News from Harold Katcher’s Lab

The big news: (1) the active ingredient in E5 is exosomes. (2) Young exosomes retain their ability to rejuvenate across mammalian species lines. Work that remains: (1) calibrate a new generation of methylation clocks. (2) Combine removal of old exosomes with addition of young exosomes. Optimize dosage and timing of treatments.


What is in E5, Katcher’s fountain of youth? From the beginning, it was described as a “plasma fraction”, leaving us guessing. Blood plasma contains thousands of chemical species in tiny quantities that nevertheless have powerful, systemic signaling effects. I thought once the patent was applied for, there would be no more need for secrecy, and the patent would reveal the formula; but the patent application was for a process, not a substance. Katcher and Sanghavi claimed rights for a broad array of substances extracted from the blood in different “embodiments” of the patented technique.

Then, in last month’s preprint posted on BioRxiv, the authors casually revealed the secret, almost parenthetically. “the exosome fraction of the plasma, which we term as E5”. So E5 is not large proteins or small proteins, it’s not non-coding RNAs — according to this document, E5 is made of exosomes and exosomes alone. Independently, a group from the Smidt Heart Institute published in July their finding that exosomes from embryonic plasma had a profound rejuvenating effect, while plasma without exosomes had no effect. (Akshay alerted me to that paper when it came out.)

This new preprint reports on experiments using E5 from piglets to rejuvenate a total of 14 rats. Epigenetic markers, cognitive, physical, and metabolic markers all improved impressively. Some of the limitations of Katcher’s experiments remain to be resolved. Because of small numbers and killing of test rats to obtain tissue samples, we still do not have a strong indication to what extent the restoration of all these markers of age translates into increased longevity.

A major finding is that exosomes from a young pig can rejuvenate an old rat. It is remarkable, very lucky, and certainly not to be taken for granted that such specific signals can cross species lines. What a horrible situation we would have been in if it turned out that the best anti-aging treatment available depended on harvesting the blood of babies.

Results

There were only 14 treated rats in this round of experimentation. 6 males in round 1 were sacrificed so that methylation of their organs could be tested. 6 female and 2 male rats in round 2 were kept alive long enough for their blood to be drawn for methylation testing, but not long enough to determine their lifespans.

Methylation clocks: For 6 treated rats, age 109 weeks, these were the average epigenetic ages by tissue:

Tissue Epigenetic age
liver 28 weeks
blood 39
heart 58
hypothalamus 82

The preprint does not provide error bars for these average epigenetic ages, but I expect (based on the published minimum and maximum, and the small sample size) that the 95% confidence intervals are broad, corresponding to rejuvenation by anything between 30% and 70%.

The fact that various tissues are rejuvenated by exosomes indicates that exosomes are a medium for transmission of age information through the body, and that young exosomes in the blood are able to induce system-wide changes in gene expression.

It’s common for medical gerontologists to frame their work in terms of their favorite evolutionary theory of aging, which is always the 70-year-old, conservative theory that won’t raise any hackles. I’m grateful that Dr Katcher is unabashed about framing his medical research in evolutionary terms, and that his evolutionary theory agrees with mine. Aging is a timed program of self-destruction, coordinated through the body by chemical signals, and evolved because ecological communities are more stable when individuals have predictable lifespans.

The hypothalamus is a neuroendocrine region of the the brain that has been associated with circadian timekeeping (day-night) and also with tracking biological age. The fact that gene expression in the hypothalamus was rejuvenated to a far lesser extent than the other three tissues suggests that hypothalamus plays a special time-keeping role, and that perhaps the hypothalamus is a source for these exosomes in vivo. It may be that the hypothalamus gradually erases half the epigenetic gains from the E5 treatment over time. From this and previous tests, we still have limited data on long-term benefits of E5 treatment.

Blood markers:

“we measured the levels of the following biomarkers on 30, 60, 90, 120 and 155 days from the start of the experiment: bilirubin, serum glutamic-pyruvic transaminase (SGPT) and serum glutamic-oxaloacetic transaminase (SGOT) to monitor liver function; triglycerides (TG), HDL and cholesterol to monitor risk of atherosclerosis and heart disease, and liver function as well; glucose to monitor the pancreas and diabetes; and creatinine and blood urea nitrogen for kidney function. The levels of all these biomarkers in the treated old rats were altered towards the values of young rats, without exception.”

(Note: Blood sugar and triglycerides in treated old rats was reduced to levels indistinguishable from untreated young rats. In humans, Type 2 Diabetes is a primary means by which the body destroys itself. — JJM)

Cognitive recovery: Treated rats learned to escape from a Barnes maze faster than old rats.

Oxidative stress: ROS markers were restored close to a young state. Antioxidant enzymes GSH, SOD, and catalase were increased close to youthful levels.

Inflammation: TNF-α and IL-6 were reduced close to the levels of young rats. Nrf-2 was increased close to the level of young rats.

Sarcopenia and muscular fitness: Grip strength of treated rats rose within a week to levels comparable to young controls, and remained strong for at least 30 days.

A new/old biological clock based on glycation of antibodies

IgG is a blood component, another name for “antibodies”. These bind specifically to viruses and bacteria and tag the cells for attack by phagocytes, another immune component.  Antibodies are not cells or globules but individual Y-shaped protein molecules with both arms of the Y able to bind to the (same) antigen, and the stem of the Y modified to attract a phagocyte only when the other end signals that it’s “got one”.

Immune aging is a big deal, as it was identified as a prime cause of aging by Roy Walford already in the 1960s. Greg Fahy pushed the theory into a therapeutic concept four years ago with his TRIIM program. With TRIIM, the idea was validated that a rejuvenated immune system can signal rejuvenation to epigenetic age and, presumably, other aspects of aging.

