Cellular Reprogramming, and the Goal of Separating Dedifferentiation from Epigenetic Rejuvenation

Rejuvenation takes place very early in embryonic development. The germline cells that go into the creation of an embryo are well protected and maintained in comparison to the average somatic cell in the adult body. Nonetheless, there is an accumulation of age-related epigenetic changes and molecular damage. Cells purge themselves of as much of this change and damage as possible, in order to ensure that the young are born with young somatic cells and tissues. This is primarily a resetting of epigenetic controls over gene expression, decorations on the structure of the genome that control shape and access to specific genes by the molecular machinery responsible for producing proteins from genetic blueprints.

A cell is a state machine, largely governed in operation by the matter of which proteins are produced, and in what quantities. Not completely governed: some damage, such as mutations to nuclear DNA, is irreversible. Some molecular waste cannot be managed even by cells in a youthful epigenetic state, and will degrade normal function. In a collection of replicating cells, that waste can be diluted via cell division, or even passed off entirely to a sacrificial daughter cell in a process of asymmetric division. So long as no one cell or small number of cells are vital, even serious mutation can be evaded by replication, provided that mutated cells are rejected. This is how single celled life, such as bacteria, can continue indefinitely. Further, a few lower organisms, such as the hydra, essentially a tiny bundle of stem cells in which every structure is replaceable, use this strategy in order to achieve individual immortality. Higher animals, with complex central nervous systems that include many non-replicating cells that cannot be sacrificed, cannot use this strategy, and so suffer from degenerative aging.

Embryonic rejuvenation is a process that can be understood, induced, and manipulated. The creation of induced pluripotent stem cells from normal adult somatic cells via reprogramming is one example of what becomes possible given sufficient knowledge and technical aptitude. This combines, in the same way as occurs in the early embryo, both an epigenetic reset and loss of somatic cell state, such as the shape and function of a skin cell or a brain cell, producing dedifferentiation into a pluripotent stem cell state. Researchers are presently looking beyond experiments in cell cultures towards the application of reprogramming in living animals. An epigenetic reset is a desirable outcome for somatic tissues throughout the aged body, likely able to reverse to some degree many age-related issues, such as loss of mitochondrial function. Dedifferentiation of somatic cells in an adult individual, on the other hand, is a roadblock and a challenge. It will lead to cancer where it occurs to a lesser degree, and it will cause pathology and death if prevalent. Differentiated cell state is vital to normal tissue function.

Thus an important question currently under investigation is whether or not these two aspects of reprogramming are inseparable. Is there an approach to reprogramming that will produce maximal epigenetic rejuvenation with minimal dedifferentiation? If so, that could prove to the the basis for a very useful approach to the treatment of aging. It likely cannot help much in the case of stochastic nuclear DNA damage leading to somatic mosaicism, and it cannot help with the accumulation of some forms of persistent molecular waste in long-lived cells, but it could nonetheless be beneficial enough to be interesting.

Cellular reprogramming and epigenetic rejuvenation

A recent addition to the anti-ageing strategies being developed comes from cellular reprogramming approaches. Induced pluripotency studies provided evidence that age-related cellular phenotypes such as mitochondrial morphology, function and number, as well as nuclear envelope integrity, are not irreversible. However, developmental cellular reprogramming turns a cell to a pluripotent state, where it has the potential to generate any somatic cell type. This process is not appropriate for an anti-ageing therapy in vivo because it requires not only the loss of the original cellular identity, but also the re-establishment of self-renewal capabilities. Therefore, induction of pluripotency or the direct injection of pluripotent cells in vivo, invariably lead to cancer in mice. For a cellular reprogramming-based intervention to be considered rejuvenative (turning an old cell into a younger cell), we need to uncouple its effects from dedifferentiation (loss of somatic cell identity).

