10-15-2001 Transcribed by Jim Davies Jan: Uh, so ah, what I 'm going to talk about today, I'm going to give you the final data on PKG-Transfected cells, I presented the data before I set that, I did another set of clone. Just to make sure, that, um, things worked with more than one clone.You haven't seen this data before.And then I want to talk about a few other things. And I'm actually going ot start with the other things. And, because, ah, we always have this problem where, these people, I guess there actually aren't that many people who don't know the background. Maybe I can skip the background. What I did, though, for a little bit of background for the people who haven't heard what I do before, but in the upper right hand corner for you local veterans I have some local information that you can just watch while I ah, all: [laughter] Nerem: I thought maybe that local weather affected your ability to make constructs[?] Jan: Well it's a sunny day, so that's good. Nerem: Well on a sunny day you're usually out bicycle riding instead of [garbled] All: [laughter] Jan: Actually, I guess, I mean, almost nobody who doesn't know what I do but everybody knows that, except Josette, at least there's somebody.. And you're all familiar, at least you're all soon be very familiar with our construct made of muscle cells of collagen, the I work on uses rat muscle cells, the big problem that I'm addressing is the fact that these cells, when you put them in these constructs are actually when you take them the side of the body they change their phenotypes, so they change their function very dramatically. They go from what's called a contractile phenotype which is what's generally found in vivo, and that's the major, the major function of that kind of cell, is just to contract and dilate in response to the proper signals in your body, so, um, constrict and dilate. When you take them out of the body they turn into what's called the synthetic phenotype, which, is, ah, characterized by protien synthesis, higher proliferation rates, basically a different kind of cell. The goal in my whole project is to characterize these kinds of cells and to see if we can shift it between these two phenotypes. The shifting, mostly what I'm trying to do is to shift it from this phenotype to that phenotype. [refers to synthetic and contractile cells on screen with laser pointer.] because From here to here happens automatically. [refers from contractile to synthetic.] So, ah.. Nerem?: So how come you don't have the [garbled] score up here? Jan: This is, ah, this is local. That's bowling. [laughter] Jan: ## It was not a good weekend for the atlanta teams. The braves just ##. I'm not going to be discussing that. ## Um, and so, the way I try to affect phenotype in our constructs is to use biochemical and mechanical stimulation and also genetic modification which is what I'll talk about at the end of today. and for those of you who are brand new. in terms of biomechanical stimulation, I compare a control constructs to the ones that have been cultured with ##-beta, with ## growth factor. And uh, I've presented all those results before, I presented them most recently at the ##. In terms of mechanical stimulation, there are probably also mostly familiar with this system that we use where the cyclic ## inside of our constructs in order to mimic the hostile pressure that's um, seen in vivo. And this is my last background, I think. The way I, the way I characterize our constructs for today's talk is um, is either gel compaction, which is easily done, cell proliferation using ## DNA which most of you have already heard of, the expression of ## or SMA, that's a, which is a marker of the contractile phenotype, in that and a major component of the contractile apparatus.. Many people use that as a marker of the contractile phenotype. And I'm going to start out with a tiny bit about western blotting and why it doesn't work for me, [laughter] and also histology, which I'm going to start with histology-- so this is under the heading of other things. I actually presented this at BMES, got a good laugh. pretty good, you think? [laugh] The reason it got a good laugh was because I'd mentioned that I'd just done it a few days ago, which is completely true. But this is kind of my first good stab at histology and I need to see is more reproducable. Histology isn't really a big part of what I was doing. but I did take just lots of samples to see what the constructs looked like. And judging on ends of like one or two or three, I think basically one, in the first case, two here, and, actually, probably two constructs here I've looked at so far-- well, two different experiments. Um. If you look at control constructs you see what ah, Drawer[?] and other people have found, uh, before me, as you mechainically cement[?] the constructs, you get, uh, an alighnment of the cells in the collagen fiber circumfirentially to oppose the mechanical forces that are being applied. Um, what I saw with PDGF and again, you know, n of 2 kind of thing, is, the matrix doesn't appear to be as dense. I actually thought it would be denser, because, uh, PDGF is very stimulatory, but it, and I don't well, you see these kind of folds, and it just doesn't seem to be a very dense matrix. And I don't think it's a cutting artifact, which I thought it was at first on the first one, but I did the second one. And all of these are from the same experiment. I don't know why there'd be a cutting artifact on one and not the others. So that's my preliminary, it looks like there's kind of a more open structure. Which is possible there could be more, you know, protonaise[?] or something, being, ah, produced. Because of PDGF. PDGF does a lot of different things. and then with TGF beta, what was very striking was that, uh, and I'll use those famous words that it's difficult to see in this slide. But, uh, you see a lot, actually you see more, kind of uh, small, uh small fibres. But if you look under the scope you can actually see tiny little microfibrils which I didn't see in the other ones. Instead of these bigger, kind of, um, you know, big structures here you see a lot more small stuff. Which, uh, is possibly due to the cells secreting their own matrix. Uh, which in TGF beta has been known to cause, but, you know, that's just speculative. But I just thought I'd just show you guys that. So you can critique it. The next couple things-- Nerem?: Were they laughing at your data or we were laughing at the fact-- Jan: I think they were laughing at my good joke ## female?: I think they were all relating to the fact that they had just been there ## as well # the last few days. Jan: Right. Am. And ## in order to get fresh data. Um, the next couple things are what you'd call retrospective analysis, data mining. Where I just took a look at the effect of passage on a couple things to do with these cells. Um, and what, basically, when I do experiments, I set it up, I usually get ## six experiments for every kind of treatment that I did. I kind of start cells at passage four or five, and then I would, uh, use them, ah, for, you know, four or