Dr Fahy also subscribes to a programmed theory of aging, and he personally offered me my first opportunity for a wide audience in the anti-aging community in 2010.   Mitteldorf_Evolutionary-Origins-of-Aging.pdf

Hence, “immune age” is a promising target for measuring success of anti-aging interventions, independent of methylation age. This idea has been realized with a Glycan-Age clock. Antibodies in the blood are coated in complex carbohydrate (sugar) molecules, or glycans, and the structure of the glycans carry information, analogously to the way that DNA or proteins carry information via their sequencing. The particular sugars attached to antibodies change with age, and they become important signals promoting inflammation later in life. Clocks based on glycation of antibodies are as old as methylation clocks, though they have been developed to a lesser extent.

It is surprising, perhaps, that glycan age was never measured in the TRIIM study, but Katcher’s group added glycan age to the tests on his treated rats.

“a significant (p < 0.05) reduction in the relative abundance of the pro-inflammatory agalactosylated IgG2a glycoform (G0) was recorded. This was accompanied by a simultaneous upsurge in the antiinflammatory digalactosylated glycoform (G2)”

Is there such a thing as “biological age”?

The preprint includes the statement,

“However, in the context of aging and rejuvenation, it is crucial to differentiate between improved health or organ function, which could be achieved via medication or surgery, and genuine molecular age reversal.”

Intuitively, we all feel that this must be a legitimate concept. In our experience, we can look at most people and form a pretty good idea how old they are, and this translates into reliable expectations about their stamina, resistance to disease, and ability to cope with a fundamentally new environment.

But this is because we’ve mostly encountered natural humans. We know, for example, that movie stars can have expensive surgeries and skin treatments that make them look decades younger. We don’t expect that people who have had a face lift will live longer or have more youthful vitality.

It is perfectly possible to imagine anti-aging treatments that rejuvenate the liver but not the nervous system, or that slash the risk of heart disease but increase the risk of cancer. Hence, it is advisable to report a variety of functional and metabolic tests rather than relying on any one measure of “biological age”.

What is measured by the methylation clocks?

For more than a decade, I have been committed to the paradigm that the body’s gene expression changes with age, and that changing gene expression is a driver of aging. Methylation, as a convenient surrogate for gene expression, would then seem to be a reliable measure of biological age.

But in the last four years, I’ve entertained questions about this paradigm, and I am no longer absolutely committed. The crucial question is: Does gene expression change as a driver of aging, the body destroying itself with inflammation and by scaling back autophagy and repair? Or does gene expression change in response to damage accumulated with age, scaling up repair functions to mitigate damage?

As recently as four years ago, I wrote confidently that the former greatly predominated. Epigenetic changes drive aging. On this basis, I was confident that if you change the methylation clock, you must be changing the driver of aging, and this was bound to lead to longer lifespan.

I have since become convinced that both kinds of changes in gene expression accrue with age, and that there is no easy way to tell the drivers from the responses. I first blogged about this in response to the GrimAge clock. GrimAge is the best predictor we have of remaining life expectancy, but it is based not just on drivers of aging but also on the body’s distressed response to toxins and cell-level damage.

The bottom line is that epigenetic clocks are the best measure of life expectancy that we have, and that they are improving every year, but while uncertainty remains, it is well to supplement methylation clocks with specific tests of metabolic function, stamina, inflammation, and cognitive performance.

Six new methylation clocks

Prof Horvath (presumably it was Horvath) trained four new clocks on rat tissues plus two new clocks that were cross-trained on humans and rats. Since rats have a maximum lifespan of 3.8 years and humans 122.5 years, one logical way to train a clock that works for both species would be to scale the rat ages and human ages so that one rat year is about 32 human years. This is just what Horvath did.

But in addition, he trained a second combination clock (rat and human) to read in absolute years. This presumes there are changes in epigenetics of a very old rat (say, 3 years) that are parallel to epigenetic changes taking place in a very young human (also 3 years). A strange concept, indeed! What kinds of changes would we expect to take place in a 3-year-old rat and simultaneously in a 3-year-young human? My guess is that the training program is selecting those sites that behave very differently in rat and human, and that results derived for rats with this clock will not translate.

But I would suggest that even the time-scaled clock (based on 32 years of human life = 1 year of rat life) is suboptimal. This is because different events in development and aging may occur at different points in the life cycle. A male rat reaches sexual maturity at about 8 weeks. Using a 32x multiplier, this would correspond to a 5-year-old boy. But in fact, boys don’t reach sexual maturity until well into their teens.

Prof Horvath has been developing methylation clocks for more than a decade, and his work has had a deep impact on the science of aging. To my knowledge, all his clocks are based on straight-line changes in methylation over a lifetime, and I have counseled that this is an unnecessary limitation on the clocks’ accuracy. More realistic clocks can be constructed based on parabolic curves, or even the simple expedient of grafting two straight lines together to create a “spline”. He has been creating cross-species clocks for the last few years, and to my knowledge these have all been based on a single scaling factor for each species. My recommendation is that here it is even more important to project the life cycle of one species onto another using a flexible mapping, pegged at several intermediate time points which are determined in a mathematical optimization process. Cross-species clocks will be of increasing importance as we translate mouse studies to develop experimental protocols for human medicine.

Unanswered questions

A few paragraphs up, I referred to a major issue in methylation clocks: A given intervention affects a set of methylation sites that are all associated with younger age. Which of these are actually beneficial, leading to life extension; and which represent reversion of the body to a younger state when it had less damage to deal with? For the latter, the younger state corresponds to repair mechanisms that are less active (presumably because they are less needed), and “rejuvenation” to this state could actually lead to shorter life expectancy.

To date, the best methylation clocks have been calibrated by how well they predict future life expectancy. This is, of course, very useful information, but it is all based on naturally-aged individuals. Our interventions that reduce methylation age may not increase lifespan.