Cellular reprogramming has demonstrated potential not only in regenerative medicine, but also in the ageing field through the amelioration of both physiological and cellular ageing hallmarks. While partial reprogramming might be used as a catch-all term to describe this type of rejuvenation, it does not reflect the fact that the described interrupted cellular reprogramming techniques are applied with the aim of (epigenetic) rejuvenation as opposed to inducing pluripotency (loss of cell identity). Reprogramming-induced rejuvenation (RIR) is a better term, capturing the nature of the utilised process and final aim of the interventions.

RIR has shown promise as a treatment to safely reverse ageing whilst retaining the ability to revert to or maintain original cell identity, both in vivo and in vitro. However, the precise nature of RIR still needs to be fully understood before it can be safely implemented as an anti-ageing treatment. For example, tracking any traces of pluripotency in partially reprogrammed cells (particularly in vivo) is a necessary precaution to minimise long-term cancer risk. Additionally, can rejuvenated partially reprogrammed cells be cultured long-term? The rejuvenated phenotype of some OSKM-treated cells lasts at least four weeks, but does this phenotype remain stable or eventually start to deteriorate at a rate faster than normal ageing?

Other important RIR safety concerns include how the reprogramming factors are introduced in vivo. Retroviruses are commonly used to integrate reprogramming factors into the genome. However, this method bears risks, such as insertional mutagenesis, residual expression and re-activation of reprogramming factors, and retrotransposon activation, all of which could increase cancer risk in vivo. Non-integrative delivery methods, such as transient transfection, non-integrating viral vectors, and RNA transfection are safer alternatives. For example, researchers have successfully used mRNA transfection to non-integratively conduct RIR. Another safe alternative is chemical-based reprogramming, which involves direct conversion of a somatic cell to a pluripotent state by use of small molecules and growth factors. It is conceivable that, in the future, chemical-based reprogramming could be adapted to achieve rejuvenation, however, this reprogramming approach currently only works for mice.

While RIR applied to skeletal muscle stem cells appears effective in improving regenerative capacity and muscle function in immunocompromised mice, further analysis is required regarding the somatic mosaicism of partially reprogrammed stem cells. Somatic variants at a stem or early progenitor cell level in turn can cause lineage bias, reduced stem cell function, and increased risk of developing haematologic cancer (e.g. age-related clonal haematopoesis). This can lead to the development of pre-malignant cells, which have a higher propensity to transform to a malignant state, the effect of which could be attenuated or exacerbated by RIR.

It also remains to be further explored whether and how RIR would work on post-mitotic terminally differentiated cells, such as neurons, cardiomyocytes, or adipocytes, but also other non-dividing cells such as quiescent or senescent cells. Pilot work has been done in the latter two states, demonstrating that a rejuvenated phenotype is achievable after restoration of cell division. These results may point to a scenario where proliferation is an essential requirement for rejuvenation. Indeed, induced pluripotency of postnatal neurons was only possible after forced cell proliferation via p53 expression. Coincidentally, the natural rejuvenation event in the early mouse embryo spans over stages of very active cell proliferation.

Overall, RIR is currently the best prospect to achieve epigenetic rejuvenation. Further studies are required to fully determine its limitations and efficacy.


are there any gerontologist who thinks that other approaches will give more lifespan increase then damage-repair? Do they think other approaches can give us centuries?

Posted by: Gekki at September 18th, 2021 11:57 AM

Hi Gekki! Just a 2 cents.

I doubt there are many other approaches, the largest problem is the efficiency/result being much less than hoped or even temporary...and there is always the 'cancer' problem in the background, looming...it's like cancer wants to overtake us - any chance - it gets (it will). It a evolution/natural selection response - if you are damaged/compromised - you compromise the specie..therefore, you are unselected - and selected - for removal/elimination. It's the 1 vs Many thing; single (invididual) vs collectivity (specie). Kind of like 'get the vaccine or not' (individual choice vs collective choice), it's you vs everyone else. The way it proceeds - is with cancer, cells become rogue and compromise you; therefore you can't continue 'Being' (living)...anymore (for, you compromise the specie/collectivity).

