Rhonda: Right, right, a lot more to learn. Epigenetic age reversal, that's a big interest, of course. And I'm sort of curious about, like, some of your thoughts on some of the...there's been some recent studies. So, we were talking about programming, right, we were talking about, in a way right, with the developmental program and the epigenetic clock sort of really tracking that well, being part of that, in some way connected. I don't exactly understand why [inaudible 00:42:11]. But I don't think anyone knows. Okay. So, some of this work with interrupted cellular reprogramming, or the partial reprogramming, as it's called, a lot of the works come from Juan Carlos Belmonte's Group where they can...maybe you can explain like what this is to people and how that affects epigenetic aging or what's known or not known?

Dr. Levine: Yeah. So, this really came out of work, originally, from Shinya Yamanaka who discovered, what we call, these Yamanaka factors, which are four transcription factors, we just call them OSKM, which, when expressed, you can actually take a somatic, so, an adult cell, and convert it back into what looks like an embryonic stem cell. So, we call these induced pluripotent stem cells. And then you can use those to make a number of different types of cells.

But the interesting thing and why aging researchers got really invested in this science is that, not only are you making it embryonic-like in terms of its stem-cell properties, but the epigenetic clocks seem to be almost completely reversed. And we've actually shown recently they're not completely reversed but you can take a skin cell that has an epigenetic age of 40 and do this, it takes, you know, a few weeks to do, and basically get back to an epigenetic age of zero in those cells.

Rhonda: And you keep it the skin cell, it doesn't lose its identity?

Dr. Levine: Yeah, so, it loses its identity...

Rhonda: Okay, when [inaudible 00:43:45] back.

Dr. Levine: Yeah, so, this is considered kind of this full epigenetic reprogramming. And then what Juan Carlos Belmonte and others have done is look at this idea of partial reprogramming. So, can we push the cell back a little bit? Because actually what we find is that this age reversal happens first prior to the cell losing its identity. So, can you do that part without pushing it all the way back, what we consider up or down the landscape, to this pluripotent stem cell? So, can I just make an old skin cell a young skin cell but still a skin cell? So, that's the goal.

Rhonda: And with some of the recent work, at least out of his lab, they're using a premature aging mouse model, a progeria model, and have shown...I know there's a new publication I haven't read, just came out, but the older one, the first one, 2016 or something, cell paper, I remember they showed, in multiple different organs, it seemed to reverse some of the hallmarks of aging, you know, and the organs were performing functionally a little bit, you know, younger than you would imagine, at least in this premature aging mouse model, and I think even health span of this mouse model that's prematurely aging, it seemed to be improved. I mean, what that means for humans, that are not mice, with premature aging syndromes is to be determined but the epigenetic clock was also reversed as well. Right?

Dr. Levine: Yeah. And I think the new publication, which is done in more of a wild type, not a progeroid mouse, does show kind of some reversal of the epigenetic clock. And you can do this just cells in a dish, we can partially reprogram them and show reversal of the epigenetic clock and other functional improvements in the cells.

Rhonda: So, to you, what does that mean, like, that you can do that?

Dr. Levine: Yeah. No, I mean, I think this is the most fascinating thing. Again, I don't know in terms of translation, like actually making this a therapeutic, and I don't even think people were at the point where [inaudible 00:45:41]. But yeah, I just think it's so amazing. I mean even the original thing, that you can take, you know, a skin cell and turn it into an embryonic stem cell. And just we always think of, you know, like, this is one direction, cells are going to only move, you know, what we consider this landscape, in terms of their states, and they can only go from this state to that state. The idea that it can go back I think is amazing, and I think just understanding how that process works...

And then the other thing we're really interested in is what are the features of this programmed cell? Like, does it truly look like a young cell or is it a totally different type of cell that, in nature, maybe we haven't even seen? And what does that mean for how it's going to function?

Rhonda: Totally. The questions I have on my mind are, "Okay, well, you take this, you know, 40-year-old skin cell," as you mentioned, "and let's say you're going to completely reprogram it to a stem cell and your epigenetic age goes back but, like, what happens to all the damaged mitochondria? Are they still there? Like, what about the pieces of DNA that, you know..." Like, is that stuff still there? Like, where does it go? How does it go away if it does?

Dr. Levine: The exciting thing is actually the mitochondria seems to also be kind of rejuvenated. I mean, I don't really like that term, rejuvenated, but it seems to be kind of set back to a better functioning state.

Rhonda: Oh, really?

Dr. Levine: Yeah, again, it's not clear how all these things are linked to each other. I think the other question though is, you know, cells also build up kind of these aggregates and other, you know, nasty kind of byproducts that accumulate. What happens to them? I don't think we know that. But it's an important thing I think to figure out.

Rhonda: So, I mean, if the epigenetic clock, because it's controlling gene expression, it's like, well, maybe the nuclear coded mitochondrial proteins, maybe everything's just bouncing back to how it was and, so, you're building better mitochondria. Right? I mean...

Dr. Levine: Yeah, I have some colleagues who argue that it starts with the mitochondria, getting rejuvenated, and then that's how everything else...but yeah, I think...