In two previous rounds of Katcher’s experiments, males experienced more epigenetic rejuvenation, but they were killed before their lifespans could be measured; while females experienced less epigenetic rejuvenation, and their lifespans were measured to be only modestly extended relative to controls. Unexplained is the fact that the controls lived far longer than Sprague-Dawley rats are usually expected to live, and that a single treated rat lived far longer than 7 others that received the same treatment. These are all curious observations that warrant further investigation.

The failure of Katcher’s rats last year to live as long as their methylation age predicted should be a motivator for future research, studying not just the interventions but also the clocks.

We can try to classify different methylation sites based on theory; but if we want to address the question head-on, we need different experimental interventions that affect different CpG sites. Then we can see which CpG sites, when artificially methylated or demethylated, lead to longer lifespan. Hence, Katcher’s experiments provide a unique opportunity to determine in practice: which methylation sites, when reverted to a younger age state, actually extend lifespan? This is an experimental solution to the stickiest problem in aging clocks: which epigenetic changes correspond to drivers of aging and which correspond to responses to damage?

Do we have methylation for Sima, Katcher’s record-setting long-lived rat? If so, we might compare methylation profiles for Sima to his cage-mates that were treated the same way, yet died many months earlier. This is a start, but what we really need is an experiment in which about a hundred rats (M and F) are treated with different E5 protocols, their methylation profiles are monitored over time, and they are also permitted to live until their natural end. Manipulated methylation profiles can then be correlated with life expectancy, and from this a uniquely useful methylation clock can be constructed.


For me, the bottom line is the same thing I’ve been saying since the beginning: Katcher’s research is the most promising line of investigation in the field of anti-aging medicine, and it is a travesty that it is taking place under a single investigator’s direction, and with very limited resources. It will take extensive lab resources to optimize E5 dosage and timing, and to try combining addition of young exosomes with removal of old exosomes. This same research can be the basis for the first methylation clock that is known to predict lifespan for rejuvenated animals.

I would like to see the abundant money that is now in anti-aging research re-directed into rejuvenation via exosomes, and I would like to see Katcher sharing his techniques openly with the research community.

80 thoughts on “News from Harold Katcher’s Lab

  1. A counterpoint, if you will. What do we know experimentally, what we may know, but not yet released, and what we simply don’t know.

    We know that E5 exosomes affect some many tissues, but not all of them. This implies that there is no post natal “master” clock. We also know that in rats, the life expansion is limited, while there is a rejuvenation effect to many tissues. One tissue that has a limited E5 effect is the hypothalamus. But another signally protein (Platelet Factor 4) has experimentally shown to rejuvenate the hypothalamus. Neither seem to rejuvenate the thymus (which is directly involved in the immune system maturation). This leads me to surmise that the “master” aging clock does its job early in embryogenesis, setting multiple separate clocks, each of which has its own timer and control feedback system.

    What we don’t know yet are the following – the post mortem results from the aging-to-failure experiment. What part(s) of the rat’s system failed? That is a starting point to look for the other clocks.

    Also, we don’t know the results of the topical E5 human experiment. Was there any effect at all on human in-vivo testing. An important point.

    We need at aging “test-to-failure” of the E5 EVs combined with PF4. Does adding something that rejuvenates the hypothalamus actually increase lifespan? If not how many (and which) pathways need to be altered for the effect?

    Just an alternate viewpoint.

  2. Thank you Josh for the review of our paper. While we are analyzing the results of E5 treatment I wanted to share my thoughts on lifespan. It was important to understand how E5 was affecting each organ but as there was an interest to see if this translated to any improvement in lifespan we also conducted one lifespan study where the female rats were allowed to live to their natural deaths. What is being missed here is the import of one of the rats Sima breaking the world record of maximum lifespan for her species. I have two questions to highlight this achievement of Sima: which other rat/mice longevity study has created a world record for maximum lifespan? Second question: in our 5,000 years of recorded history have we ever seen a human crossing the maximum lifespan? Nature guards maximum lifespan very strictly. Breaking through that is something extraordinary. It is like finding out one human in a longevity experiment lived to 140 years. Even if it is only one human that manages that it would be for the first time amongst 10 billion lifespans.

    • Akshay, appreciate all the good work you and Dr. Katcher are doing on this. Not sure if you have already answered this but what is your thoughts on how the body is enforcing this lifespan limit even when the epigenetic age is backed up like that. Is there some clock that just overrides all that once a certain point is reached or is it something like the heart giving out because the stem cells eventually run out at that advanced age to replace the cells that are lost.

    • Hi, Akshay, first, contgrats on the progress Yuvan has had! It’s without question my favorite project in the longevity space. I have a couple of questions.

      1) Did you try to figure out why E5-treated rats in the lifespan experiment died and if there were any differences compared to the control group?

      2) Have you checked whether E5 rejuvenates the thymus, too?

      If it doesn’t, it could potentially be a good idea to test a combination of E5 and Fahy’s thymus rejuvenation treatment. If most rats die of cancer because their immune system without the thymus can’t fight (some) cancers off, then it may be the reason why E5 hasn’t been even more successful.

      Also, if Josh is right in his hypothesis that there are multiple aging clocks, then the state of the thymus may well be one of them.

      • Hi Daniil thank you very much. Yes we have conducted a necropsy and the report will be in the Lifespan paper we will be uploading on bioRxiv soon and submitting to a peer reviewed journal. We have not yet had the chance to test for thymus but we are assuming it should also get rejuvenated. But by any chance it doesn’t we have the world authority on thymus rejuvenation on our Scientific Advisory Board: Dr. Greg Fahy. We did not find any can solid tumors in the necropsy of treated rats.

    • @Josh “Inflammation: TNF-α, IL-6, and Nrf-2 were reduced close to the levels of young rats”

      This must be a typo, as Nrf-2 activation is reduced in aging.