DNA DSBs (Deoxyribonucleid acid Double-Strand Breaks) are causal to aging, for they activate the 'haywire (loss of)' process of epigenetic drifting (with time passing/aging). Thus, they create 'epigenetic/epigenom(ic) compromising'...that leads to change of the epigenetic landscape - the clock/epiclock, to move/tick forward...until the whole system can't work anymore (too much damage (DSBs), too much telomeric DNA loss, too much disarray (cell senescence))...etc.

Very few therapies do anything about that/those problems. Epigenetic reprogramming (partial) and as you said, DNA repair...but it is limited (sadly), so is epireprogramming, let's hope it improves...because there is basicaly almost nothing else that can make 'Centuries' lifespan...

Possibly (but unlikely): Redox shifting (animals that live ultra long have improved redox, it is an important part...but not the total decider, just one part), Lipofuscin removal (doubtful, but it will help..it will be limited..just like progeria progerin removal/fast-aging syndromes that accumulate ceroid/lipofuscin/progerin...etc...thus, this won't clogg anymore the proteasome/lysosomes, but I am wondering how much of an effect on lifespan...I think it may not be enough and just some thing elese with have to do - I.e. weaker result than epireprogramming, DNA repair or Calorie Restriction)...Telomere Increase (it's ambivalent and ambiguous...), you need to keep them (tall enough and not run out of them with age), the only thing is telomerase/telomere increase (which can cause cancer); telomere extension therapy is kind of 'sorta forgotten...whatever'...because inconclusive results; you need tall telomeres but cancer just around the corner, and it has been shown that the Taller the Telomere - the Faster It shrinks...it's very funny like...I mean it should shrink Slower as the telomere becomes taller...but it's the inverse that happens..it makes sense.

Evolution found a way to 'nullify' excess telomere - by accelerating its erosion - as it increases in height = null = no loss no gain. It's why BioViva telomere therapy is great but I wander how much telomeres are Really Gained (Net Gain)...because tehre is No Gain - No Loss...Null. As telomere increase in size, they Speed Up in loss - to Compensate the Increase in height. Catch22.

What else? Mitochondrial DNA/protein/gene export from/to nucleus, that SENS therapy...where they reproduce 2 3 mito genes...don't know much about this, mitos are also consequential to lifespan (ROS producers, (excess/unquenched/unbalance) ROS -> DNA DSBs), but mitochondrial therapies/studies and anitoxidants have Always been weak and just don't hold your breath. It seems Not Enough.

Same thing, for 'Stem Cell INjection'...stem cell replacement/niche replacement...with age we 'run out of adult stem cells/the niche gets old (telomere loss in stem cells)' and thus, tissue can't be rebuilt anymore...I have mixed feelings about this. Stem cell studies in mouse give 20% lifespan extension, notthing to write home about (mice have big results, humans no).

Total Replacement of the Body (become cyborg, doubtful), replace heart, muscles, liver, lungs...with 'young' new parts...it's wishful thinking.

It is Clearly the Nucleus (nDNA/telomeric DNA/epigenome), the Biggest Target to live Centuries. I would wager it is the only way, 99% of the rest will Only make 'health improvement'..meaning you will live healthier (like senescent cell removall...), you will be happier but you will die. 120 tops.

Just a 2 cents.

PS: sorry for not better news, I'd love/wish to say : 'YESS! We got it! finally, the golden chalice - we will live 500 years like a clam'...alas. Let's keep fingers crossed.

Posted by: CANanonymity at September 18th, 2021 8:22 PM

Turn biotech is doing well with partial reprogramming. Don't you think they could bring effective therapies for epigenetic rejuvenation to the market?

Posted by: Alex at September 19th, 2021 10:27 AM

Hi Alex! Thank you for that. Just a 2 cents.