Rhonda: Yeah, it's back to that hallmarks of aging, you know, mitochondrial dysfunction...I mean or, as you mentioned, it's probably not just one thing, it's a combination of all these factors together combined where your proteins are misfolding and your mitochondria is functioning and, you know, your DNA damage is accumulating. And so, yeah, I mean it's a fascinating area. And the programming part, like, I was just curious what your thoughts are in terms of the basic science? Like, to me, it goes back to, again, that program. Like, there's something going on, we don't quite understand, but it's something.

Dr. Levine: Yeah, this comes back to this whole idea that I don't think what we see with aging is just random stochastic damage or errors. I think we've always thought of aging as just accumulation of errors but it really might just be a program that kind of goes wrong and there's nothing evolutionarily that needs to prevent it from doing that because it doesn't benefit, you know, fitness to prevent that program from going wrong. But the idea that it can be reprogrammed, again, using the operating system kind of analogy, that you can just take an, you know, operating system that's not doing well and do an update and take it back to this better state...and again, we need to figure out exactly what that means, but I think it's really exciting.

Rhonda: It is. And like there's no doubt that accumulation of damage does play a role in aging but like maybe it's not the cause or the only thing, maybe it's just the feed forward loop accelerating it. Who knows, right? I mean, it's also interesting.

Another really interesting area is the plasma exchange. I'd love, like, you know, for you to kind of explain to people what some of this interesting research is in the aging field, plasma exchange. You can start back to, you know, their original parabiosis studies maybe.

Dr. Levine: Yeah. Well, and actually I think this relates exactly to what you just said is there is accumulation of damage and there are, you know, these things that are accumulating in our systems. And it could just be the program responding to that damage in a way that, you know, it was set up to do. So, this idea of parabiosis, I mean, this is, what, a century...really old. Like, people were doing this in like the early 20th century, where basically you can take two mice and connect their circulatory systems. It's not a pleasant procedure if you're one of the mice but they'll do, what's called, heterochronic where they take one young mouse and one old mouse and connect them and then just say what happens to the aging. The young mouse, you know, is now having some influence from the old mouse and vice versa.

And what we find is that the young mice has accelerated aging compared to one that's paired with another young mouse, and the old mouse is somewhat rejuvenated compared to an old mouse, compared to an old mouse. And then, more recently, people said, "Okay, well, maybe you don't have to connect them, you can just do this whole plasma-exchange method where you can put young plasma into an old mouse," and it seems to, in some ways, again, rejuvenate them. Not to use that kind of snake-oily term but that's the best we have.

And actually, we've been doing this with cells in a dish. So, we actually buy serum from older individuals versus younger individuals and we can grow our cells in these two different conditions. And we, again, can age even fetal cells using old serum, the young serum seems to be not as problematic.

Rhonda: Very interesting. I know some of the recent work out of Irina Conboy's lab at UC Berkeley, what was interesting to me about her research, or her recent research, was that they were able to take this plasma and, you know, basically, it was just the saline and albumin...they took old mice and, like, it was essentially diluting out their old plasma and it rejuvenated these mice. Whereas they did it with the young mice, there's really no effect. Yeah. So, it really indicates, like, there is, as you mentioned, there's something accumulating, at least in the bloodstream, with age that may, in some way, be accelerating the aging process. So, what do you think the epigenetic age would do, like, if that was measured? Is that going to be measured?

Dr. Levine: Yeah. So, this is what we're doing in the cells. So, actually the Conboy's were the first ones who did this in-vitro experiment as well, so, that's how we knew that it would work. And now we're looking at the epigenetics of those cells and also the RNA. And people have started to do this too in terms of the mice. I don't know if they've done it in terms of just saline-albumin exchange but, in the normal kind of parabiosis context, it does change the epigenetic clock. And so, the question again is, "Is the methylation patterns that were capturing the clocks just a response to the accumulation of these kind of problematic factors?" Because people always wondered, "Oh, is there something magical in young blood that's rejuvenating?" versus, "is it just this problematic things that accumulate in old blood?" and it seems to be more of that. And yeah, the idea that you can just dilute it out and get the whole program kind of responds and rejuvenates itself I think is really amazing.

Rhonda: So, I guess the main questions would be, you know, at least if I remember from the Conboy study, like, even the brain, like, I think it was the hypothalamus or something, I don't know how significant it was in terms of like they were measuring, whatever biomarkers they're measuring for marking aging, it seemed to be better, even in the brain. The question would be for the epigenetics is like, "Well, is it again one of those things with the cancer chemotherapy experiment where there's just inflammation, there's something causing damage, and it's a transient thing, like then you have to keep getting these plasma exchanges, you know, which isn't yeah sustainable really." Well, I don't know, at least I think I don't think it is." You know, or is there something that does it long-term effect the other organs and stuff? Like is it something that's going to be a permanent thing, you know?

Dr. Levine: I mean I think we don't know. Actually, I'm stealing this from my husband, so, I'll give him credit for it. He talks about...it's kind of the like climate change in the body. Right? So, the cells are in a problematic climate and they're going to not behave the way that, you know, they should be. And then, if you remove that, you know, everything kind of gets better. But if it's not sustained, how quickly is it going to return? And I think we don't know that. My guess would be about as transient as kind of the effect...you know, if you could dilute and that's maintained for a while, it would probably be maintained in terms of the cells' kind of features and epigenetic measures. But yeah, if it returned really quickly, then I think it would be more transient.