Web Conference with Dr. Derrick Rossi

- 00:00 To the first point, career path, I don't know if you've all got maybe three or four hours to spend because I took some sort of very strange circuitous career path to ending up working on this subject. So I, just to let you know, I did my undergraduate, my graduate work in Toronto, Canada. I'm Canadian. And then I had the opportunity to go and do a Ph.D. in France. So as a young man when you're given the opportunity to go to France you should definitely go to France. I went to Paris for a couple of years and was in a Ph.D. program there. I was, first let you know, I was in a Ph.D. program in Toronto so that's Ph.D. program number one. Then Ph.D. program number two was in Paris. Then I had to leave Paris because I ran out of money. In Texas for some strange reason, and then I had an opportunity to go to Finland and yet another Ph.D. program at the University of Helsinki. So again as a young man you get the opportunity to go to Helsinki, you should go. And there I started to really get serious about science, not that I hadn't been before but I was kind of too young and maybe too immature to really focus. So I got down to business in Helsinki and did a lot of science. And then that I had a lot of success there. I published a lot of papers and that gave me great opportunity as a post-doctoral fellow. I could basically go wherever I wanted. So I had great offers to go to really fantastic labs. And I had not been in stem cell biology but I had been sort of paying attention to the literature and reading the literature and it was clear to me that stem cell biology was about to hit a golden age. So I thought I would go into that field so I started to do some research as to what would be the most optimal setting for that. Went to a lot of post-doctoral interviews, got a lot of great offers at a lot of great place, but when I got offered a position at Stanford University in the lab of this fellow named Weissman who basically is one of the founding fathers of hematopoietic stem cell biology, that was clearly a great opportunity so I went and I started to study hematopoietic stem cell biology. And I had, I was there for 4 1/2, five years studying how hematopoietic stem cells change during aging. We know that actually the hematopoietic system deteriorates quite dramatically with aging. You get hematopoietic malignancy. Your lymphoid, your adaptive immune potential goes down, become So I wanted to sort of find out how much of that decline of the hematopoietic system was due to the aging. Web Conference with Dr. Derrick Rossi

The stem cell compartment it turns out number of studies that we did, quite a lot. So the stem cells really matter there. That was the focus of my post-doctoral work. When I started my lab I was working on truth related to hematopoietic stem cell biology mostly. But then Yamanakaalong and this was terribly exciting for everybody. Basically it was a see change moment in regenerative medicine. The reason for that is quite simple. Whereas human embryonic stem cells were great in that you can make presumably any type of cell or tissue that only you could imagine putting back into patients, they have a big problem that they're derived from embryos that aren't ones itself. So if you're going to transplant those cells or tissues back into patients you're always going to have to think about immune rejection and histic compatibility issues. So what was so exciting about Yamanaka was that now it was this ability to create personalized pluripotent stem cells from a patient who had been one you'd say derived cells or tissues from those cells you put back into the patient without a fear of immunorejection and this is really, really dramatic. Moreover you could basically make iPS cells from anybody in the room there with a skin fibroblast and within a few weeks you can have a pluripotent stem cell tailored to one's own cell so that amazingly the promise of that is enormous. So I think Michael also asked me give a sort of historical recap of pluripotency reprogramming, let's say. Actually it really all starts the 1950s in a frog called Xenopus with studies by guys named Briggs King and then later in the 1960s by Sir John Gurdon that showed that you could, well let me back up one sec. So what you need to know about reprogramming is how completely fantastic it actually is because normally developmental processes are completely unidirectional. You go from more primitive cell to less primitive cells to differentiated cells and that's a one-way street in biology. There's really no going back. And this is believed for many decade and centuries that really you couldn't turn back the developmental hand of time. So what these experiments in Xenopus by Gurdon and others showed is that that's actually not true. So they basically took oocytes from these Xenopus, large oocytes and they took out the nucleus of them and then they put in the nucleus of somatic cells of some differentiated cell type they introduced into the oocyte and amazingly it was something about the Web Conference with Dr. Derrick Rossi

cellular milieu of the oocyte that was able to reprogram that introduced nucleus back to a pluripotent state. And the ultimate demonstration of that, in fact in the 1960s was the generation of cloned frogs and this is where cloning started. But really it's As I said it turns back our developmental pathway which up until that point had been unidirectional. So subsequent to experiments in the frogs somatic cell nuclear transfer, it's what it's called when you enucleate an oocyte by putting another nucleus in there and reprogram it. This was done with a number of different including most famously Dolly the sheep obviously you've heard about and cows. Basically anything could be reprogrammed in this setting but this doesn't get us superb stem cells. That kind of or it does but not in the way that Yamanaka does it. Yamanaka does it is particularly falling on the shoulders of work that was done in the late 1980s by Harold Weintraub and colleagues that showed actually that transcription factors are important for mediating cellular identity. So basically they did it with fibroblast cells and they isolated a transcription factor called MyoD and when you introduce this factor into these fibroblasts you birth them into myogenic lineage, muscle cells basically. And a single transcription factor could do that. So this started a whole field of research where people were looking at different transcription factors that they thought would be important for specifying the fate of different cell types and a lot of work in the 1990s showed for example that you could reprogram one type of blood lineage to another type of blood lineage. Work from Thomas Graf and colleagues using single factor, a transcription factor, ectopic expression one cell over another. And this culminated in this really spectacular discovery by Shinya Yamanaka that, you know, and I don't know if you've covered that original reprogramming paper. Have you done that in class?

