On Cellular Reprogramming and Cellular Rejuvenation

The commentary linked below takes a look at some recent work on the topic of cellular reprogramming and the rejuvenation it appears to cause inside cells. In the grand scheme of things, it really hasn't been that long since researchers first discovered how to reprogram somatic cells into induced pluripotent stem cells. These artificially altered cell populations have the same characteristics as embryonic stem cells, able to generate any type of cell in the body given the right stimulus and environment. Reprogramming is so easy to carry out that it swept through the research community with great rapidity, and the improvements and further experimentation started almost immediately. Along the way, numerous researchers have found that reprogramming old cells in this fashion appears to revert a number of characteristic signs of cellular aging. Damaged mitochondria are removed, some epigenetic markers are altered in the direction of youthfulness, and so forth.

It is understood that cells are, in principle, capable of rejuvenation. Something must happen to repair the damage and epigenetic changes of aging in between that point at which aged germ cells get together and the point at which a young embryo is growing. Parents are old. Babies are young. A range of intriguing research on early embryonic development suggests the existence of a program of cleansing and repair that operates when the embryo is still only a handful of cells. It is not unreasonable to think that cellular reprogramming as it currently exists is triggering some fraction of those developmental rejuvenation mechanisms as something of a side-effect. The interesting question is whether or not there are useful near future medical applications that might result from a greater understanding and control of cellular rejuvenation of this nature.

The most obvious application is that any sort of cell therapy using the patients own cells is probably going to be improved if the cells are more rather than less youthful. Since reprogramming has this effect, and researchers are working towards using induced pluripotent stem cells in therapies, this will probably happen by default at the outset, and then be improved via degrees of optimization as the field of regenerative medicine progresses. On the other hand, safely inducing some form of rejuvenation-like repair or alteration of cell state in site in the body and brain sounds like a much more challenging proposition. It isn't at all clear that such an approach is even possible or plausible; a greater understanding is needed when it comes to exactly how rejuvenation is being achieved in reprogrammed cells. For example, it may well be the case that some of what appears to be rejuvenation is in fact a selection effect. Reprogramming typically has a low rate of success when you look at the number of cells in a sample that are converted, and perhaps those are all less damaged examples. But see what you think of this commentary and its references:

Stem cells for all ages, yet hostage to aging

Researchers showed that aging transcriptional changes in fibroblasts were reversed in induced pluripotent stem cells (iPSCs) derived from donors across the lifespan. Subsequently, when iPSCs were induced to form neurons by direct induction (iNS), the aging transcriptional signature was also absent. In contrast, when aging fibroblasts were directly programmed to iNS by a similar protocol, they maintained an aging transcriptional signature. Remarkably, much of this signature was not the original signature of the fibroblasts but a new age-associated signature more closely allied to neural related gene action. Thus, fibroblast-derived iNS retained an "aging state" on direct cell programming, but not a hard wired, age-related transcriptional signature. The potential for fibroblast rejuvenation extends to 'senescent cells': from the same 74 years old individual, iPSCs were derived from either primary fibroblasts or replicatively senescent fibroblasts after serial in vitro passaging: both differentiated into normal embryonic lineages. Surprisingly, given the huge attention to regulatory mechanisms underlying iPSC generation, there has not been extensive comparison of iPSCs by donor age.

How do pre-existing problems such as DNA damage relate to these processes? Mutations accumulate in aging skin as in all other mammalian tissues. Primary fibroblasts from breast skin of donors aged 20-70 showed exponential increases in double-strand DNA breaks against a linear doubling of chromosome structural abnormalities, 10% to 20% across the adult lifespan. Are their corrective mechanisms as part of the reprogramming process, and if so, how do these work? Alternatively, reprogramming may select against damaged cells within a mixed cell population, which might be estimated by the efficiency of reprogramming. Future studies may define a threshold level of DNA damage that is permissive for iPSC generation. It has been proposed that iPSC generation with extensive cell proliferation would "dilute any accumulated molecular damage" which could not occur during iNS generation under conditions that limited cell proliferation. While replicative processes may weed out protein damage, it is not clear how these would remove DNA damage. As well as selecting against damage to nuclear DNA, selection is also likely for mitochondrial function. Other groups have shown remarkable mitochondrial rejuvenation in iPSCs generated from aging donors.