      @Akshay, are you planning to include suPAR measurements in future trials?

      suPAR is a better measurement for systemic inflammation than CRP, IL-6, and TNFα alone, as it is not affected by acute changes and short-term influences

      https://pubmed.ncbi.nlm.nih.gov/34925360/

      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7812430/

    • It’s plausible that there would have been an exception where something went unexpectedly right, but it would be very hard to find a record, and if one existed verify that it wasn’t myth, mistake or fraud since both poorly supported optimism and incredulity exist on this question even today.

      People said that Jeanne Calment lived to 122, but years later some have said that was fraud. Maybe, but I don’t think it’s that hard to believe given what we know about other supercentarians. Then there’s this person from China with an even more extraordinary claim, 135 years, who I’m guessing isn’t the exception to the rule, but maybe there’s some tiny probability. There’s not much to go on what to trust on this question even in modern times. https://www.scmp.com/news/people-culture/china-personalities/article/3160416/chinas-oldest-living-person-uygur-woman

    • I think at this point it is settled that aging is both programmed cycles of growth and, after completion of development, switching many repair systems off, up to exclusion of many repairs, combined with some repair processes that were never developed in our species and thus constituting from the biology point of view hard ‘physical’ limits. And both amplify themselves in positive feedback loops (as it is known that switching off some repairs speed up deterioration processes running independently of clocks). Some of these hard limits are still not addressed, like cessation of production of elastin, which is switched off before or early on maturity.

    • Hi Akshay,
      I’ve been following you and Harold’s research for years. I’ve never posted before, but I have a question, and please forgive my ignorance as I’m just incredibly curious and don’t have a background in science.
      I heard Harold mention that the amount of E5 given to rats is way more than would be normally be found in blood. So you’re effectively flooding the system? Is that correct? I’m fascinated by the idea of conserved cross special cell signaling, as it puts me in mind of Michael Levin’s work. That somehow there is a universal development program, which leads me to my next question. Harold mentions “Youthening” of the rats. What does this mean exactly and what does it look like. Is E5 reversing the methylation age of the cells or is it fully rejuvenating them? I noticed that the weight of the rats stayed the same, which made me wonder if it was the former. Are the rats simply healthier or do they still carry their damage? Some of Michael Levin’s work seems to point to a preprogrammed physiological blueprint is that what you’re tapping into or you resetting the program further downstream?

      • Hi Paul,
        Yes flooding the system because it’s battling against existing regulatory transcription which is causing the age related changes. We have so far not tried to remove old exosomes and their cargo before injecting E5 but could be something we hope to try in future. Answer to your second question is that E5 is resetting the epigenetic configuration closer to that of the donor’s lifecycle stage. But each dose has a life. So there is a fluctuation between epigenetic patterns of old and new. This may be the reason that during the peak of the dose the rats are 3x stronger, more lively, 30+ age related biomarkers are showing a systemic turn towards a younger biology/body. But then the legacy transcription will again begin to dominate resetting the epigenome back towards its actual age. We want to create a steady state reversal and not the fluctuating one. That should solve the mystery of why the treated rats died. Would love to do a lifespan study after we achieve the steady state reset. Hopefully the rats would remain young for a really long time.

        • I wonder if the hypothalamus might be the source of a lot of these age-related exosomes. Claudia Cavadas has been experimenting with rejuvenation of the hypothalamus, but it’s tricky, not least because it’s surgically inaccessible and behind the blood-brain barrier. https://joshmitteldorf.scienceblog.com/2016/01/29/is-aging-controlled-from-the-brain-npy-and-alk5/

          Is it possible to grow hypothalamic tissue in a test tube and harvest exosomes?

          • Josh,
            It’s so tempting to believe that some central intelligence is guiding and sequencing these trillions of gene controls of activation or silencing or methylation or acetylation, etc. But such a complex central control tower would be unmanageable. Controlling millions of complex processes in trillions of cells is impossible to achieve from a single source like a part of our brain. It would be 100000 times more complex than managing all the flights in the world from a single air control tower. So we can infer that the origin of the instructions for development and aging are decentralized. In that case the only place in a cell that contains such data is our DNA. There is no doubt it’s a programmed. This program is executed through timed global epigenetic changes. Let me try and show a sequence of collapse we see that is sudden after puberty but manifests gradually as our systems fight a losing battle in trying to cope. Josh I will start by citing a paper mentioned by you in your 2015 post: The Mystery of Aging, Solved at Last! The paper you mentioned was: Repression of the Heat Shock Response Is a Programmed Event at the Onset of Reproduction by Labbadia and Morimoto – probably the most profound finding with regards to the source of aging. In this paper the authors share their finding of a global epigenetic change occurring in cells through H3K27me3 which is trimethylation mark, an epigenetic modification to the DNA packaging protein Histone H3. Caused by regulatory molecules secreted by germline stem cells through their exosomes. We don’t question the physiological changes we undergo during puberty but just after puberty in that same sequence is one important change that launches aging originating from germline stem cells. This epigenetic mark collapses multiple stress resisting support systems all of sudden by 40% to 80%! They highlighted heat shock protein collapse which leads to increase in protein production errors. But they also tested other such pathways: endoplasmic reticulum UPR (UPRER), oxidative stress response (OxSR), and mitochondrial UPR (UPRmito) and found all of them having a sudden decline after puberty of 40% to 60%. The nuclear architecture is tightly organized and accurate transcription/gene expression is a result of spatial orientation. So any loss in this would initiate cascade of errors in transcription and cause aberrant gene expression leading to further collapse of repair pathways and misexpression of genes. This important tight nuclear organization is dependent on certain proteins like the WASH protein: https://www.fredhutch.org/en/news/center-news/2015/03/study-reveals-how-nuclei-maintain-shape.html
            As the protein production is affected so would the production of WASH proteins. Unraveling of nuclear architecture leads to major errors in transcription: https://www.nature.com/articles/s43587-022-00317-6
            Leading to decrease of longer transcripts with aging. These longer transcripts the authors found are associated with longevity genes. But why only longer transcripts are affected? Explained in this paper: https://www.nature.com/articles/s41588-022-01279-6
            So as we can see in this chain of sequences what started from the germline stem cells leads to collapse of stress resistance pathways and gradual loss of repair pathways which led to protein production errors which led to loss of the tight assembly of the nuclear architecture which led to transcription errors and aberrant gene expression which would lead to deterioration of the cell, its tissue and organ and finally end in our death. This is just one chain of events which are triggered after puberty. There are many others. Citing some more interesting papers related to loss of nuclear architecture and its effects in aging: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3414389/
            https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253059/
            https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8673774/