- I didn't assign it but I definitely talked about the work involved.

- Yeah, you should assign that next year because it's so breath, I mean it's just spectacular science. It doesn't get any more spectacular than that if you want to be inspired. So as you know basically what Yamanaka did was he had this idea that if he took Web Conference with Dr. Derrick Rossi

all the factors that they could imagine might be important for specifying a pluripotent state and introduced them into fibroblasts that somehow you could convert these cells back to a pluripotent state. And I can tell you that if he actually presented this idea for example to a granting board prior to actually doing it in '63 he probably would have been thrown out the window. I mean it's such a wild idea it's amazing that it actually worked. But it worked because they took a large-scale approach. They didn't just use one factor. One factor wouldn't have done it. They identified 24 factors, transcription factors that might be for specifying a pluripotent state, introduced these, and then as you know sort of found that this worked and then they whittled back 24 factors down to four factors. They're now the canonical Yamanaka factors, this Sox2, Klf4, c-Myc, and Oct4. Subsequent to that, might be a long answer. Sorry for that. Subsequent to that, others have now followed on the heels of that, show that various combinations of transcription factors could move the identity of one cell type to another. For example in the pancreatic lineage Doug Melton and colleagues show that ectopic expression of free transcription factors convert these exocrine cells to endocrine beta cells. Quite fantastic, it's been done in the neural lineage as well, possibly in the cardiac lineage as well. So it really is all the rage because many cell types though clinically useful cell types, they're actually really hard to get ahold of or to get in large numbers. So for example we work on hematopoietic stem cell biology largely and these cells are exceedingly rare and if we could have a way of making lots of them from some readily available cell type by breaking the code for what sort of set of transcription factors would specify that lineage, that could have really great clinical benefit. So hopefully that's not too long of an answer to sort of set the stage for what we did. And at this why did we enter this business in particular? Well we were excited by Yamanaka as everybody else in stem cell biology was but we realized that there was really significant hurdles that needed to be overcome before iPS cells could ever enter the clinic. And foremost amongst these in our mind, we started this project almost three years ago, was that the typical vector for affecting reprogramming was viruses be it retroviruses or lentiviruses. And viruses we know really have been great for clinical translation Web Conference with Dr. Derrick Rossi

because they're great into the genome and sometimes they land in spots where they ectopically drive the expression of nearby genes and cause cancers. This is what happened gene therapy trials in the 1980s. So we set out to try to find a way around for hitting the nucleus basically. And wethis idea of doing via an RNA intermediate. We know that basically intermediate probably means normally DNA encodes proteins but it does so via an RNA intermediate so we thought what It's not such afantastic idea. And I'm sure many others had the idea and have actually had the idea for many decades and the reason that you don't actually see RNA being used as a tool for molecular biology is because normal what happens when you introduce RNA into cells, the cells think they're being infected by viruses, nucleic acid, and they respond with a very robust antiviral response. And that's what we found when we first started this project but we found a way around it and I guess you just covered the paper there so you know how we did that. So I'll sort of, that's how we ended up in this field in particular. Basically inspiration was pretty spectacular for in the clinical promise that

- 14:06 Hi, I'm Rachel. I had a question concerning the oxygen content and I was just wondering why does reducing the oxygen content as you have in Table One increase the efficiency of the reprogramming? And then also so is there an oxidizing factor? And also why does the presence of LIN28 sort of rescue the loss of efficiency in higher oxygen environments?

- 14:30 Okay, well good questions. So basically the issue with the oxygen content it's actually universally true of almost all mammalian cells cultured in tissue culture incubators or incubators because pretty much by and large they're all at atmosphere oxygen tension which is about 20% O2. Well guess what? Cells in the body are not exposed to 20% O2. By and large the body is a relatively hypoxic environment and cells have evolved for millennia under living withoxygen. And so to pick cells out and to culture them at high oxygen, what that actually does is it causes oxygen distress. And just because oxygen tension it's more free radicals which basically bang up against your cellular macromolecules causing them to Web Conference with Dr. Derrick Rossi