five passages. So that's probably four experiments. And then I'd start another set and I'd do one or two more experiments, with a new set of cells. Uh, and by taking that data from a whole bunch of different experiments, and just plotting it out looking at proliferation, it looks like as they go higher in passage x and start to increase in proliferative capacity a little bit, and then it looks like there might be a plateau. For what it's worth that's my retrospective analysis for that. And in terms of , ah, SMA expression, again, uh, the passage, we can see that it gradually declines as these cells get older in culture. Which is, kind of what you expect. So it's not exactly a new finding but it's just interesting that things seemed to happen that way and that the cells ##. Now, to take that a step further, uh, and this actually came out with, came out when we were talking to Tom Lincoln ah, at dinner, when he came to visit, um, and ah, we thought ## acid flow through the ##. And I'd had a couple glasses of wine ## [laughter] Jan: ## And uh, the reason that we started doing this was that when you look at, um, ah, gel compaction amongst uh so non-transsected cells these are kind of standard rat smosa cells vs PKG transected cells which actually compact the gel, in this case, or in this experiment, slightly better than the untransected cells. You notice that the transected control cells which are transected with an empty plasma, so they're, ah, this will become clear when I talk about the PKG transected cells again but they're basically fake tras ##. : mock? Jan: Mock-transected, thank you. Um, you notice that they don't, ah, #tract the gel very much at all and, one of the questions was is that because, well, there's several reasons that could be. One is, because, the obvious thing is is maybe they're not expressing PKG and that's why they're not compacting gels. Um, the other thing however is that since these are clonal, uh, isolation can't be ## basically 1 or 2 cells that are expressing the ##, or expressing the gene, and them grow them up. Basically starting a whole hot ## cell population from a a very very small number of cells, so it's a huge number of passages. So we kind of wanted to see what happened when we take our normal cells and subject them to a huge number of passages. Do we see the same kind of ## effect in terms of gel compaction or, whatever. Um, so the experiment I did was I took my kind of standard p5 cells, uh, and you know I made, started up making #naise and constructs with those, I passage them 5 times and each time I split them, I would split them at a ratio of 1 to 100, which is huge. I normally do 1 to 2, 1 to 4 or 1 to 6 for my experiments. So this is blatant abuse of my cells. ## So I went up to what was called p10, althought it's I put a star there meaning that it's not proven p10, its just, I didn't know what to call it. So then I made more constructs and did the same thing again up to p15 and I looked at gel compaction, cell proliferation and SMA expression. And what do you find? Lo and behold, these guys still compact the gels very nicely all the way out, so they do lose their capacity to compact the gels. um, now, I'm not sure how well, I mean it's very difficult to simulate this clonal, ah, selection thing unless I actually just took one cell and ##. Which in retrospect maybe I should have done. I didn't. ## [laughter] Jan: I didn't. I'm not even sure how I would do that because I pick one cell, with a pipette.. ## Anyway, so they still compact the gels.In terms of proliferation, in ## you actually see a rise in that proliferation-- proliferative capacity. And you actually see a similar rise in the gels it's just that you can't see it on this particular axis. So they actually start to proliferate more. When you go, and remember you saw that kind of plateauing thing with that passage between like 5 and 10. But we're way out in ah, nowhereland, and what has been suggested in the literature at least is that basically these cells become fiberglass after enough time in culture. So the ah, one thing I haven't looked at yet is SMA expression, ah, and I'm doing that actually doing western blotting because, um, for various reasons I couldn't get close ## on those samples. And I want to do them all at the same time. So I have the samples, and I can do western blotting. Speaking of western blotting, I have been doing some, and my results, ah, one thing I find difficult to-- well, I'm not sure what to do about is the fact that most people, and what I've been doing as well is to load the lanes in the western blot based on total protien. The problem with that is that you then assume that, well, you either are only looking at relative expression of proteins, ## you're looking expression one protien ## all the others in the cell. Or you're assuming that the total protein content of the cell is constant among cells, which is what, kind of, most people assume. The problem with that is, as I said at the very beginning, these cells ## phenotype ## release a whole lot more protein than they do in vivo. So it's going to be difficult to say that one cell has the same amount of protein as another. And in fact if you look at just some of the biochemical stimulation that I did you can see that there are, especially with ## about a 50% increase in, in the amount of protein in a cell. cause what I did here was I counted the cells, I pulled the counter and I then I did the total protein ##, and you can tell that these cells have 1.5 times more protein. So normalizing by protein is not necessarily the best thing to do. And, so I have to figure out how to get around that or how to incorporate that into my western blotting cells. And uh I think we need to talk about ##. [female laughter] Jan: How much less of that ##. ok, so finally, ah, let me get on to what the main topic was which was the data on the genetic modified cells. For those of you who, um, don't know, we were working with Tom Lincoln and his group at the University of Alabama at birmingham, they study a, uh, called ## or PKG, it is a member, or a player int he nitric oxide pathway. Uh, if you've been doing your reading you'll know that nitric oxide is a very potent cause of ## cell relaxation. Um, but what we found is that when they were trying to study this so they, and they found that the levels of PKG would disappear in culture. So they decided then to modify cells to put the PKG back in so they could study it. But when they did that, they also found that the cells reverted to, very much, ah, a contractile phenotype both in morphology and protein expression and a lot of different things. So they kind of recognized that this is also involved in ## cell differentiation.And, ah, we've been working with them using their ## transfected cells which data I'm going to present today this is kin dof the final data on the ## transfected cells. And just as an example of these when you place these on a monolayer and let them get confluent[?] they really do produce a lot of ## and that's much more ## on transfected cells. So. Gel compaction data. I think in all