the cells have the ring chromosome phenotype uh in after reprogramming the majority of the cells the vast majority of the cells now had a normal 46xy kyte so how did this correction occur or can we look at it by other molecular methods so what Marina did was use snip genotyping to look at copy number variants the ring chromosome fiber blast this would be all of chromosome 17 the P arm the croome the Q arm and this uh cluster of salmon colored dots shows that there's a a 50% uh deletion at the tip of chromosome 7 17p in the ring 17 fiberblast that was now corrected after uh the uh uh uh in the in the IPS cells that were made from there and IPS CS were made from the two typically deleted lines and they maintain their deletion again showed by the salmon colored region at the tip of 17p it's an important experiment because one possibility is that there are genes in this region when deleted would not allow reprogramming to occur but in fact since they were able to be reprogrammed that suggests that that's not the case in fact the deletion in this IP mds3 line is very similar in extent to the ring chromosome line so it doesn't seem that there's a genetic cause for this as well so Marina then looked tried to think about what potential mechanisms could have occurred and two occurred to us that were easy to test one is in the original cell with the ring chromosome there would be loss of the Ring remember I told you there's anupy in the ring chromosome lines and impatients and then by uh sematic non-disjunction there would be complete duplication of the uh uh normal chromosome another possibility is that there will be a double strand break occurring in the ring chromosome and after mitotic uh homologus or combination there would be then repair of the tip of the chromosome using the other normal chromosome as a template this is relatively easy to test especially with the snip array that we had before because in this case the entire chromosome would be homozygote because it would be duplicated and in this case only the tip of the chromosome would be now homozygote the rest of the chromosome would be heterozy we could use the snip genotypes that I showed you in the previous slide to test that out so here just shows the results from chromosome 177 uh and this would be from the fiberblast uh in the ring chromosome patient uh the the representation here is for the three potential genotypes so Snips are either A or B you could be heterozygous AB homozygous AA or homozygous BB and in the ring fibroblast line there was a mixture of all three genotypes in the IPS lines deriv from The Ring chromosome patient now they're completely homozygote either AA or BB while is while in the two different typically deleted lines that were made into IPS CS though those IPS C had a similar distribution of heterozygous and homozygous genotypes those patterns were also seen in every other chromosome in the IPS cells from The Ring chromosome line of the fiber blasts or from the typically deleted patients so it seems then like the mechanism of uh rescue is that in the fiber blast where there's a ring chromosome during reprogramming there's loss of the abnormal ring chromosome and duplication of the normal chromosome to restore a normal genotype uh the same mechanism works for other chromosomes so in this case we looked at ring 13 two different patient lines and again the fiber blast had ring 13 and multiple IPS clones derived from those fiber blasts from two different patients with two different Rings now had normal kype and normal uh distribution of of genes and by using the snip genotype that I showed you before also rescued by uniparental isodisomy so uh in summary then for this part of the talk there's ring chromosome correction in IPS cells that involves non-disjunction ring chromosome loss compensatory uniparental isodisomy and selection so what we think happens is that occasionally a cell loses the ring chromosome as it's going from fiber blast to the very proliferative State that's occurring during reprogramming and an early passage then if some of those cells lose the ring they may die but at later stages when the ring chromosome is lost and then reduplicating the other chromosome those cells will then be selected and after time either the ring chromosomes are lost or or the normal kot type is now over overgrown in the cells and it's again due to unial isod only so you can correct then the ring chromosome defect in fiberblast during reprogramming uh into induced Flur poent stem cells so again the Miller deer syndrome can be modeled uh ring chromosomes are frequently lost in IPS cells although I only only showed you information for ring 13 and ring 17 others have modeled tried to model ring 14 and ring uh uh 21 and they've also found similar uh uh phenotypes in that the the unipal isodisomy rescues those ring chromosomes the duplication of the wild type chromosome rescues monomi and compensates for the ring loss contemp cons compensatory uniparental isod diomi restores the normal kype and the normal copy number of deleted genes we think dynamic mosaicism in the IPS cells leads the preferential survival of the kot typically normal cells so that's all well and good for the 150,000 patients that have uh that have ring chromosomes but we're interested I'm a human geneticist and seeing this may have broader