oxidize. It's the free radicals that do this and by reducing the oxygen tension you can reduce this. It's actually not something we discovered. It's been known for a really long time and it amazes me today that anybody is culturing mammalian cells in anything butoxygen because pretty much any cell type you want to handle ex vivo does better at oxygen. So what oxygen distress does is itfree radical generation, causes macromolecular damage, RNA, proteins, lipids. Anything in the cell is susceptible to oxygen damage. And usually what this does is it elicits the upregulation of genes called tumor suppressors. Though what tumor suppressors did was basically say okay, they're damaging the cell and we're going to stop dividing until we clean up this damage or take care of the situation but we're not going to continue to divide this setting which is suboptimal, potentially mutagenic. So tumor suppressors get turned on and thatslows the cell cycle down. Well we know that the processes of the reprogramming requires cellular division. There needs to be an awful lot of epigenetic reprogramming that goes on during reprogramming which involves and requires actually multiple cell division. So by culturing at low oxygen you reduce oxygen distress, you reduce the elicitation of tumor suppressor genes and therefore allow the cells to pass through cell cycle more readily meaning more quickly to reprogram, more quickly, more efficiently. What LIN28 does, the second part of your question, it doesn't directly impact pathway as far as I know or maybe that anybody knows in terms of oxygen distress but what it does do is it acts as kind of gas pedal. So LIN28 actually expression leads to stabilization of c-Myc amongst other things since c-Myc is something that drives cells through the cell cycle sometimes even if they don't want to go through the cell cycle so even if they're experiencing oxygen distress. So I think that if you put all, put all of that together, that very long answer, you can see why A) low oxygen increases the efficiency of reprogramming and why at higher oxygen LIN28 would actually kind of have a similar effect. But as I said if you're going to take home message here, think. Because if you're fin' to go out and do, you know, culture cells and for, in your, you know, if any of you are going into careers in biology, buy yourself a low oxygen incubator and get your because Web Conference with Dr. Derrick Rossi

by and large pretty much all mammalian cells much prefer to be cultured and are maintained at a much better state at low oxygen. Good question though.

- Thank you.

- Xian, you have a problem?

- 18:59 Hello, I'm Xian. The follow question is what'll be the difference between the reprogramming efficiency of RNA and virus KNS under low oxygen concentration? I mean like meaning like hypoxic conditions.

- 19:13 Yeah, well it's, you're going to get a beneficial effect whether or not you're using RNA or viruses. The method, the vector of reprogramming doesn't matter here. Basically you're basically introducing those vectors be it RNA or virus just to get the expression of these Yamanaka factors going. That to my mind isn't any different at lower oxygen. In fact it's been shown at enviral experiments that if you go to low oxygen you'll get an increased efficiency. So the vector doesn't really matter here. It's the oxygen tension itself.

- Hi, my name is Sylvie and--

- Hi.

- 20:00 So in your paper you compare the RNA-induced pluripotent stem cells to fibroblast human embryonic stem cells and virally derived iPSC lines. There are several comparisons that seem to me that RiPSCs and human embryonic stem cells were the two best stem cells to be used and you describe how the RiPSCs are very similar and comparable to the human embryonic stem cells in many ways. How would you compare the potentials and the hopes of the near future of both stem Web Conference with Dr. Derrick Rossi

cells and also would you say whichever one is better than the other? And can you also briefly tell us about the main differences between the two types of stem cells?

- 20:46 Okay so that, I think you asked just quite a bit there but let me see if I, let me see if I, I've got it. So from the get-go I think that there's nothing sort of intrinsically different about reprogramming by RNA or viruses other than the fact that at the end of the day you've got IPS cells that either have viruses in their genomes or nothing in their genomes. So we know that viral reprogramming actually involves silencing of the viruses. So during pluripotency reprogramming the viruses actually get reprogrammed, or silenced but nonetheless they remain in the genome. Moreover, viral integration is not entirely random in the genome but it's pretty random. So you've got viruses scattered around the genome that have been epigenetically silenced. It may well be that that silencing has local effects that affect more than just the virus itself, the integrated virus. So when we compare the whole transcriptional profile of RNA-derived iPS which we call RiPSC cell by the way, RNA-derived iPS cells, versus viral, I don't find it terribly surprising that the RNA-derived iPS cells a molecular profile that more closely recapitulates human ES cells. Human ES cells don't have viruses in them scattered around the genome various levels of silencing similar to the RNA-derived does in contrast to virally- derived. So I think from the global molecular profile that doesn't surprise me at all. Could be that virally-derived iPS cells that have their viruses been excised, there's various strategies for doing that, might have a profile that more closely looks like I would predict, that have a profile that more closely looks like human ES cells. But we didn't have those in this I can't speak to that. And then okay, so I also mentioned a little earlier that from the gene if we're ever going to derive cellular therapywith patients you really want to get rid of the virus instead of the genome if you can because gene therapy trials from the 1980s showed us and it's basically due to the fact that viruses integrate the genome relatively randomly. Sometimes they integrate in sites that are really, that you wish they hadn't landed in front of an onc or a proto-oncogene that all of a sudden gets turned on by the viral sequences and becomes an oncogene and leads to cancer. Another important Web Conference with Dr. Derrick Rossi

issue which I don't think is discussed enough is that told you that the viruses get silenced during reprogramming but guess what? We don't really want to use iPS cells patients, not unless of course you want to give the patient a teratoma. Really what we went to do is derive clinically useful cell types from those IPS cells. Well guess what happens to those silenced viruses upon differentiation? Oftentimes they become unsilenced and now all of a sudden you're trying to differentiate towards one lineage or other lineage and you've got ectopic expression, Oct4, Sox2, Klf4, and c-Myc to deal with which is clearly not a good thing considering at least two of those genes are again proto-oncogenes and may interfere with the differentinto effect. So I think that if you were going to at face level show that virally-derived iPS and RNA-derived iPS were, had the same developmental potency you'd probably want the version without the viruses. Now what about ES cells? Well ES cells of course are the sort of what we've used as the standard pluripotent cell. The problem with ES cells, you know, I mentioned this a little while ago, is that they're not derived from anyin particular. They're derived from justblastocyst. So whenever you're going to use those in a clinical setting if that's the hope, the problems with these cells, you're going to have to consider histocompatibility and immunorejection. And so if you could completely comfortable with the fact that iPS cells have the same developmental potential as ES cells and you'd want to use iPS cells if you're going to put them back into patients because you could deliver that without immunosuppression. Yeah, I think that answers your questions.