the next graphs untransfected-- well, from this graph untransfected cells are the gray bars ## PKG transfected. What we originally found was that PKG transfected cells actually do a better job at compacting the gel than the untransfected cells. However one of the things I needed to do after that initial thing was to use another clone to make sure that that was a steady kind of observation. And if you combine the data of the first clone and the second clone the fact actually this.. it might in fact be three clones-- I haven't actually.. they haven't told me what the last 2 cell sets they gave me if its the same clone of it if it's the ## or just two batches of the same clone. If it's a different clone or two batches of the same clone. But anyways, um, when you put that data all together they don't, in fact, compact the gel quite as much as the, um, untransfected cells. That could be just because of genetic modification or various other-- or it could be because of PKG. It's hard to say exactly why that would be. When you do that in tubes, you see, a pretty standard result which is you don't see, uh, well, you do see a difference between the static and mechanical stimulated tubes. But you don't see a big difference between untransfected and ## tubes. Looking at cell proliferation, this is now in gels, generally our untransfected cells, um, well, our untransfected cells which is actually this-- this gray bar increases the number by about 10% over 6 days in culture. ## And then, ah, these PKG transfected cells in tubes, they actually decrease in number. And uh, just as a reference, in the untransfected cells also increase the number in the tubes as well. Uh, and this is really surprising because these cells don't take down as readily and ## the gels they probably don't survive the process as well so I think some just die, and they don't proliferate back to the normal ##. Whereas our untransfected cells all of them lived ## and they might, they, um, proliferate very slightly. And then finally in terms of SMA expression, again so this is the marker contractile phenotype that I'm looking at. Ah, untransfected cells, what I've established quite thoroughly, I think, is that they lose a lot of expression-- they lose their expression of that protein from ## cells in gels. When you use the PKG transfected cells you get some expression back. So I guess you should say you lose ## of the expression so you have about a 5 or, a 4 or 5 full increase in static ##. In tubes, ah, because of the fact that you don't actually lose as much expression in the untransfected cells, the amount that the PKG transfection helped is only about, ah, twofold increase. And you see a similar thing in mechanically stimulated tubes. And mechanical stimulation doesn't seem to affect SMA expression either up or down in either the the untransfected or in the PKG transfected cells. Ah, and then this is the same data right here, this is the data we were just looking at. One thing to keep in mind however is that I'm comparing everything to these um, monolayer controls, uh, these are untransfected monolayers you can see that we're losing expression in the gels but these guys, you know, do really well in monolayers-- the PKG transfected cells do well in monolayers but they lose a lot of their expression in gels. But they still have no ## untransfected cells. What is this? ok. Um, So I had, you know, biochemical stimulation, mechanical stimulation and genetic modification. I combined biochemical and mechanical. I've also combined biochemical and genetic, ah, so here, and so here, this is basically looking at the effect these different, ah, ah, ## biochemicals on PKG transfected cells. And this is gel compaction and you see the same kind of trend, where, PDGF and TGF beta both cause increased gel compaction. Also with these cells. And this is just the-- this is basically just looking at the final data part on the previous slide so this is the day six data, you see that, um, as I said, you have increased compaction caused by PDGF and TGF beta regardless of which cells you're using. And, again, the-- these cells don't -- don't sorry, these cells don't compact the gel quite as much as these. In terms of cell proliferation, these , um, this is all PKG transfected cells, various monolayers and over six days you only get something like a 1.8 or something fold increase in cell number. Whereas with untransfected cells you get something you get something like a 10 to 12 fold increase. So these guys proliferate much much more slowly than our untransfected cells which makes sense because that is one of the markers of the phenotype we're looking for, but transfected. And then, ah, in gel, uh, again, well, I'm sure I should just talk about the biochemicals. if you add the biochemicals PDGF seems to start to cause, um proliferation. And TGF inhibits proliferation. That's the same thing that you see in, um, untransfected cells. But actually the only thing that's statistically significant on this graph is the difference between PDGF beta monolayer and control monolayer. None of these are statistically significant. But it's only an n of 3 experiments. So, hypothetically if you did-- if somebody else did it more times, then ah, you might see significance. ## Anyone? [laugh] Probably not. Probably not me. I mean, it's not, I want to see if there's any response in general. I don't think it's a big enough response to make it worth you know, doing a lot of work on. And then finally, ah, looking at SMA expression, um, again, in monolayers, you see this-- again, very similar to what you see in nontransfected cells: PDGF causes a decrease in expression, I'm not actually sure if that's statistignificant, probably not. And then PGF beta, doesn't seem to cause much of a difference here but it in, in mono-- in untransfected cells it causes an increase. Again, that might be what's happening. Although it doesn't seem to be as pronounced here as it does in nontransfected cells ## TGF beta. And in monolayers we see a very similar trend, again it's harder to see ## but again, I don't, and, in monolayers-- I'm sorry, in ## gels there's no, ah, statistical difference. And then finally, [laughter] Jan: I did a couple of diff-- 2 experiments. Using, you know, the whole ## match of things I can do. So the PKG transfected cells TGF beta ## mechanical stimulation. This is about as much as I got out of this because I got some nice pictures, but I did analyze these because this is-- this, concurrently with me doing these experiments Tom, they had some problems with ## cells and, um, I stopped using the first batch and that's when I started using their second batch of cells. I never actually ## but Nerem?: These are the first batch? Jan: This is the first batch, yeah. And it's just interesting, and kind of tantalizing, that, you know, just in terms of gel compaction, you know, these are um, at TGF beta those are usually in in in disks doesn't cause much of a-- I'm sorry, in tubes doesn't cause much of a increase in compaction you can see that these are probably a little thinner. Not much shorter, but kind