These findings have broad ramifications for the field of regenerative medicine. Whatever the mechanisms at play, the loss of aging signatures in iPSCs is good news for autologous iPSC directed-cell therapies where the aging population will be the major target for personalized regenerative medicine. However, while iPSCs and their direct derivatives may be rejuvenated, the host's aging environment is problematic. For example, grafts of embryonic neurons into older Parkinson patients show donor cells acquire features of diseased host neurons. Inflammation related to Alzheimer disease, and to basic aging itself, can also attenuate grafted stem cell function. Thus, prospects for rejuvenation by iPSC may still remain hostage to the aged host.

Comments

''Are their corrective mechanisms as part of the reprogramming process, and if so, how do these work?

...It has been proposed that iPSC generation with extensive cell proliferation would "dilute any accumulated molecular damage" ...
...While replicative processes may weed out protein damage, it is not clear how these would remove DNA damage...
...Other groups have shown remarkable mitochondrial rejuvenation in iPSCs generated from aging donors.''

Hi there!

This is most interesting.

I'm trying to put this in perspective with SENS approach of rejuvenation. Although, they are different, they are both rejuvenation.
Those iPSCs that do get be to correctly reprogrammed increase many important factors to rebuild/rejuvenate such as pluripotent 'immortal/god-like' genes such as Oct, Sox, Nanog.
Which, they themselves, alter other important genes and enzymes such as Telomerase/hTERT and also telomere shelterin proteins such as POT/TRF1 and
also, Ki67, an important positive telomere DNA regulator.

Baby skin that is wounded and heals without any scarrification; or a gecko/lizard that rebuilds its cut-tail after stem/cell transdifferentiation, make use of what i'd call stem-cell-like 'rejuvenation',
in the case of the baby's open skin wound, instead of macrogranulation and rebuilding dead scarred tissue, it litterally rebuilds the skin - diluting the damages, completely,
including undilutable/uncrosslinkable, by Matrix Mettaloproteinases MMPs, Glucosepane itself must be removed somehow and may other AGEs (Advanced Glycations End-Products).
The baby skin's ECM (Extra-Cellular Matrix) is rebuilt from the ground up (collagen/hyaluronate/decorin/perlecan/other ECM layer degrading and production enzymes are all in full motion during that process),
effectively 'diluting that damage' to no damage at all - and back to exactly the same skin pre-wound time; total rejuvation. With aging, it would be the same thing, only an elder's skin tissue would be back to that
baby's skin (of course, the ECM would be dramatically altered (collagen I, II, III, IV plumped up, long-chain hyaluronic acid increased, AGE crosslinks removed, fibroblasts fully replicating and in young 'spindle' shape (rather than flat, enlarged, senescent, mutated, with aberation, undividing fibroblasts of elder skin, etc).

One other effect of rejuvenation in baby skin, like iPSCs, is the activation of a stem cell gene zincfinger. This gene activates telomerase sporadically and confers 'stem-cellness' to cells, rejuvenating them back in a fraction of time (just like telomerase activation, only even more powerful and removing damages too, as in the baby skin total healing to prior skin).

mitochondrial bp deletions (large base pairs chunks of DNA removed from DNA) which accelerate mitochondrial mutations (which in consequence affect
the mitochondrial Complex ETC (Electron Transport Chain) efficiency and reduce the mitochondrial ATP output production; this making the
mitochondrial incapable of proper respiring/respiration - in turn generating further ROS and giant dead effete mitochondria (which produce ROS too)).
Increased ROS is benefitial only in terms of mitohormesis effect, priming the system to increase its antioxidative systems and reducing the TOR/mTOR
CR mechanism which is at the center of this (for many many genes, if not almost all, are modulated via mTOR/PK3/Insulin signaling. It is at the crossroads
of damage, growth and repair/survival).

mitochondrial and nuclear DNA damage (lesions, 8-oxo-diguanosine, 8-oxodG) and y-H2AX telomere foci
DNA Repair Enzymes (BER, NER, NHEJ), polymerases, topoisomerase, Telomerase itself, and others would be capable of reconstructing/repairing destroyed DNA (DNA deletions, breaks (DSBs/SSBs) and lesions) in mitochondria/telomeres/nucleus during DNA synthesis/mitosis.