- 25:59 Practicality of your methods, how soon do you think your methods will be applied in the field? Do you believe that your current methods are practical in the consumer sense to patients and whatnot? And if not, how long do you think it will be until someone improves upon them even further to the point where they are practical?

- 26:19 Yeah, that's a good question. Well actually how soon are they going to be used practically? Well I know that there are really many dozens if not hundreds of labs around the planet that are already starting to work with our technology. The Web Conference with Dr. Derrick Rossi

problem, the problem, problem. It's a new technology that we developed and it's actually a little bit complicated and it's not so easy to work. So I think that that's as true of any new technology that ever gets introduced into the field of biomedical science. It's always there's going to be some growing pains as other labs sort of adopt this technology. You have to learn how to make good quality modified RNA. You have to learn how to transvect it, get it in the cells. You have to learn how to reprogram using this technology, maintain thisfor days and weeks. So yeah, there's going to be some growing pains for sure butthere are literally hundreds of labs that are at it right now. So it'll take a while but they'll get good at it. We'd hope to facilitate that as much as we possibly can, get a public protocols, papers, and the like. So let me just look at the question again.

- 27:39 Can you elaborate a little bit on your technique? So you said it's kind of complicated. Can you give us a sort of slight step-wise detail of the major issues

- Yeah. that you think could be complicated?

- 27:54 Yeah, well I mean it involves some molecular biology from the get-go. So just compare it to viruses for example. People have known how to make viruses and lentiviruses relatively. Mind you I'm sure that the first time lentis and retroviruses came out on the market lots of people had a hard time adopting those too and it probably took a few years before that became commonplace. So it's probably the same thing but the first thing you've got to do is you've got to clone a cDNA of interest or in this case it's Yamanaka factors. But if you're going to use the technology for directed differentiation which we also show in the final figure of our paper then you have to clone in whatever other factor that you're going to use and you have to clone it in with the proper biprime and three with the poly-A tail on there. That's pretty straightforward molecular biology. Somebody who's reasonably proficient at molecular biology can do that. Then you've got to put it into an IPT reaction. This is a cDNA template that you have now. Then you've got to make an RNA out of it and that's again, it's pretty much a straightforward transcription reaction. But what Web Conference with Dr. Derrick Rossi

we do is we swap out the nucleotides so we don't, you know, with two out of the four nucleotides we put in sort of specialty nucleotides. These could be uridine and 5-methylC, 5-methylcytosine. But again you can order those nucleotides partially, by the way they're very expensive, and synthesize modified RNA. But then actually the real challenge is that the thing that people don't realize. Getting them in the cell's not so easy. There's a lot of cell type dependence even amongst for fibroblast layer cell types. There's definitely dosing effects. So you've got to use different transfection reagents potentially which have their own toxicity associated with them. And for reprogramming you've got to unlike viruses where you put your virus on once, transduce the cells, and then go away and come back three weeks later and you've got colonies on your plate, here you've got to add in the RNA every day. But the whole sort of routine that we got this to work was basically rendered the RNA nonimmunogenic. As I told you, normally if you put an RNA into a, and you see that in Figure One of our paper, if you put a regular old unmodified RNA in the cells, the cells really don't like it. They think they're being infected with a virus and they mount a really robust antiviral response. Basically turns off translation and if robust enough will kill the cells. So we essentially abrogate this response but not entirely. So you have to be vigilant and watch that your transfection is still transvecting robustly each day. Reprogramming takes several weeks to occur. You've got to have these reprogramming factors expressed at robust levels and it's kind of dependent on the cell density so, which is a good and a bad thing. So as the cell number increases your per cell RNA content decreases. So you have to be cognizant of that. So it's really actually very challenging for all of these reasons again as I said, unlike viruses where you put a nice, high, tighter virus on in the cells and walk away and come back three weeks later and you've got colonies. So I advise many, many labs around the planet and it occurs to me but it's completely obvious to me when somebody emails me and I get, you know, it was many a day for months of people trying to do this. And it's clear to me that some people are going to be hobbyists when it comes to using it. They just think, well I think our paper's a little bit deceptive in that the title of the paper is, I can't remember what it is exactly. Web Conference with Dr. Derrick Rossi

Something like Highly Efficient Reprogramming. Well the human mind is a funny thing. People read highly efficient and they make a little equal sign in their brain and they think that easy.