of thinner. or, what do you call it... ## or something. When you add mechanical stimulation, when you start to get you know, increased compaction they start to shorten up. And in these guys, ah, I promise I didn't squish them together although it looks like ## but I didn't touch these. [laugh] They definitely show a major increase in gel compaction. And I did it twice ## and that's where it's at. ## forty ## cells. It would take ## takes more time. So the only question is whether I can do that before my dissertation. I mean, well, I mean there are pros-- I mean, there are reasons to do it ## Nerem?: ## so what's the reason? Jan: Well, because I think one of the -- I guess, one of the things I I didn't really mention is that using these ## transfected cells there's going to be big differences between different clones. So I think it's interesting that PKG expression causes certain things but because every time you use a different cone your results might be different, um, I think it's better to go ## a viral transfection and then do these studies. Nerem?: ## Jan: Potentially. ## Nerem?: It just seems like a very interesting-- Jan: It is interesting and you know, that, you know, it killed me, [laugh] cause I did and I was really happy, and then, I talked to Tom and Aaron problem with the cells, and these cells might, I did to the, ah, alpha ## and they don't they didn't ah, produce much in these particular constructs. And, ah, I have no idea why. So-- Ann: Do you have any feel for the time course of the compaction? Did it happen all at once or is it slowly over 6 days, ## you've got it, a lot of compaction ## have a feel for it? Nerem: You've got some time-course data. Jan: Yeah, that was, um, well, I mean, I'm sure you know the most of the compaction occurs in the first two days. And you're talking about the mechanical stimulation kind of thing? Or just these cells? Ann: Like in other words, when you put them in the bioreactor, do you think they contracted a lot more? I mean obviously, from static to the mechanical stimulate they did. But is that an initial effect? Or is it, I'm just curious. Jan: Meaning, the effect of the mechanical stimulation? Does that increase happen very quickly? Uh, I actually don't hvae time course data on that because, you know, I have to keep it sterile, but my impression is that it actually happens pretty steadily. Like it's not like overnight they're different and then they stay what way. It's there a little bit-- Ann: ## change. Jan: Who else does this? Steph: Yeah, I agree. But it's like about two days ago. Jan: Yeah, I only did four days of mechanical stimulation and that, you know, yeah, probably after the second day or the third day they ##. Ann: Yeah, ## curious. wall: With your PKG transfection when you see less compaction you also see less proliferation so there's less total cells in the construct. So you have some idea of what whether it's because it's transfected or becaues there's not as many cells. Jan: Good question. Um, one of the things I don't really have a good handle on and uh, ## been studying this a lot is, you know, is gel compaction a function of, ah, to me it's ah, in my experience and what I think I see is that it's more of a function of having a very active cell type. With more of a synthetic where you're getting a lot-- it's more like a migratory, and proliferate response versus, ah, being a contractile response where people say that the cells are contracting the gels in the some way as if they would be contracting in vivo which I think is a very different mechanism. In terms of the numbers of cells, that's a good question. I don't really know the answer. wall: I always thought that it was because they were using some of their cellular energy just spitting out this protein, so then they wouldn't have enough energy to do it. But if there's less cells total, that could also be.. Jan: True. Well, Chris has some new data using 2 million and 4 million cells. And you don't ## see less compaction with the 4 million, right? Or-- : or no difference. Jan: Or no difference. It's not like a whole lot more cells is going to compact a whole lot further. And then we talked about why that might be, you know, maybe there's an optimum density where once they reach a certain cell density they don't const-- they stop compacting it. table: You talking about 2 meg per or 4 meg per mil, or-- Jan: 2 million-- : 1 million cells or 2 million cells? So that one million cells from now, 2 million cells from now? ## gel dense-- you know, protien density. Or uh, 2 meg per mil. Jan: Right. Well, didn't you find that.. I'll have to look at the data again.. But with 2 and 4 meg per mil you didn't find a difference in compaction. table: No, there's definitely a major difference. Jan: In 2 and 4 megs per mil? Table: But not in the number of cells. Jan: But not in the number of cells. Table: there was no difference in the number of cells. Jan: ok. Table: And the number of cells, though, that was just, we just looked at them, and measured the diameter and we had a picture of that the last time I did a presentation. And uh, the diameters of the gels weren't any different in disks, when 2 million-- 1 million cells per milliliter ## 2 million cells per milliliter. People must have done this before that. I remember discussing that with Ann, I think, because she found very different results and she had more cells. Ann: I definitely saw more compaction ## in tubes. ## Jan: ok ## Table: ## preliminary data was in disks and that's sort of what we were discussing, ## one or two. Ann: [laughs] ## Table: The tube data, though, with the compaction has all the same number of cells just different protein concentrations. Steph: Have you done live dead ##? Jan: Ah.. Not recently. I probably did. Steph: cause that would be neat to see. Jan: Yeah, you're right. Well, I don't know, 6 days later, how many-- I'm not sure they're dying, over time, I think they're just not making it into the gel, they're coming up dead. Cause that's what happens when you take them down in monolayers. You know. In untransfected cells you get like 90, 95% plating. With PKG transfected cells you get like 60, 70%. So, I don't know six days later if you actually see the nuclei. But maybe. And I probably have done that, but that would have been a lot of work. Probably something.. we should have done. [laughter] Jan: Um, so, currently, I do still have more data to analyze. Mainly, with the western blotting data and a little DNA data still ##. But I'm hopefully going to start writing full time. Tomorrow. [laughter] Nerem: Full time means 8 hours a day? Jan: Full graduate student time. [laughter] Jan: Could be more, could be less. [laughter] Jan: I'm not saying. [laughter] Jan: Now I'm, you know, I think I have the data to write it up, it's been, now the question is what am I going to try to defend. And finally, ah, The only work that I have in the planned, ah, the near future is what we just talked about which is to go to identify the transfection of these cells ## Just to get away from the problems we see plasma transfection. You know, I think, this