They could, definitely, I believe - in them, and any other companies that are for the epigenomic reprogramming; at least, they are trying. I was just only reflecting on/of the recent results...which are Impressive...but, yes, it's not the 'kind' of impressive we all hope (obviously). But it is Great. Because we all know the other options is nothing/antioxidant/CR/exercise...the stuff we know (and that does not make miracles). So, Power To Them.

I am just being realistic (optimistic - enough, but with a sprinkle of pessimism, jsuit a grain (a realism grain)). Being deluded is bad, being optimistic is good/important, it's just we getting 'Thin' of the hope /running (thin) out of patience/hope a bit. I know 'You Can't'...it's like 'if you had run out of it - you'd be dead'. That's applicable to me (atherosclerosis/thrombosis/embolism) if I had run out of it and just decided to lay on the floor and say: ''the pain - too much, prefer to end'', I woould not be talking to you, I would be inexistent.

I just fear (and have human doubt/worry a little) about the Result of it, like a recent study of epireprogramming that 'looked at everything so far' and made a résumé of it all...said, basically, we are facing limitations..that we will have to overcome...be it teratoma formation (Cancer), be it temporary 'reprogrammed' - you need to do this daily...not 1 per year..it's a thing you'll doo all the time - to prevent aging. I am not 'against' that I am Even OK with that..so long as it defeats aging, I would do it in a heart beat - Everyday..because it is like 'Breathing Air' or going to a bathroom to do #1 or #2...you gotta do it, or you don't exist anymore. Like eating, breathing, 'living'. Some things are essential to our life, epireprogramming may end up so and so essential, that permanent/every 5 minutes needs be (done). A 'constant 'pitstop' tuneup'..just like racing cars after 1 lap...need pitstop. Every Lap -> pitstop -> nonstop (TuneUp). Or else, Tune->Down.
Repair, same thing, need constant repair of the usaged-ing car accumulating too much mileage..(AdG car analogy...pitstop/garage tuning).

It is scary that Full Reprogrammation is deadly, while Partial Reprogrammation is (thankfully) not, but because Partial -> Partial Result...-> Weaker? Result. One more limit. Partial because we tryinh to circumvent the 'problems/limits' (cancer/temporary thing/reprogrammation comes back to senescence soon enough if not repeated...etc).

It's a Script/a Scroll...and we just trying to scroll back to top of the 'internet page' (scrolling up)...but it keeps scrolling down until reach end of the page - the end (of the story/paper scroll/webpage/'Code (epicode)'). Have crack open code, and that's epireprogramming (programming/coding), and so far it's limited; I so hope that the reversal (of 30 years of the epiclock) is substantial enough and 'Robust enough' (and we can repeat daily this therapy) - that the 30 years - would stand,,and you could Repeat This On and ON..to keep 'your 30 years'..but I have a fear that the 'limit' of this is that - you Get 30 - no more. And thus, you Continue To age...I.e. you are back to 20 years old...but You Continue to age...from your 'new 20 biobody'. In essence it is not True Reversal (Repeatable) reversal of aging..only partial reversal, you are effectively younger...but you continue the process, and you Need to repeat this daily epireprogramming to 'keep' your '30 bonus'...if you stop..you Immediateliy go to your 'current 50 bio body'...Again. The studies are not saying : ''we bring you back Again to 20 years old on your epiclock''..well, at least I am not sizing/getting that; if that would be possible then Indeed we could repeat this Ad Vitam Eternam (and that is pure LEV), just go back to your 20-year old 'self/clock'...but I did not get that from the recend studies.