  3. Things do seem to be pointing to young exosomes being the best thing out there for healthspan. We probably have many years to go to figure out what seems to be causing the limits on lifespan extension. You see the rats looking way younger and being more active than the controls at those advanced ages but once they are at a real old age they seem to go downhill quickly and die anyway. I think one of the controls in Goya’s recent work outlived all the treated rats who received young blood despite the treated ones living longer on average and having lower epigenetic ages. When you look at the pictures of the rats at times before they died the treated one’s looked a lot better as the controls looked haggard compared to them.

    • Mike true, these mysteries of biology of aging. As Josh said we inherit a program that ensures through multiple cascades that our cells unravel. Interventions like E5 are fighting these legacy forces. It’s like killing us slowly through a thousand cuts. E5 tries to bandage lots of them but death requires even just one cut to end life. Nature does not design without cause including the enforcement of a maximum lifespan limit. That’s why it’s so exciting to overcome such an impossible block in a mammal. Shall we find ways to make all treated continue beyond maximum lifespan in youthful health? I think so. Work in Progress.

      • It might be worth considering starting treatment at earlier ages, Akshay. A murine model is an accelerated aging model that by even middle age is a storm of inflammation, aging and incoming death! And you are trying to hold that back with youthful (but not completely understood?) signalling from exosomes. It’s a tough ask. The success (on lifespan as well as much easier healthspan) depends on overcoming the inertia of a system moving rapidly on a downward trajectory. In detail that means you are trying to reprogram cells in a way that will ‘stick’. You need them to ‘flip’ to your side and start producing ‘good’ secretions. It may be that there is a subset of cells that are refractory and will stubbornly continue to pump out aging factors. It may even be that restoring youthful activity could increase some aspects of harmful secretions. We should be able to work out how profoundly you are influencing the trajectory of aging by measuring the time for the benefits to fade (say on a marker of inflammation or some other measure) between earlier and later iterations of E5 treatment. If the gap between treatments can be increased (maintaining the same benefits), then you are winning. If the gap needs to get shorter to maintain the benefits, or the benefits cannot be maintained, then you are losing, though lifespan extension is still possible.

  4. It seems to me that there is more of an emphasis on lifespan as opposed to healthspan in the “community” or articles here (like this one), not that people don’t want improved vitality. Is that because older people, who are beyond 50s and maybe not in all that good shape (or never were) physically, see less of a purpose for it as opposed to longevity? Is it the longevity idol that is seen a lot among humans, but mostly in the west? I’m curious. As a younger or middle aged person, I would wager anyone with means would take treatments for healthspan and just live healthily to whatever the natural age might be, and either be fine with that or even markedly prefer that. Any thoughts on this, my friends? Thanks.

    • My view is healthspan is huge. If things worked in humans like rats you could literally have a 75 year old like an in shape 50 year old. People would love that even if most still died at 90 – 95 years old.

    • Healthspan is important. I’m currently a relatively healthy 65 (played 2.5 hrs of tennis is 104F heat today), but would love to carry that ability another 20 years or more into my mid 80’s.

  5. Josh I believe you are too focussed on a theoretical intracellular methylation score when you should be more interested in the type of cells that are being measured, i.e. inter cellular changes. This is what IMO determines methylation age in practice.

    It is my (well founded) belief that it is much easier to reverse methylation age than real age. I and many others have reversed this measure by 5-10 years, and I even know of 1 case of reversal by 28 years! I myself have 14 methylation age results, and in the space of 3 months I was ‘able’ to increase my epigenetic methylation age by 7 years and in the next 3 months to decrease it by 8 1/2 years. When you submit a sample the most important factor is the composition of the cells in the sample. It is relatively simple to increase stem cell proliferation such that the reading is hugely decreased. It is also relatively simple to use telomerase activators to make all those new cells divide many more times than would normally be the case, and in that instance your epigenetic age is hugely increased.

    In conclusion, this is a fairly malleable clock and I suggest that it probably only suggests the rate of aging at that point in time (at best) and not an absolute measure of where you are in the progress of aging.

    This is not to suggest E5 is not causing profound regeneration (after all they looked at ‘real’ biomarkers too), just that methylation aging cannot be used as a reliable proxy of this regeneration.

    • Horvath makes the distinction between “intrinsic” and “extrinsic” methylation age. Intrinsic is the methylation changes within cells; extrinsic is the change in proportions of different cell types. What you are describing is just what Horvath calls “extrinsic” methylation age.

      Tru Diagnostic claims to be able to tease these two apart with an algorithm that separates intrinsic from extrinsic methylation age.

      I agree with you that the epigenetic rejuvenation that Katcher is achieving may not translate into commensurate longevity. I haven’t given up on the possibility of better methylation clocks that can be relied up to predict lifespan when interventions are made. My message at the end of this piece was that I think an extension of Katcher’s experiments might help to give us the data we need to create the better clock.

      • I’m a little sceptical. Even if you could eliminate the contribution to methylation age due to the change in the composition of cell types, that still wouldn’t solve the problem (completely), as the cell types typically measured turn over quickly, and therefore intracellular epiAge acceleration due to extra divisions (for example), which is easy to influence, will throw off results. This is a complex system and we can’t reduce it to a single measure so easily!