- Easy. Highly efficient does not equal easy and it's very that a lot of people think oh, it's going to be really, really easy to do this and it's totally not. So the advice I give to people is if you're prepared to go into this be very prepared to go in. You're going to have to work at this. It took us about two years of optimizing this to get it to work. I mean we knew how to make modified RNA two years ago but really getting it to work, really just sort of finding the right sort of combination of transfection reagents and medias, media very important, cell density, everything very important. So I think it's going to be very challenging as people go forward. So I think your question was well is there any way that this will, there'll be new advances to make this easy actually in the end and I'm not sure about that. It helps that we're constantly working on things in the lab, differentapproaches to this technology which might make it easier. Yeah, the one thing I'll add actually is that to be honest I see a lot of utility for this technology in other aspects. Are you guys, can you hear me?

- [Audience] Yeah, yes, yeah.

- 33:43 Okay, yeah, so to be honest, in the sort of cell engineering domain I actually think that the most valuable aspect of this technology is actually not making iPS cells because you could make iPS cells by other methods which are actually even transgene-free. There's various strategies for doing that without hitting the genome. I don't think there are many greater or worse or better than our technology. Ours works at very high efficiency, yes, when it's working well and it's in hands that know how to use it but actually I think the real utility comes from, you know, as I said you really don't want to put iPS cells in patients unless you're interested in giving them a teratoma. You really want to take the fate of those cells clinically, so cell types, and that's where I really think the technology is going to be most utilized becauseextremely versatile. You can make a modified Web Conference with Dr. Derrick Rossi

RNA for basically any cDNA, make any protein. You can add in multiple proteins from anywhere from one to probably a thousand. You can dose titrate those proteins. The more RNA you put in, the more protein you get. The less RNA, the less protein. You can do sequential programs so you can put RNA A, B, and C in before days one through three during your differentiation protocol and then you need different factors tomove that cell that now made towards another cell so you swap out those RNAs for RNA DNF. So it's amazingly powerful as a tool for cell fate engineering. In fact in collaboration with many colleagues here in just that is using this technology to specify various cell types from pluripotent cells which really is what's lacking in the field. We've had embryonic stem cells for mouse embryonic stem cells for many decades. Human embryonic stem cells are already going on second decade and there are not many typically useful differentiated cell types that we could derive from those because it's challenging take a pluripotent cell from ground state to a differentiated state. So I think that this technology proceeds most of its use in that domain. It has other applications too therapeutically but here's Michael, hard Michael.

- That's fine.

- 36:24 Hi, I'm Christina. So can you discuss what one means when one says that ES cells are the gold standard in stem cell research? How do tests that are used to compare ES cells to reprogrammed cells differ in humans versus mice? And what would you say is the most stringent test for stemness and how much trust would you place in the cell that passed this test?

- Okay so--

- A lot of questions, I know.

- 36:52 A bunch of questions. So the gold standard, well it's the gold standard because pluripotent, everything we know about pluripotency and a cell that is pluripotent Web Conference with Dr. Derrick Rossi

is derived from our knowledge of embryonic stem cells pretty much. That's actually not whole entirely true but at least embryonic stem cells of the various pluripotent cell types has emerged as possibly the most useful. They can differentiate in principal to all lineages. They have relatively stable genomes. They are relatively easily derivable and handleable. That's certainly true of mouse ES cells. Human ES cell's a little bit more challenging because of the ethical issues involved with deriving pluripotent cells from discarded embryos. That becomes your positive control in this field. It's a really important positive control. That is the bar to which we measure all other attempts at a cell that might have these potentials. So the reason that we use those cells in our study is exactly that. That's what we aspire to as, when we're trying to make a really bonafide functional pluripotent cell. But the importantsort of, I know you didn't ask this. Venture here anyhow. I said there's ethical issues in some people's minds about ES cells and certainly with iPS cells you bypass all that. But I would argue that it's very important to continue to use embryonic stem cells because as I said, it acts as the positive control. We wouldn't have known we had made a good pluripotent cell if we didn't have those cells to compare them against. So there's a lot of debate about this and I think to be honest everybody long term wants to see it make sense but we don't really use ES cells anymore not because of the ethical issues but possibly also because of that but more because of these issues compatibility and really if you can make a cell that's equally as potent but now is tailor-made to a patient and introduce cells into those patients without human rejection issues that's obviously where you want to go. And I think all scientists would say that. Let's see, what else did you ask me? There was a lot of questions there. Oh and the assays. Okay, so you know and think you had a review paper also that you studied but you can be more rigorous with your confirmation assays with mouse ES cells. And the reason is you can make whole mice derived from pluripotent cells by using the tetraploid complementation assay. So you basically use extra cells that for the developing embryo contribute to the extra embryonic tissues, trophoblasts and the like, and you can make it so that the ES cells or the iPS cells alone can give rise to the embryo proper. So this is what you can do with Web Conference with Dr. Derrick Rossi