All, I got is you are reversed 30 years...and you need to repeat this to 'keep your 30 years reversal'. if you stop, you lose it (back to old senescent self). I so hope I am wrong and that we can reverse the clock infinitely and postpone aging/death at howevery many times/repetition we wish (for however long We decide to live or not). Just the fact we reversed the epiclocl 30 years is such an Achievement..now we need to repeat it continously (and makes sure damages/residues clogging/mutations/Cancer...etc are circumvented/fixed/repaired) and then LEV (would) comes (hopefully, we got still another 50some years, I just fear that when I loooked at papers from 1970 1980s...it's an illusion..it was talked about then...some 40 yaers ago...why would we Necessarily believe 'because progress - reprogramming 'ahppening now'' we will have the epireprogramming all working in the next 40some years, it may still not be. It's a reality/we have to face. I hope not; I just hope people realize taht we hit limits constantly and is why it's not happening (Sooner, but later/very later)). But, nothing else to do, than 'keep'on keepin'on' (cause other thing, is just accepting defeat/end it there/lose hope and abandon ship).

Just a 2 cents.

Posted by: CANanonymity at September 19th, 2021 5:00 PM

There is a significant amount of difference between "cellular reprogramming" and "whole organism reprogramming", and "full versus partial" is somewhat less important than what needs to comes next..

There are no fewer than a dozen companies already focusing on some angle of cellular reprogramming (all based on learnings that go back to the original 1952 cloning experiments by Briggs and King, modified by John Gurdon in the 1960s - co-winner of the Nobel with Shinya Yamanaka, Bea Mintz, Rudi Jaenisch, etc.)

But cellular reprogramming for rejuvenation is NOT the same thing as organism reprogramming for rejuvenation

The regenerative biology world has actually known this since before the discovery of DNA, in the study of these complex dynamics at the organism level

In organisms which use reprogramming for rejuvenation purposes, many other events simultaneously must transpire including (but not limited to): ECM histolysis / remodeling, activation of pro-regenerative parts of innate immune response, membrane potential changes, morphogenetic gradient formation, etc. etc.

It is in the context of these physiological regenerative micro-environments that these organisms are capable of utilizing this reprogrammed cellular flexibility for complex regeneration, tissue remodeling / disease reversion, and yes, age-reversal

So cellular reprogramming is just one small part of an important rejuvenation cascade which must be recapitulated in humans to yield this desired pro-longevity result

So it would be nice to see this groups focus not just on optimizing the cellular reprogramming step, but the 90% of everything else that must happen after to make the integrated organism younger - cellular rejuvenation and organism rejuvenation via reprogramming are two very different things.....

Posted by: Ira Pastor at September 19th, 2021 6:09 PM

Hi Ira! Thank you for that. Just a 2 cents.

Thank you for this precision. It's something important to remember, indeed..organismal 'whole micro-environment' reprog. not just cells; ECM, cytosol, large organs, cartilage everything in between 'the meat' etc...
What I am wondering, is, if the stem cells/germ cells (primordial cells) could be reprogrammed (like iPSCs), and thus, could rebuild tissues and micro-environment/outside cell...i understand that it is not just cells that make the microenvironment be what it is - it's the collection of everything, making it; but when I look at embryo formation (of course where are not small embryo anymore..we have organs and everything), maybe the rejuvenation/reprogramming of certain stem cell niche...might? be enough to rebuild microenvironments; stem cells when differentiating can rebuild a lot; but as you said, it will depend on the microenvironment/status...

Maybe I'm being Even Too Optimistic about reprogrammation and organismal/whole organism reprogrammation is still pipe dream and is mandatory for lifespan extension; cell reprogramming is just enough on its own - because of everything else/environment 'the problem'. You are right that companies definitely need to axe their research on this - take a broader approach and see the big picture 'whole environment/'whole micro and macro structure env'', not just the organs' cells...the organ is made not just of cells. Still, thje cells are important for organ function; but as you stated it's a collection of so many things; that it's very possible cells on their own (reprogrammed) either in vivo or retransplated would have limited effect (like stem cell injection). I could understand that large 'wholebody/whole animal' organismal reprogramming is just infeasable/impossible/too hard...I wonder about that, what would be the solution (if any) and which companies (if any) even are tackling this. It puts a(nother) nail in the coffin and another wrench in the life cogwheels.

Just a 2 cents.

Posted by: CANanonymity at September 19th, 2021 7:26 PM
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