        But I may add Tru Diagnostics as a comparison to the other providers (MyDNAge and TruMe Labs) I have used and see if there is a difference.

    • Mark, If you don’t mind me asking what were you taking to reduce methylation age? Is there anything out there besides Ca AKG that can have a big effect on it? I take 1 gram of Ca AKG a day and though I have never tested it I am pretty sure its had some effects as my eyesight at 54 went back to what it was in my early 40s. The change just hit me all at once at about the 6 month point of taking it. Are there other supplements that affect it that much and would taking more than 1 gram of Ca AKG a day give much more of an effect you think?

        • Pro Health longevity Calcium AKG. I get it from IHerb but I think they have their own site also. That stuff someone is selling is a ripoff imo because its like triple the price and you get no more AKG than with this. I dont buy that claim that more gets absorbed either than other Calcium AKG sources.

          • The bioavailability of AAKG (arginine alpha-ketoglutarate) is about 50%. This means that the body can absorb about 2.5 grams of 5 grams of AAKG. The remaining 2.5 grams are excreted unchanged.

            There is no scientific evidence that calcium alpha-ketoglutaric acid (CAKG) is more effective than arginine alpha-ketoglutaric acid (AAKG). In fact, AAKG may be more effective because it is better absorbed by the body.

  6. i just hope there is no attempt from over zealous governments to control blood products. With the links being suggested between livestock and global warming etc. It would seem an excuse to make inroads in this direction. I am sure they will not condone life extension. Healthspan perhaps ? I am not a pessimist, generally. Well done to Yuvan. I have every faith.

  7. Akshay, do you agree with Josh that the Hypothalamus could be the most pivotal one producing time regulating signals?

    If so, then the already rejuvenated hypothalamus should produce more youthful exosomes to create a viscous cycle?

    Too interesting.

  8. Hi Leo, I would love to agree but I am of a different view. There can not be any central clock. It’s globally distributed in every cell. Every cell carries spatial and temporal information in the DNA. Spatial tells that particular cell where exactly it needs to be in the 3 dimensional collection of cells that make a body. And also to live up to its position in the collection it has information to switch on specific genes and turn off all others. But there is also temporal information that makes each cell follow sequential trend of changes. For example around the age of six our milk teeth fall out and around 13 puberty sets in and in 50s menopause. These are not stochastic changes but programmed temporal changes. After puberty there are also a chain of sequential changes that begin to unravel the cell’s tight nuclear assembly culminating in what we begin to notice as aging. But the process starts just after puberty – we go about our daily routines not knowing how inside us every cell is slightly loosening it’s assembly. There is master controller – it’s global and every cell is born with these programs embedded in them.

      • It seems clear that aging, measured instantaneously as a snapshot, is the just sum total of the signalling in the blood. All cells contribute to it and sample it in turn. We can certainly intervene in the contents of the blood, as you are learning to do. How profoundly this affects bodily tissues already aged is the big question. E5 results are promising such that I’d be suprised if at minimum, you are not stopping things getting worse. It’s just that an old rat’s mortality is so high that stopping.aging, even reversing it somewhat, might not equate to big lifespan gains.

        • I should have said aging can be measured accurately using a blood sample. That doesn’t mean the blood is all there is to aging. Clearly the blood is reflecting the state of the body. And as Josh rightly points out, once you intervene, the clocks you used to measure natural/normal aging, may no longer apply.

          • Akshay and Mark seem to me to be accurate here. Mark, do you suspect that relative improvement that you can get (in humans) will be closer to slowing the aging process (diminishing pro-aging signals in the blood), or rejuvenation? It is a technical but important point, and you rightly question it in the sense of “what does it do to already aged cells/tissues?”.

            That is, if you had to guess, would you say that it just keeps people in a more streamlined state (they get to live “longer” in their 40s or 50s), or they actually appear to be rejuvenated and/or feel that way?

            Good stuff, my friends.

          • Mark and Palamas, blood is reflecting the state of the collection of cells in our body. Can we stop aging or actually reverse it? I would like to cite a seminal paper published Professor Hayashi of Tsukuba University in 2015. Josh has written a blog on it. It shares so manny nuggets: aging phenotypes are caused by epigenetic regulation, rate of cellular damage is the same between young and the old what changes is the rate of repair and most relevant to this conversation he shows that this age associated damage is reversible. He demonstrated this by reversing mitochondrial respiration defect in the old cells by infusing glycine. Of course it is only in vitro and of course it’s only one defect but it is nevertheless provides evidence that such damage is reversible. We all know the importance of mitochondrial health especially in relation to aging. So in principle the condition of aging should be reversible and we have repeatedly achieved that in old rats resulting in stronger, livelier, youthful rats including one breaking the maximum lifespan barrier. Now we need to figure how to extend this youthful reversal for all the treated. Work in progress.

          • Hi Palamas,

            I personally feel that signalling via exosomes or something similar in the blood will restore health to a more youthful level, but probably won’t extend lifespan by the amounts we want. By that I mean people will live longer – as you tend to do when you are healthier – but it will be in the order of years or a decade, not centuries of indefinite youth. I believe this because of the damage that has already been done to the genome and because of certain hardwired in limitations, like lack of telomerase, which have yet to be adequately addressed. The proximal cause of our ‘inevitable’ demise, the genomic damage, we might be able to overcome, if enough remaining (stem) cells are pristine enough, and those cells that aren’t can be encouraged to die and are properly replaced. It is a little like diluting aging factors/increasing youthful factors in the blood, but you have to actually do it with cells. So, its a longer term project and won’t happen overnight.

          • Hi Akshay,

            I like that Hayashi paper too, but we shouldn’t assume epigenetic suppression of SHMT2 leading to mitochondrial ETC defects is a cause of aging. It is tempting to jump to that conclusion, but as Josh likes to point out, untangling cause from consequence is a tricky business. In old worms, glycine rises with age, but giving them more makes them live longer! Totally plausible to me that SHMT2 sabotage could be an adaptation to something else. For example, SHMT2 (or whatever the equivalent gene is in worms) is 2-way and it might be that mitochondria are putting an export ban in place.