applicability it turns out that uh you may be aware that chromosomal abnormalities are actually quite frequent in about 50 60% of spontaneous miscarriages uh 4 to 11% of still births 5 to 7% of neonatal deaths half a% of live births and of course low frequency of ring chromosomes are found so it's actually quite a significant uh abnormality throughout the lifespan uh now if you think about correcting a varieties mutations in cells for for the purposes of uh of uh of uh regenerative medicine strategies then one way to do this if you have just point mutations is that you could take the somatic cells with the disease genome that is small deletions or point mutations reprogram those cells with the disease genome and IPS cells and then correct the disease genome uh with a variety of different genome editing tools such as crisper or tailin and now one will have mutation corrected IPS cells to give back to the patients um but in the case of of of chromosome aberration such as Miller deer syndrome how might you do that and so what we're thinking one could do is perhaps uh if a cell has large deletion or of course ring chromosomes that we shown then perhaps during reprogramming uh and selection and screening you can correct the IPS cells if you make the large deletions into ring chromosomes and then the ring chromosomes will either be lost during reprogramming or during the propagation of those cells and of course there's a lot of problems before we could ever consider using this and I'm not even attempting to say when we do this this is just a thought experiment at the moment because there are a lot of uh issues that might result in in problems such as genome instability imprinting or recessive mutations but I think some of those can be corrected at least by uh the genome editing and so one example I don't think tun's here but tahun Kim is a postto in my lab that's trying to do this he's trying to use genome editing to insert locks P cassettes into the ends of a chromosome that's deleted in this case the Miller deer syndrome chromosome region the cassettes uh will contain are containing a fluoresence marker as well as a cdna for another fluoresence marker TD tomato on the other chromosome there's a fluorescence marker and the the um promoter an atg for the TD tomato construct these are locks P sites that are also introduced in the same orientation on each end of the chromosome in two genes that appear to be uh uh not responsible for hin sufficiency so once those are introduced then tahan's introducing cre combines with the thought that if cre combin a will properly Rec combine and form a ring chromosome these cells will then Express TD tomato and these can be selected even if it's a rare event so tahon has gone through all this experiment he actually has has some TD tomato positive cells now that look like they're promising candidates for having a ring chromosome the problem is that they're pretty low in frequency so we're trying to find ways to purify those cells that we can do carot type make sure they do in fact carry the ring chromosome and then what's going to happen is if he does have cells that are TD tomato positive generated from the locks P insertions a cyan and cyan and Venus positive cell that now becomes a TD tomato tomato positive ring chromosome containing cell then further propagation will result in the loss of that ring chromosome loss of TD tomato and then perhaps duplication of the normal chromosome to rescue that phenotype now this may happen during Repro uh in propagation of the IPS cells we're hoping that that's what would occur if not we're going to have to do this correction to form the Rings in fiberblast or other sematic cells and in addition to trying to correct chromosome deletions we're also thinking to try to use this to actually model ring chromosome defects so there's some phenotypes that you might like to examine in in cellular model and one of those is ring chromosome 14 syndrome which even if there's no genes are deleted in ring chromosome 14 there seems to be a problem with gene expression on that ring and the children have very severe phenotypes particularly EP epilepsy so one would want to make a model of this but of course this correction mechanism foring chromosomes has so far not allowed this to occur so the idea would be if we can introduce lock P sites into chromosome 14 at the ends can we make an inducible model of ring 14 and then finally can we correct extra chromosome copies as alen was talking about such as tromis by perhaps again taking one of the extra copies say in triom 21 introducing lock P sites in the end forming a ring and perhaps uh growth selection will allow us to lose that ring uh and have restore a normal diploid complement to those cells in culture and the idea would be that these cells can be used either as models or perhap perhaps for regenerative medicine strategies in the future so uh again what I've tried to tell you about is there's a novel mechanism during reprogramming for the rescue of ring chromosomes we're going to try to exploit that for making disease models for rings as well as for potential cell therapy in the future and I'll just acknowledge the people did the work I pointed out Marina and tahan's major work again this is a collaboration with shinya lab particularly Yohi Hayashi and shinya lab other collaborators