the mousenot that with the human because you'd be making one human by doing such and probably do an experiment and ethically nobody condones doing that except for possibly the Raelians. So the assays are more rigorous in the mouse but we are left what we are left with ethically for human cells. So sort of the highest standard though I don't know if I really buy it is this teratoma assay. So listen to me. The reason that it's kind of considered the highest assay at least for human. When you make a pluripotent cell you can look at the markers and the molecular profile and the like but really the only thing that really distinguishes pluripotent cells is its ability to give rise to any and all cell types. And the best way to assay that is in vivo and really the only way you're left to do that with human cells is the teratoma assay. So you put these, you know, a bolus of pluripotent cells into a immunocompromised mice and you wait a few months and little tumor forms. And if you do histological examination of the tumor you can see that those pluripotent cells gave rise to tissues of the three different layers, mesoderm, ectoderm, and endoderm. So that's considered the gold standard. On the other hand, well not the gold standard, the highest level of validation that one can make. To be honest though even partially reprogrammed cells will make teratomas or have the ability to make teratomas. So we need a better assay. And some would argue, and I think it may be true, sort of looking at the sort of genetic state of the reprogrammed cells might be the best way of doing that, sort of doing it a global epigenetic profile. And if you see because we know that when you reprogram cells back to pluripotent state sometimes they retain some evidence of the cells that they were derived from, something called epigenetic memory. And I think that probably the best assay would be in the future for human would be to say okay, do a sort of like if there was a sort of relatively inexpensive whole from epigenome analysis that one could look at and say the most reprogrammed be genetically? Therefore it probably had the most developmental potential, the greatest potential to form all cell types. I think ultimately that might be the assay get.

- 43:22 So you mentioned how you don't entirely buy teratoma formation because even partially reprogrammed cells can form those three germ layers so I was wondering how Web Conference with Dr. Derrick Rossi

you knew those cells were, why you considered those cells to be partially reprogrammed if they were able to form the three germ layers.

- 43:45 Well I mean we didn't actually end up with any partially reprogrammed cells in our study but by other markers be it epigenetic or molecular some colleagues actually here and others met. What is textbook fully reprogrammed involves expression of a bunch of cell surface markers, nucleus markers and sort of epigenetic remodeling at certain loci. And it's very clear that some cells haven't met all these criteria and so these are called partially reprogrammed clones. Yet you take some of those clones into a teratoma assay and they'll make teratomas. So you could argue that possibly during the teratoma formation they fully reprogrammed then made the teratomas. I don't think that's a very good. That's probably not the case. So that's why I and actually people in the field, we all kind of know that teratoma is, you know, it's got its limitations for this very reason. But is an in vivo assay. It does allow differentiation of multiple style types from three germ layers and probably very importantly cells won't make teratomas very well but at least some that closely approximate fully reprogrammed cells actually do make the teratomas. But I think that as I said if you did a full epigenetic profile of such cells there's some clear differences between those and fully reprogrammed cells and possibly also ES cells.

- Okay, thank you.

- Sure.

- Hi, so somebody in our group

- 45:42 Hi. found a recent article that was titled Copy Number Variation and Selection During Reprogramming to Pluripotency and in this paper the investigator, the researchers claimed that reprogramming can cause genetic rearrangements and copy number variations which are alterations of the DNA in which a region of the genome is Web Conference with Dr. Derrick Rossi

either deleted or amplified on certain chromosomes. And how would this impact research or the therapeutic uses of these iPS cells and have you seen any of this in your iPS cells?

- Yes, so you're referring to a paper that was published recently from Timo Otonkoski--

- Yep.

- In Finland.

- Mm-hmm

- 46:27 And also there was actually another paper in the same issue of Nature which someone in your group may come across but a similar study from Kun Zhang at UCSD and actually we're on that paper as well because we contributed some of our cells to this and that is what they did there was they did beet sequencing of basically sequencing genomes from reprogrammed iPS cells of various types and the fibroblasts and they found actually that kind of similarly mutations occurred sometimes during reprogramming, sometimes in coding regions So I mean I don't find in either of these studies to be terribly surprising. Culture human cells ex vivo or any cell ex vivo long enough and you will accumulate genomic insult particularly as I said if you're culturing them as 99% of the planet is doing at atmospheric oxygen tension. Most people don't culture their cells at low oxygen. So you carry cells out for long enough you will introduce mutation, the nature of cultured cells. Actually interestingly that Otonkoski paper showed that these copy number rearrangements were actually only an early passage iPS cell. And when they carried the cells out to later passage interestingly they sort of lost the genetic aberrations and became actually cleaner with time. So in other words there was a selection against these sort of evolution. Most mutations that occur in the genome are disadvantageous to the cell or the organism. In human or the history of all living things mutations are all over the place. Almost all of them are bad and only Web Conference with Dr. Derrick Rossi

occasionally you get something selected for an evolutionary advantage. So basically that's what Otonkoski and colleagues found, was even though these copy number variations were to kick her toward term they would be selected against which of course is good because if you're going to want to use these cells you want to have something that's as clean as possible. But as I said, you know, he's going to be always an issue related to culturing the cells ex vivo unless possibly we can find ideal culture conditions that eliminate any possibility of a replication error or oxygen distress or various types of insults that occur. It's part of the deal. It's true also of human ES cells or mouse ES cells so I don't really see that iPS and ES cells differ there particularly. ES cells maybe if they've been established long enough to be able to sort of select against these bad clones. But if you continue to passage any cell you will introduce new mutations successive weeks So that is going to be, you know, that is part of something that we're never going to get around when we think about cell-based therapies that involve ex vivo manipulation of cells.