is annoying when you use different clones and very different results. ## All the cells are transfected to begin with. ## That's it. wall: I have a question. Jan: Yes. wall: With the PKG, I mean you talk about being able to switch back to the contractile phenotype but you also talked about the desire to switch back and forth depending on what sort of environment they have, so once you transfect the cells, put them down on this one way road. Jan: Actually, that's a good point and that's another reason a ## viral transfection might be good because you don't have to transfect them until you want to. So you could envision not you know, having them untransfected and then being very synthetic or whatever in vitro while you're... While you're creating a construct. And then transfecting them to become contractile I mean, that's oversimplified, obviously, I mean obviously you can't implant ## transfected cells, at this point. But, you know, if other things come along in terms of, ah, gene transfer, that's one thing you could do. Or you could have, uh, when I was writing the proposal I had this thing I forgot the other day about [laugh] putting in a, um, maybe I shouldn't even say this, in case somebody makes me do it. [laughter] Jan: But putting in a, um, an on/off ## motor, which would be great, so you have, you know, you could have ## off or something like that ##. And then you'd get um, infection of the gene, and switch it that way. That would be great, but that's, um, ## Right, of off, you could have tetracycline in the culture and then as soon as you put it in the body ## off, whatever. But, ah, that's a URS project. [laughter] Jan: That'd be a good URS project. [motions head in Josette's direction] ## [laughter] : But ## viruses are only transitively expressed. So, would that be a problem? If ah-- Jan: Well, ah, first of all, we could try I think we're going to-- last time I talked to Tom he wanted to use an ## associated virus ## A, D, which are from the-- so that's one option. Another option is, again, hypothetically, pass the in vivo environment is suitable keeping the ## in the contractile state so you just get them contractile in vitro transiently, and put them in vivo ##. You know, pro ## Um, but also, again, you have to, you have to rely on the gene transfer techniques getting better too for any of this to be real useful. But that doesn't mean it's not working out right now. Nerem: You want Andrew to look at it ##. Want to set a record for longevity for a URS. [laughter] Nerem: Any other thoughts for Jan? : ## [laughter] Jan: Something like that, wasn't it? Steph: Endothilial cells are always ##. [laughter] Steph: Endothilial cells are always more important than ## cells. ## Nerem: ## your version of your title of your talk. Jan: yeah, we thought we should, um, I don't know if you every bought a book from Amazon.com but they send you emails telling you what else you might like. : Research who got this also got ## Jan: Right. [laughter] Nerem: Any other suggestions on other experiments Jan ought to do before he graduates? [laughter] Jan: I'll hang around, just to-- whoever suggests one we'll hang around just until you graduate just to suggest some for you. [laughter] Nerem: Well, we're going to try to meet later? Jan: Yeah, I think we should. What's your schedule like? Nerem: I think its pretty wide open. Jan: ok. : Is Tiffany ##? Tif: Yes I am. Jan: She's grabbed the initiative-- she's learned something at BMES. Tif: I did. ## Ann has kindly donated me her construct disscussion ##. I thought I'd take the opportunity to, ah, get some ideas, bounce 'em back off of y'all that I learned at BMES. So, just a couple slides. First of all, um, there was a talk at BMES, a study done at the University of Acron and it was ## and their motivation was ## but they did, um, do a number of microarry studies of these endophilial cells, under shear stress. Which was very similar to, um, what I'm going to be looking at with endophilial cells on constructs. And we'll be looking at their expression within microarrays. But what I thought was interesting was their shear stress conditions. And I really wanted to get y'all's opinion on this. Um, what they did was precondition all of their cells for 24 hours at 15 dynes per centimeter squared, and that established, like, a baseline for gene expression under bloodflow. And then they did step changes in their gene expression, you know, kind of repeated their graph here with the step changes. Including, you know, a higher shear stress, low levels, down to a stepping change down to a static condition, and they even did one retrograde study, which was pretty interesting. And then did the, um, DNA microarrays of the endophilial cells under all of these conditions and compared the genes being expressed to those ratios. So what I found interesting was that they preconditioned their cells for 24 hours at a pretty high level of shear stress, and then instead of taking, um, cells and flowing them and then comparing them to static, which seems to be, like, the common trend, they compared them to their delta of zero. So, you're basically like getting the cells used to being under flow, benching, taking them to higher stress ## and um, using that as our model. Just for reference, um, some of Jeff's work, he did preconditioning work as well, which I'm sure you all know. But his, what I found, there may be more work he's done that I don't know about, but what I've found is that his preconditioning studies were like 2 hours at low shear stress, and then he would change it to high, and then compare, just, here's comparing the effect of this preconditioning phase and they also did a long term preconditioning for 44 hours at 2 dynes per centimeter squared and then compared with and without the conditioning. And then they also compared those cells that were totally in static culture, versus, um, cells that were preconditioned. and then in static culture. So I was just wondering what y'all thought of this model, just in particular with the step changes, and how ## cells is acclimated to 24 hours, cause I was thinking more-- I thought this is great idea. Struck me pretty well. Um I think this talk conflicted with um, like Jan's presentation or overlapped I think I might have been the only one that saw it. So ## [laugh]. Um, But, I'm open for suggestions, I know all of you have a lot more experience with a flow studies and ## than I do. So what are your thoughts on this? This is a good idea? ## Jon: Can you go back to ##? ## When you say high shear how high ##? Tif: This actually was ##. ## Tif: Yeah, they were starting at 15 and then going up another 10, or going down, to what? Then went down to 5, they went down to a half, they went to static. And then they had, a, um, ## Nerem: And when you say, uh, what's that bottom one? Tif: The bottom one was, um... Nerem: That was a-- that was a pulse in the flow? Tif: As far as I understood it. Unfortunately there's no-- he didn't spend a lot of time on that. so I think it may have been kind of a, like a pilot. [laugh] so, you know, we should do ## which I think is