at UCS also helped in this project as well so thanks very much I'll be happy to answer any questions so my yes Phil destabilizes inist at right yeah and um oh keep and and were you suggesting that the reprogramming regime itself destabilizes the R over just mically propagating it's hard to say so did everybody hear the question is it is it the reprogramming itself that's causing the instability or simply the propagation of ips cells so we won't know until we try to do the experiment so the experiment that we're doing now of course is to do this ring chromosome formation in IPS cells so we think that it's the rapid proliferation of ips cells with a very very uh short cell cycle very very short G1 just they synthesize DNA and and actually then just divide so that rapid cell cycle is impeded if one has a ring chromosome and so the loss of that will then allow those cells to outcompete the other cells so we think that's one potential mechanism of how that could occur another possibility though is we make the ring chromosomes and the ring chromosomes are stable over many many generations in which case that would suggest it's due to reprogramming so if that's the case we now can make some other sematic tissues out of the IPS cells and then re- reprogram them and see if that's how that's happening but the key thing is to have a marker so cells that have the ring chromosome with TD tomato that we can fish out of a whole population and ones that are losing that so we can see how that's happening so the question is a good one hopefully we'll find out soon yes sense of how long 2us one so and can it divide or is it actually unable to divide until unless it shows some it so can how long will it take a 2 N minus one cell to divide a lot of it would depend on the chromosome presumably it will take a relatively short period of time where it can be uh 45 have a complement of 45 the exception would be the X chromosome because 45x those cells will actually can actually propagate quite quite well although not completely well um so so it may be that it's happening all at once that you lose the ring chromosome and duplicate the other chromosome it could be that the duplication is occurring first and losing the ring chromosome until we have a way to look at those cells by by sorting or other mechanisms that have a TD tomato positive cell we won't be able to address those so another possibility of course for making for doing these experiments is we can find more about the mechanism of how this is occurring and maybe even Pathways it might be responsible for the loss of the Ring chromosome yeah so if you with a ring chromosome and you partially dilize the spind with drugs do you lose the ring chromosome can you destabilize with drugs the the spindle in a way to uh change the kinetics of what's going on this so I think in general if IP in IPS cells if you slow down the cell cycle by a variety of means it's going to impede uh the plur potent state so that's what about fiber the original so they probably grow slower we didn't measure how fast that the fiber blasts are growing rather rather than other fiber blast lines because they're all primary cells and so there could be some differences there with passage um probably the more interesting question is why is this such a problem in IPS cells we can have human beings running around with ring chromosomes and I suspect it's just because one has uh so many divisions that are occurring in these cells in culture that you can't sustain ring chromosome for very very many passages uh and so they're lost in culture in a way that they're not lost in humans but that's another question that will need to be addressed hopefully we could do this in models as well is a phenotypic varability among individuals who are affected by ring chromosomes because if there is a loss of hyos would expect that you should get all sorts of things there's a lot of phenotypic variability in patients with wiing chromosomes for one it's uh it can be secondary to the loss of genetic material when the ring forms others it's because there is mosaicism among patients that have ring chromosomes so some normal cells some ring chromosome some cells in which they're breakage products a lot of it just depends upon the chromosome that's involved and as I described ring 14 it's a very interesting story because whether there's deletions or not the phen type seems to be the same there seems to be something because of the Ring 14 formation that may be silencing other genes on that ring chromosome and may be contributed to the phenotype so it depends upon the chromosome itself perhaps it depends upon what genes are brought together during the ring formation there could be position effects and it probably certainly depends upon the chromosome itself whether there's Hao insufficient chains on the ends or whether they're genes like ring 14 where there's suppression or some other mechanism that's causing that to happen yeah bill you any idea how fre DS are happening at the moment we don't and at the moment we don't we haven't tested that again I think we're going to need to purify uh in some way what's happening we can look at what happens after the ring forms by purifying those cells and then having those cells to examine later at some point I think that's what we're going to have to do okay thank you | ↗ |