- So as a follow up to that you didn't surmise anything from those papers and, well, one of your papers that it had anything to do necessarily with the production of the process of making an iPSC but actually it's just inherent in normal culture conditions?

- Well yeah, I mean that's, one never knows of course. I mean for some of the cells used or many or most of the cells used in the Otonkoski paper or the other paper in Nature, back to that paper, they were virally-derived iPS cells. So viral integration of course involves integration of the genome. That is a mutagenic event. You are completely, permanently altering the genome. Now it may be that during viral integration at one site it destabilizes the genome at other sites to facilitate a greater or lesser mutagenesis. I don't know the answer to that a priori. I don't know why that would be but who knows.

- Well was there less in your-- Web Conference with Dr. Derrick Rossi

- And also, yeah--

- Was there less in your RNA-induced cells versus the viral ones?

- 51:14 There actually was although I would say that we only contributed a couple lines and I didn't do that much new sequencing other than for whatever reason on the original fibroblast. There was very few which we would have been happy to see but I would attribute that less to viruses versus non-virus. I would just say that we've always cultured our cells at low oxygen. Most people don't so I'll bet you that experiment's proper control and there's, you know, cultured cells have 20% versus 5% O2. You will inevitably find those cultured at lower oxygen will contain less mutations. So yeah, I mean it might be that we didn't integrate the genome and this contributed the very low if not negligible sort of mutation frequency found in our net ARC but I'm convinced that we know the answer to that yet. The other thing about reprogramming is it is tremendously a struggle for a cell. Think about it, you take a fibroblast or a blood cell or whatever. You know, it's sort of content with its identity. And then all of a sudden you added a complement of transcription factors that completely mess up identity for that cells. It's a very stressful The cell doesn't know what it isn't I don't know, they're you know, maybe the mentally deranged without their medication, just don't know who they are. So it is a very stressful state and there's a lot of epigenetic sort of remodeling that's going on. And with that may come genome instability as well. I don't know the answer to that but it wouldn't be unreasonable.

- 53:03 Recently a new stem cells which is induced conditional self-renewing progenitor cell has been introduced and according to like Kim et al. these iCSP cells have their pluripotential and can overcome the limits of iPSC cells as well. So I'm just wondering what is your aspect on this new approach and this new stem cells which is iCSP cells.

- iPSC cell... Web Conference with Dr. Derrick Rossi

- If this is a follow up question this isn't on the sheet.

- But I'm not sure. I'm not quite sure I caught the question. Would you repeat it?

- 53:43 Oh okay, so recently a new stem cell which is induced conditional self-renewing progenitor cells called like iCSP cells has been introduced by Kim et al. And basically they used a v-myc which is conditionally expressed by the tetracycline and according to their paper these new stem cells, they are pluripotentials and can overcome the limits of iPSC cells. And I'm just wondering what is your aspect on this new type of cells and this new approach.

- 54:21 I don't know, I actually have to say that I actually don't know because I haven't read that paper closely. So I don't think I'll comment on it simply because I'm not familiar. Not only that but a new cell gets introduced, a new pluripotent cell, once every sort of six to eight months in the literature and then it requires a lot of validation by other labs to know that we're talking about something different or something that's reproducible. A good example is something called these MAPC cells, these multipotent adult progenitor cells that were sort of introduced to a lot of fanfare in the literature pending their publish in Nature I guess it was about 10 years ago by Catherine Verfaillie and in Belgium everybody got very excited. And in fact, you know, the right. So again they were derived from adult cells and that they appeared to be pluripotent and they could make all embryos, make but turned out that these cells were pretty much almost impossible for anybody else to ever get ahold of and to work with. So, you know, yeah, let's see. The great thing about Yamanaka is this has been validated by thousands of liveslast four years since the paper was published so this is extremely validated. So yeah, I don't know, I can't speak to that paper in particular.

- I'm Sophia.

- Hi. Web Conference with Dr. Derrick Rossi

- 55:55 So my question is regarding the current court case do you think that human embryonic stem cell research subtracts from the funding that could go towards iPSC research?

- No, I don't think so. I mean I don't have the numbers but basically stem cell research in general, you know, you're talking about funding, you know, government funding, NIH funding.

- Mm-hmm

- 56:18 But much stem cell research be it embryonic stem cell, adult stem cell, iPS cell is really on the rise across the board in all fields because as I said maybe at the beginning, it's really the golden age of stem cell biology and there's so much promise and how with all the increased manpower new discoveries are being made all the time which is fueling the excitement all the more. But I wouldn't say that one particular type of stem cell research is detracting from other types of research. In fact I know that actually across the board the funding is increasing for now. Might stem cell research be detracting for funding for other fields? That probably but not specifically human ES cells. I know this has been sort of an argument made in this court case, that... But it's actually the numbers really don't support that at all. Actually stem cell research in general, be it adult stem cells or iPS cells through human ES cells, they're all really dramatically increasing. Mind you now the federal government's about to take a bigin all of them across the board but that's nothing to do with the type of research that's being done. That's just with the budget of the U.S. Government.