  9. @Akshay,

    Have you guys moved most of your operations to Mountain View? I saw an interview with Harold that said he would have continued treating Sima, but the experimenters in India made a different call. Hopefully, we can get that with the next batch to keep going.

  10. Hi Adam, we have labs both at Mountain View and Mumbai. That decision was taken by our PI at Mumbai lab out of ethical reasons rather than lack of resources. Each injection is a painful experience especially as their age progressed. Whether that was a good decision from science point of view is debatable. That didn’t seem to stop Sima from creating a world record for maximum lifespan. Still can’t stop feeling incredulous about that. It’s like what we would feel when the first human crosses 140.

  11. Not sure if it has been mentioned , but there has been a recent interview with Dr Katcher on YouTube channel Modern Healthspan. Apologies if duplicating this information.

  12. Mark, Prof Hayashi does claim for it to be the cause of aging. My understanding was that he meant to prove a point that such damage can be reversed.

    • Lol for some reason the word ‘not’ seems to be eaten up by the Lord of Typos. I meant Prof Hayashi does not claim for it to be a cause of aging.

      • yes, Hayashi did establish very important things, like mitochondrial decline being epigenetically regulated rather than due to genetic mutations (either of the nuclear or mitochondrial genome), as was previously believed.

        I am a little suspicious of the results of ‘rejuvenation’ from epigenetic reprogramming however, as only a few cells are actually rejuvenated, many die. One of the biggest problems in aging research is the difference between cell culture and a live animal.

        • Mark, totally agree with you in that: I too do not believe that cell culture derived therapies or in vitro experiments truly represent what will happen in vivo.

          • Akshay, I was curious if you think E5 could have the same issues that Yamanaka factors seem to have. If I remember correctly in one study the oldest mice were resistant to its effects. That it turned back the epigenetic age on the 50/60 year old human age equivalent mice with no problem but the 80 year old equivalents weren’t getting the same effect and seemed resistant to the rollback.

  13. Akshay, I have a testing business question.
    Having seen Dr. Katcher’s hand results from topical application, have you considered using topical E5 for the scalp? Even with Rogaine, there would be a huge market for a treatment that would raise a full head of hair. it might be worth a test subject or two during the trials.

    Hope this gets past the Recaptcha

    • Yikes. I had the hair idea for months and all I did was think there was probably some complexity that I wasn’t aware of preventing the decision to test that — like it getting into anatomically different pores requiring greater FDA approvals, the need for the patients to go bald so the dosage could be applied evenly to see if it really works, etcetera. But George was right, and I should have just asked.

      Most men start losing their hair just as their earning potential is starting to increase. It’s a time when many aren’t married, have a lot to prove, and so care a lot about their appearance. For higher earning careers that increase tends to happen several years later when the problem is more advanced.

      Also, provided you don’t need regulatory approval or it was very easy to get for a topical and the affect is fast and obvious, I would not be surprised if you could get a Silicon Valley VC to try it themselves and get them very interested. Though I suppose even with the skin cream you could just carry around a sample or a few individually packaged does with the Yuvan name and contact information on it. Or maybe two doses so they don’t throw away the container with the contact information until after they notice the affect.

      • Adam giving it to VCs to try it themselves is a great idea. As soon as we have positive human clinical trial results we could do this.

  14. Hi Akshay, the authors of the following preprint on UK and chinese population state that “plasma proteins are a reliable instrument for prediction of multiple common diseases in diverse populations and can be used as a robust biochemical aging signature to improve early detection and management of common diseases”.
    Given Harold, you and the team’s deep expertise on blood proteins, could draw your interest.
    https://www.medrxiv.org/content/10.1101/2023.09.13.23295486v1

  15. Hi Engadin, plasma proteins can be used as a diagnostic aid. We used the Glycan clock which measures Glycans on IgG antibody proteins circulating in plasma. I am more interested in the nucleic acids which are bound in vesicles for their role in resetting the epigenome in a recipient cell.

  16. Hi! This is very interesting:

    In the article by Josh: “E5 is not large proteins or small proteins, it’s not non-coding RNAs — according to this document, E5 is made of exosomes and exosomes alone.”

    In a podcast, Sandra Kaufmann stated that “80% of the positive effects from stem cell therapy is in fact caused by exosomes.”

    From the testimonial by Garrett Mcnamara at https://www.wellbeingint.com/ regarding actual exosome therapy it seems that there is a “there” there.

    So, lets say Akshay and Harold now have a good basis in the E5 and maybe “thinning” of old exosomes can be sufficiently acheved by blood delution. Then we are on to something, and Akshay, since companies are “selling” exosomes already, why would you need approvals etc when other already sell it (not as good as E5 i presume though).

    • I do wonder if most of the effects the Conboys got with their plasma dilution studies was due to the thinning of the exosomes for a period of time. I believe Dr. Katcher said they were giving the treated rats like 4 times the number of young exosomes a young rat would have in its blood. That probably explains why their team isn’t worried about diluting the plasma fists as that many would mostly override the exosomes that were already in the blood.

  17. Hi Are,
    Yes EVs and exosomes are truly marvelous. Actually E5 does contain proteins and non coding RNAs: they are in those exosomes. FDA does not allow plasma based therapies that are injected without its approval.

  18. All of this information is fascinating. I have a question about the origin of the “young exosomes” or where they can be produced. Is it true that porcine exosomes can work, or can they be synthesized or extracted from elsewhere?

    Also, is part of the therapy or longevity of blood donation thus possibly related to removal of “old exosomes”? Thanks again for all of your work and insight.