very important, but we'll get to that a little later. And uh, their general results were that they didn't see a lot of changes between in these ## -like-- I should also mention these changes, they only lasted for 6 hours. So they preconditioned for 20 hours and then um, once they changed it they left it for 6 hours. And there's actually a question to the speaker why six hours and he was saying well I think that the genes the endothilial cells will have modified their expression after six hours. He conceded that maybe they should have been done a little longer than six hours. Nerem: And the marker was gene expression? Tif: [nods] They did microarrays for 12 genes and then was comparing the ratios of their expression. So I saw -- in this 12 minute presentation [laugh] They did see some changes that they thought were interesting which were kind of glossed over a little bit, but uh, with respect to this kind of retrograde motion, and also the positive 2.5, which would be the delta of minus 12.5 in the gene expression um, but I'm hoping that ## so that people ## more thoroughly. But I thought, just the idea of your, having a control basically be ah, a precondition instead of a static feature was-- Jan: But that was really Jeff's idea except that you're using different levels. Tif: Mmm Hmm. Jan: That was Jeff's-- wasn't that-- ## Jan: basically doing what other people hadn't done? Tif: Was preconditioning the cells-- Did he precondition them at low shear? And I wasn't really sure-- ## Tif: Right, yeah. He was looking at 1, 2, 4 hours. Really short time courses. Nerem: I mean you could argue about preconditioning at 2 versus 5 versus 10 versus 15, I'm not sure there is an answer. Uh, the only important thing is to be consistent. I'm not quite sure-- they seem to have a lot of conditions. They precondition at one level but then they have a lot of conditions after that. Tif: Right, that they change. Nerem: Yeah. By the way the second author on that paper is a former Ph.D. student of mine. Tif: Oh, really? [laughter] ## Tif: Yeah, it was the student ##. So with that I was thinking more like if you preconditioned at 10 instead of preconditioning at 15 ## you precondition at 10 for 24 hours and then ## step change up to 20 ## move down to 2, like a low value and then like a static value. And look further out, like a more like a more ## course. Cause I think in the body you're more worried about you know, the ## gene expression as opposed to the shorter time periods, just due to changing the blood flow level. Um, and then, like ##. Jan: That's a lot of microarrays. Tif: [laugh] yes, it is a lot of microarrays. Jan: Cause you have to do it a bunch of times. Tif: Right. ## Tif: [to Josette] Oh, I'm sorry, microarrays are um, a way to look at kind like a transcriptional profile of a gene, basically, I've just started looking at this. So keep that in mind [laugh] ## has like thousands of genes-- known genes on it, and um, you take the ## out of the cells you're working with and then um, you hybridize them the, the gene on the microarray. And then you can kind of tell the expression of different-- instead of looking at one gene like you would with PCR, that you've identified and you could-- might be changing your whole range. So it's a pretty powerful-- it's pretty new, and there's a lot of controversy that analyzing the data and that kind of thing you have to be really careful. ## Wall: Are you interested in looking at the transient effect after these step changes? Or of what the effect is at those shear levels? Tif: I think more of the effect at those shear levels over a long period of time. Um, just because, I think with respect to the body-- Nerem: But I think the transient effect is also.. Tif: ok.. Nerem: I mean I think that was one of the things that Jeff left out. In the context of MCP 1 and that is the short term response is very different that you can precondition-- Tif: Right. Nerem: -- and change the shear as opposed to if you just go cold turkey from static to, ah, shear. Tif: Right. Cause you would see a rise in the ## expression. Wall: My other question is what levels do you see ## reliability at? In terms of stress. Or shear stress. Or is it more time? Isn't there like, with the high flow, you have-- Steph: Well, I mean, ## Wall: ok. steph: So, I mean, it's more of actually a what I think of the end result of the particles in the media ## cells. With the particles. And that extenuates when you get to 72 hours, 96 hours we have ## So ## by taking out your true statics, you're doubling the number of flow loops. Ann: [laugh] on the practical side... Steph: In the practical side, if you, I mean, ## If you look at the 4 time points and the 4 different shear stress levels, like you won't be able to run all your shear stress levels at one time because then you'll have ## ## Steph: So I mean, I actually think that the preconditioning idea is really good, in reality that's really interesting ## but in a powerful sense, you want to limit the numbers-- it might be better data. Tif: ok. ok. I see what you mean. Nerem: Have you followed Zhou's work at all? Han Jun Zhou? [sp?] Tif: Uh, no I haven't. Nerem: Cause he's begun to do some preconditioning. He talked about this a little at the educational partners last week. Tif: I did see his student-- one of his students present at BMES ## I knwo he was preconditioning Nerem: ## be worthwile finding out ## ## Tif: Any other suggestions? Cause I have one other little topic I'd wanna talk about. Jan: You have minus 10 ## static? Tif: Yeah ## The other thing I'm working on right now is kind of a-- I'm actually doing in the lab right now-- my current problem is um, after, after I make the ## cell constucts you see the endothilial cells, you might see the endothilial cells ## and then I need to get the endothilial cells off. And just the endothilial cells. So I can isloate their MRA. So I'm actually going to start working on this today. But I was wondering if anyone had any suggestions lab-wise, as best ways to do this, um, ## from the endothilial cells to get them off, get their ## out before they have a chance to change it. And also need to get off, you know, as many endothilial cells as possible. Um, so some options that Jeff and I discussed were tripsonizing[?] the constructs and then scraping the endothilial cells off which he did some work with um, before he left. Another option would be, like, adding some weak collagenaise and then, same idea, like scraping the endothilial cells off the top. And then, um another option, but it might take too long, um, and the cells would change their ## would be to add collagenaise, basically digest the collagen matrix so you're left with endothilial cells and spimosa[?] cells. And then, like, separate them. So I was just wondering, functionally, if anybody has, um, thoughts on this? Steve: ## non-enzyomatic method of dissociation. Tif: ok Steve: TBGA ## Kara: And what would be the advantage of that? As opposed to tripset? Steve: Well, [cough] excuse me. Tripsonova alone, over a long term tends to cause the cells to, uh-- Kara: Sure. Steve: --lose