- 57:43 If you could possibly comment on what you feel are sort of the most pressing questions in the field of cellular reprogramming today and possibly what your lab, what kind of role your lab is playing in addressing these questions that would be great. And you know, I just want to thank you again for your time and comments. We really appreciate it. Web Conference with Dr. Derrick Rossi

- 58:00 Sure, it was fun. I'm looking forward to seeing the movie that you put together at the end of the year. Okay so well, what is the number one issue? I think it's very clear in my mind what it is. It's the elephant in the room which is making clinically useful cell types out of iPS cells. As I said, you know, both the cells for three decades, mouse ES cells, human approaching two decades and how many clinically useful cell types have we made? The answer is very few. The sort of approach people have used has been to sort of modulate the extracellular milieu of the differentiation process to try to coax a pluripotent cell from one state to another both yours substrates. This has been largely not that successful and the developmental process by which a pluripotent cellthis becomes a clinically really defined differentiated cell that basically we need clinically, a very complex So you asked but what is our lab sort of doing to move the field forward. As I told you I think really the main utility for I think is going to be the greatest utility at least in the regenerative medicine space of this technology that we've developed is to be able to actually have a new experimental paradigm now for moving pluripotent cells from being pluripotent cells to a differentiated cell type possibly by recapitulating normal developmental processes. So we know a lot about development, you know, how you get from a pluripotent cell to a let's say blood cell or a heart cell or a lung cell. And now, and we know a lot aboutplayers that move each of the pages of differentiation. So what our technology allows is transient introduction of different combinations of factors at different stages which trying to use for making blood stem cells and others that we're collaborating with are trying to make pancreatic beta cells or cardiac cells. We're doing this by knowing something about factors involved in moving the iPS cell from an iPS to a differentiated cell but the factors are stage-specific. We've never really had a good molecular biology approach for doing cell specificity before. You can introduce a virus but once you introduce the virus, that's it. It's in there. You can't modulate the control. You can't turn it off at some point or turn on another one or some combination of factors and do that multiple times. Now with this technology we've got thisability to do that. As I said, we can put factor A, B, and C in for a few days, come back for a few more days with well you still need A Web Conference with Dr. Derrick Rossi

but you don't definitely don't need B and C anymore, no D and E, and do this multiple times and in ways where, you know, that have never been possible before because we can also control the levels of these proteins. So you put in more RNA, you've got more protein. So it's amazingly dose titratable. This temporal control is really exquisite and the versatility of being able to put in pretty much any factor is really fantastic. So we're focused mostly about doing that in the lab, for making blood cells with collaborators, pancreatic beta cells. So yeah, our contribution I think is that and coming to help other labs who adopt this. But I really actually don't think, you know. You know, when we published our paper even the reviewers, they pretty much thought an iPS paper, just a little bit amazed to see that. I didn't see this as an iPS paper at all. We developed a which we applied to this problem but also it showed at the end that you could use it for pretty much in a very simple demonstration with the MyoD. But I really see that it's not an iPS paper so much as it is a whole new technology moving cell fate around and a lot of people have gotten hip to that and are starting to do exactly that experimentally and having great success. So I think that you can say that what is the, what do we really need to do in the field? Well, make cells that we can either model... The near-team goal of iPS cells is just these modeling. The far-term goal, making cells and tissue that we can put back into patients, both of which require differentiation to define lineages. So and that's really the thing we need really to get good at. And I'm kind of hoping that our technology can contribute to that.

- 1:03:19 So we're ending our segment of the documentary with each scientist saying one word about the future of regenerative medicine. So could you justdescribe your hope for regenerative medicine in one word?

- One word?

- Yes, one word. Everyone has had to go through this so I'm sorry there's like a one-word limit. Web Conference with Dr. Derrick Rossi

- 1:03:42 Well I'm going to be, I don't want to be pessimistic but I'm going to be realistic. And the word I would say is future because there's a lot of great hope and potential but it still resides in the future. We have to do a lot before we can start to treat patients. And it's an optimistic future and it's a hopeful future but it's a future nonetheless. We need to know something about the biology of these cells, cell types that we're making. We certainly need to know about efficacy and paramount to everything we need understand the safety issues involved. And so while there is great hope on the horizon it's something that we're not there yet. And I can tell you I get emails from patients or families of patients suffering from various debilitating diseases and they're looking for hope. I would do the same thing if I was let's say a parent with a child that was suffering for disease. I'd reach any way I possibly could to try to find some sort of cure loved one. But what we have to realize is that there's an awful lot of steps and the worst before we can do this effectively and safely and lively. And I think the biggest mistake we possibly make by rushing it to fast and going to clinic with something that's really not been properly validated and tested and then for example putting "stem cells" into people and let's say killing a bunch of patients because it was really a dumb idea in the first place and it was poorly controlled and understood. And that could be an enormous setback push for general because the backlash on federal funding this and that could get well don't fund the stem cells because they're so dangerous. So I think even for the future we need to be very, very careful. So yeah, I don't really mean to be pessimistic. I just want to be realistic in that regard to say that yeah, there's hope but we're a ways off.