      • Thanks for the link, that’s a good article for learning and review. It seems they both signal extracellularly, and upon integration, either activate or suppress cellular repair mechanisms via nucleic acids and proteins. It seems that the testing of this thesis would be facilitated by an overlap and “alibi” of cancer research, though what we are looking at may more appropriately be under the category of rejuvenation, which can also double as a “therapeutic” term (in a cancer treatment).

        In trying to look for, or be cautious about, side effects or unwanted outcomes, I’d wonder if in a healthy human subject, something we might not be seeing in mice (due to shorter lives) would be rejuvenation at the cost of increased risk for cancer. Do you have this worry as well, Josh?

        Best always,

        P

        • I hope I have understood your comment Palamas. Exosomes of non cancerous cells have never been shown to cause cancer. Otherwise all of us would die of cancer. However cancerous cells do spread through their secretome. In fact young exosomes improve our cancer protection systems: for example tumor suppressor genes are progressively methylated/silenced in aging but the cargo in exosomes sourced around the age of puberty could re-activate them (demethylate them).

          • Thank you Akshay. Where do you think the control mechanism comes from regarding the methylation of tumor suppression genes later in life? The “old” exosomes? This is generally the topic that I tend to agree with that Josh makes, which is that longevity is largely programmed, as opposed to “wear and tear” randomness. I always disputed that because it seemed ridiculous to me that we can take care of our bodies and repair things, as well as the fact that we have knowledge that our immune system recognizes (or can recognize) tumor cells and dispose of them. For me, not being a more “helpful” steward of your body would just hasten the program via epigenetics, but wouldn’t completely override it unless you got into really risky situations, which we don’t deal with here.

            It seems that all that remains, then, is identifying the proper and young exosomes, and seeing if they can be scaled, as Josh states. Do you think this will happen in the next decade?

            Thanks again.

  19. I believe everyone wants to know when this would be available, assuming it all pans out. I am wondering if this could get fast tracked through clinical trials given its medical value to a lot of people? And when would a topical skin product be available considering skin lotions don’t need as rigorous of a medical vetting?

    • It is already available to people who have the money to pay for it. People who can spend millions of dollars on their personal anti-aging program. https://www.bloomberg.com/news/features/2023-01-25/anti-aging-techniques-taken-to-extreme-by-bryan-johnson

      We know how to extract exosomes from calves or piglet fetuses. It’s just that at present, it’s a very expensive process with a very low yield. Maybe it’s something that David Rockefeller and Henry Kissinger have tried. Maybe Bryan Johnson will learn about this and add it to his regimen, and maybe he’ll tell us whether it makes more difference than everything else he’s doing, and we can learn from him.

    • Exosomes in food are very interesting because they are much easier to use than blood. They are somewhat bioavailable (if I understand current research correctly). Especially (unpasteurized?) milk exosomes. I don’t know if they are rejuvenating in any way. Maybe a research area (should be simple, just feed the mice this or that).

  20. Raphael,
    Human prescription medicines take 6 to 9 years to get approval-not just ours-all new medicine applications to FDA. But there are clinical trials that we hope our core group of supporters can take advantage of. As we move from Phase I to II and III the number of volunteers increase. You are right on the topical. If we get very solid results on safety and efficacy in the upcoming human clinical trial then that could probably be launched much sooner. Initially we would release a soft launch like we did for NEEL only announced to readers of Josh Mitteldorf blog. During which time we would have to build a factory to manufacture topical E5 for a full launch. That can take many months (not years).

    • Hi Akshay.

      I am currently on the NEEL and will come back on that, I also have some more thoughs regarding the Exosomes (guess by the way that your E in Elixir/E5 all the time was pointing to Exosomes).
      I agree fully that expanding the max life is a very big thing as you did. A also think that its a good chance the Exosomes are a very vital part although aging takes us “by a 1000 cuts” (or say 7+). Sometimes solutions may be simple and if we are lucky in this case maybe replacing 10% of the exosomes in a 68 year old with an 18 year old gives an age of 63 years and so on (with enhancement if we know the “bits and pieces”) .
      It seems logical that exosomes can be applied topically (Harolds hand ? ) (not 10% ish but say a smaller but “continous” replacement). A NEEL based cream with E5 sounds great. You did not by the way put any Exosomes into the NEEL ?

      Keep on the good work 🙂

    • Maxwell Biosciences believe they have found the rejuvenating factor in young blood: “LL-37 – also known as Human Cathelicidin Antimicrobial Peptide.” It is elusive because it’s so unstable. They have developed a stable small molecule they call MXB-22,510 that mimics LL-37 and are going to human trials next year.(2024). They call it a “synthetic immune system”. It causes most human pathogens both viral and bacterial to be quickly eliminated replacing nearly all current antibiotics and antivirals. They believe Immune senescence is the main driver of aging, and predict with the advent of this drug human life expectancy will be 120 yrs by 2033. https://maxwellbiosciences.com/healthspan

  21. Hi Are, everyday we are learning more about the bits and pieces and target is to make the 68 year old 35 year old. NEEL and E5 are separate products. NEEL does not have any exosomes. Thank you for your kind encouragement.

  22. I wonder if Akshay and Katcher have ever considered using bioreactors to multiply exosomes in blood on a large scale rather than relying on large quantities of pig blood. Bioreactors are already being used in the food industry to produce cultured meat, where a small piece of cells are extracted from live animals and from that sample, zillions of whole pieces of cellular tissue are produced in the laboratory:

    https://www.mdpi.com/1422-0067/22/14/7513

    Perhaps a partnership with these companies would be interesting to produce E5 on a large scale. Even using human instead of pig blood could be something to think about (we still don’t know whether human exosomes may have subtle characteristics that make them more suitable to rejuvenation than pig exosomes).

  23. -confused by the assertion this can already be done since its animal tissue
    – how much would it take to harvest ebligh to target the entire organ of the skin and how would it be programmed to target it?

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