viability and that may affect the results because you may not actually harvest the majority of the cells. You may use them to be in breaking up. When they break up before. So ah.. and then some confidence that they actually make dissociation, you know, non- enzyomatic dissociations-- ## But the commoner one that people use is the like the EGTA ## Kara: ok Steve: That they had ## And then Jonathan, you done some? Jon: Yeah. Steve: non-emblematic ## Jon: What I, well, when I tried that's the first thing that I did when I tried to isolate my cells and I found that when I used EGTA, I mean I would wait ad infinitum and the cells just would not come off. Um, and that's-- I scraped them, too. For some reason.. It could just be that I didn't try long-- you know, I just didn't try hard enough or whatever. I did this first, too, so ## but what I did and it worked real well and I'm still doing now is I use a strong collagenaise, the same amount of collagenaise that I would digest the entire um, media, um, with , but um, what I do is I place it only on the um, the lumen, Tif: ok Jon: so what you could do if you how about just have the thing open. Like this thing. you pour it on the lumen-- Tif: Well, these are already cut open. Jon: Oh, ok, well, then you just pour it on. And, uh, I think it's about 10 minutes of this-- it's really empirical, but it's about 10 minutes later you swab it off.. I don't know how resistant the gels are, but I know that the blood vessel ## will let you do that ## : I also think you might have success with maybe the ## just because he's-- Jon: right. : ## tissue-- Tif: right. : which is, I think would probably be a lot more ## gels. Ann: But I mean. I'm not sure why tripsen wouldn't be a good idea. I mean, I wouldn't leave it there for an hour, but, you know, you know, it shoudn't take you very long. Kara: Well the other thing is you're going to ## anyway so it doesn't matter if they're really, you know what I mean? if they fall ## or if they're close to losing viability, cause you're going to immediately just slice them. Tif: That's true. Kara: So. : Isn't the issue not, to like, digest the cells out but to separate the endothilial cells from the ## muscles cells? Jan: One problem you might have is ## muscle cells on the outside. If you do something, to your layer and they grow out. So you're at least going to probably have to allow this thing just to make sure it works. It might not a great validator Tif: right. Sure, but what I'm getting is the endothilial cells ## Jan: but I think if, I mean if you could just keep tripsonizing[sp?] and get them off then.. Tif: Yeah. ## Steph: You can definitely separate them. That way, so, you need to probably validate that method, with that one. Tif: Yeah. : With flow cytometry. Steph: With flow cytometry. But I don't think flow cytometry is-- Tif: the way to go? Steph: the easiest. But you can do it the other way to validate, ## Tif: That my method is working. Steph: yeah. And I could help you. But I think the other way should work. Tif: ok. Steve: I mean, the other issue that I know that Jeff encountered was the amount-- was how much do you get per construct, because-- : That's problem number 2. [laughter] Steve: --you can, yeah, you know, you can get 10 cells, great, but you're not going to ## enough for what you need to do. So that's ## Tif: I need as many of those as I possibly can. [laughter] Tif: Well thank you, very much. [laugh] For all my ## suggestions. [laugh] ## Nerem: You know back to the, the conditions. I think you initially should just focus on one thing-- one precondition level, one change. Tif: Mmm Hmm. Nerem: ## your work. Tif: Right. I was just kind of thinking long term, when I saw the preconditioning at BMES ## not my immediate concern. Nerem: I mean as opposed to changing through a lot of different levels it'd be more interesting to get into the ## flow business. Tif: Mmm hmm. Nerem: So. ## Nerem: Ok, anything else, Tiffany? Tif: No, that's it. Thank you. Nerem: What's up Steve, are you back? Steve: I'm back. [laughter] Nerem: Steve had a quasi-vacation last week. Steve: yeah. ## Break ## vacation. [laugh] But uh. Nerem: Anything you need to talk about with the group? Steve: Yeah, just a few items uh. For those of you who have lab jobs in the lab I'd like to meet with you after this group meeting in the lab just for a few minutes. And also, I have a phone list here, I just want to make sure everyone's email, phone number is correct, you can just pass it around. Just to make sure. Ah, we have the microscope finally, in the lab, uh, inverted size [sp?] microscope. And uh, it's all set to go. I did want to bring Don Aiken by who ## representative to sort of give you a training session on, you know, the care and use of the ## and everything. This is a very expensive piece of equipment, so far as microscopes go so we want to make it last. We want to do the right thing. Kara: Can we do that soon? Steve: As soon as I can contact them and we can set up a time to come out. Kara: Well just 'cause I'm thiking that the focal's down now, and so- Steve: Right. : It doesn't have flourescence Steve: If it doesn't have flourescence I can't guarantee that-- : ## flourescence yet? ## : oh, we're not? I thought we were. Steve: Well, no no no but let's clear things up Kara: [laugh] ok! Steve: I spoke with Don about trying to fit-- retrofit out flourecence that was currently on the ## by conventional flourecence, on the back of our scope in the Nerem lab. And he said he would get back in touch with me to see if that's possible to do. : Just while, you mean, just while the ## is down. Steve: Right. So. That's where we, I need to... He needs to get back in touch with me. wall: But, but, the other flourecence microscopes too. Like you can probably ask-- Garcia has a nice-- Steve: Garcia, ## Steve: They all have wall: I mean I'm not saying that you can use it but you might want to look there too. Steve: So that's all. Nerem: Um. I guess it's becoming obvious to people but um, seems like we're going to have more and more people in the lab, and that's going to produce a space problem. It already probably has, Steph's been relocated, right? Steph: yeah. Nerem: so um. ## : oh. Steph: Dunwoody. [laughter] Table: Work at home! Telecommute! [laughter] Steph: ## definitily going to have to work at home, so It's all been positive, actually. Nerem: I wish we had more space, but between now and the BME building coming online this building is really tight. Um. So, um, we're just going to have to bite the bullet and put up with it. Victoria, how are you interacting with us? Vic: Good. They're interacting very well with me. [laughter] Nerem: So it is happening? Vic: yes Nerem: ok. Wasn't sure how it was going to happen but apparently I left it to all of you and it did happen. So, um.. Debbie. How you coming on your work? Deb: Well, ah, nothing has actually worked til now. I had a lot of mistakes, or, it failed. ## no results. Nerem: But it wasn't mistakes, things turned out differently than you thought. Deb: Yeah. table: Learning experiences. : Learning experiences. [laughter] Deb: I ran into a lot of the problems, and I learn from it though. [laugh] Nerem: good, good. And Josette, are you getting into the lab now? Jos: Yeah, um, working with ## and working with Tiffany, um, I should be working with her when she ## constructs today, so I'm kinda culturing trillions of cells to make constructs and helping Ann calibrate the bioreactor. So, ## my experience so now I know how to ## cutting on [laughter] Steph: Those actually ## constctructs ## sleeves. Ann: Oh, so far so good. Um, our preliminary, um, like, looking at, like, what kind of pressures um, you get 10% ## out of ## similar. We had a lot of leaks, so Jan: ## running low. [laughter] Nerem: low on leaks? ## Jan: We bought 16 hundred feet or something like that Steph: They're in these huge spools. I mean these spools are like : wow. So they came in Nerem: Well, we got 15 hundred and 50 people I think that's ## [laughter] : More than that. Steph: So it's 7 PSIers Ann: Yeah. ## Ann: All indications is that it will be very similar as far as handling ## Nerem: Ok. Anything else? Kara: I have something. Um, I'm just struggling with this-- this kind of an issue. I have to seed cells on microscope slides for the flow experiments. But the problem is that is I have to keep these cells in the culture then for several days before I expose them to the shear stress. Well the current method right now is to put it in these, um, square culture dishes which take like 20 mils of media. And I'm changing media every day. And that's just going through too much. Cause I just can't-- I'm using up too much media, the serum's expensive, So what I need is I need something smaller I can put the microslide in and it's gonna fit perfectly-- and I don't care if I, you know, if I can't get it out with tweezers I'll just, you know [laugh] crack it or something! But I just need something smaller. And like those well chambers aren't going to work because you know the bottom of those microslide isn't sterlie and they have to be sterile. Any ideas? Tif: Are you using glass slides? Or are you making your own slides ##? Yeah, the glass. Jan: Those are sterile. When those, ah.. Kara: The chamber ones? Jan: Yeah, as long as you keep them sterile. Kara: The bottom of them aren't. Jan: Well, they are when you open them. : Yeah. Steph: Yeah, when you stick them in the flow chamber they're not sterile anymore and. Kara: Say that again? Steph: In the flow chamber? They're not sterile on the bottom anyway. They're ## Kara: Also, I-- can I ## corners and stuff aren't. generally didn't you have that little edge that's not what you write on-- Jan: That-- I think the whole package is sterile. Kara: right. Jan: Before you open it. So as long as you open it in a hood, and keep it sterile, the whole thing is sterile. Steph: I think he means put the whole thing into a dish. Jan: For example. Yeah, Don't put them in the incubator Kara: Ah, the whole thing in the dish.. : ## sterile? : Yeah, you'd have to ## flow loop, though. Because those are smaller than our slides. But you can get ## box, ## Steve: And you can get slides without the mark-- without the, uh, white part. That you write on. Kara: Wouldn't it be easier for me to make-- have them make me a culture dish ## culture dish? ## Kara: That'd probably be the easiest thing. There's not something easier I could just buy? : Have you looked? Cause I wouldn't be surprised. Kara: I looked. I found like, you know, but I was looking under those chamber slides. wall: Those chambers are hard to get off, too. ## Jan: They're not the best ## Kara: So I guess I could have-- that's a last resort.. I'm trying to find something I could just buy. ## But anyway, that's what I'm thinking about right now. So if anyone has any ideas, good ideas. Nerem: This is for the embryonic cells? Kara: Cause I'm using up too much. Too much of the media. Nerem: There goes your increased ration. [laughter] ## Kara: Well the problem-- the problem it's not actually even cost, it's taht it's hard to get the special serum that it's-- it's screened so that it doesn't induce differentiation on the cells. And so what happens is Gibgo, or whoever has to go through and all these serums so til they find one that doesn't induce differentiation so right now it's on backorder. That's because they don't have one. They have to screen a whole bunch, they have to find a batch, and as soon as they do, everybody buys it up, and then they don't have any anymore! And so that's the problem just getting a hold of this stuff. So.. Steph: What are you going to do about the flow loops then? Kara: What I'm going to do with the flow loops, I figure-- I'm trying to differentiate them. At that point. So they're going to get regular serum, and they're not going to get the ## kind which is also expensive. So, the flow loops are going to be ok. I just want to keep them undifferentiated up until that. Nerem: Hmm. [laughter] Nerem: I mean I'm just wondering about, ah, about doing a flow loop with a serum that doesn't differentiate them. Kara: And see what happens? Nerem: and see if, see if just-- ## flow by itself. Kara: Well that's what I-- that's what I was going to originally try, but I think ## that much serum. Unless Gibgo has some and we can buy up several bottles, then I think maybe I could do that. But they're not going to give-- they don't have that much serum to give me. Nerem: out at UGA. Kara: Yeah. Though I don't know whose-- I think it's actually ## or something ## all at the same lab and-- Steve: Ah, yeah. Kara: They're really nice to me, but. There's gonna be a limit on how much they're gonna give me. Nerem: Well, will they sell us some? Kara: Well, they have the same problem. Nerem: They have the same problem? Kara: You know what, right now they're trying to screen their own, and as soon as they find some, they have a deal, I think, with Hyclone-- they're going to buy up like... a lot. Then it probably won't be an issue for a while. But that hasn't happened yet. So at some point in time it's probably not going to be a big issue. But right now it is. Jan: So they don't know what ## their serum doesn't have, or does have. Kara: [shakes her head] wall: at the end of BMES, at the very last poster session they had one poster on a different sort of shear stress like-- parallel plate chamber where it was kind of like a treadmill sort of thing that created the shear on it so you could put it in the smaller container, so maybe like the 25 mils or 50 mils.. I don't know what, exactly volume-wise, but.. : You had moving fluid? : Mmm Hmm. So it's kind of like a motor, attatched to a treadmill that went under the media that would.. keep going around. So I don't know if anyone's else has heard of anything like this or whether they have feelings about it. ## : mm mm. Ann: Well my quickcell the-- where you can put in six or eight slides in one sort of chamber. : very nice. [laugh] wall: Will that stretch? ann: No. That's actually a parallel plate which everybody-- : Yeah, there's six of 'em ## parallel. ## : Cool! That is interesting. Nerem: ok. Jan: Everybody eat up. You guys too. thanks everyone.