'' In contrast, when aging fibroblasts were directly programmed to iNS by a similar protocol, they maintained an aging transcriptional signature...
...However, while iPSCs and their direct derivatives may be rejuvenated, the host's aging environment is problematic...
...It has been proposed that iPSC generation with extensive cell proliferation would "dilute any accumulated molecular damage" which could not occur during iNS generation under conditions that limited cell proliferation...
Thus, prospects for rejuvenation by iPSC may still remain hostage to the aged host. ''

This is what has me a bit puzzled and I'm trying to understand how SENS can get around this if proliferation-limited iNS could not and still featured an old phenotype/''aged''.
The more I read about this and PSCs, the more I'm telling myself that some of these cells, reprogrammed or not, carry a 'memory' phenotype if you will and such are irreversibly 'changed. Because,
really, reprogramming should erase all of that. THey say it's more the (aging/old) 'environment' in which the cells are that is the cause of them reinstigating an 'old cell phenotype' when transplanted into this aged environment.

This is showing that SENS, like AdG said, will absolutely need to be done before 40 or 50 years old, where damage is small. Above that, I don't see how it can save people, no matter how many times the rejuvenation is repeated, the effect of 'reversing' (rejuvenating) the damage will be too small to matter;
since, here, it shows that cells acquire a 'behavior/phenotype' depending on the environment itself. It's starting to sound an awful like the Extra-Cellular Matrix full of glucosepane, that we can't do anything about no matter how much we rejuvenate the cells, the junk (glucosepane/lipofuscin/AGEs) is still in and undegraded.

Posted by: CANanonymity at July 10th, 2016 10:37 AM

PS: IF SENS fails a true life extension above 120s beyond health improving and there is nothing we do can about it, this is something we face as a prospect (aka we will all get old and die...f... So be it.
We'll live the sh...out of the remainder years).

I suggest changing the strategy towards maintenance of redox, starting from young adulthood.
This technique is only good when started young. The redox milieu dictates the amount of
oxidative stress the cell is exposed to. When the redox is in the negative the cell is
oxidative stress resistant and in a 'reduced' state.

With aging the redox milieu shifts from reduced to oxidizing, going from -300 mV
up to -250/200 mV. Which means about 0.5 to 1 mV up each year. This increases the oxidizing
process as it exposes the cell to higher amounts of the oxidized thiols pool.
Damage is consequential only if the consequence - is - (seriously) consequential.
Aging, itself, is a consequence of damage accrual. But if damage is avoided, less impact and almost nil; it
becomes inconsequential. The redox is affected by the damage processes that create aging.
When the redox is maintained, - consequential - damage is almost nil/completely avoided, meaning that damage still
accumulates (if it and when it does happen), but its consequences are almost none. As such, aging is stopped.
Its 'weight', as a consequential mechanism advancing our demise, (aka making us age), is now nearly totally weight-less.
That is the oxidized burden is nill forever long we need it to be to maintain eternal life.

Many studies have shown that mammalls of various decades-lifespan difference show a dramatic difference in their capability
to maintain their redox. Those that live up a 100 years, maintain it (the same as it was during their 20s), those that don't die, very young with an oxidized redox (+100mV).
Demonstrating that the cell can 'be damaged' as long as it lives - despite that damage. This way the environment of the cell (extra-cellular) remains
pretty damage-free; because the cells keeps on functionning despite incuring some small - inconsequential - damage.

Posted by: CANanonymity at July 10th, 2016 11:14 AM

CANanonymity,

Aubrey hasn't said that it specifically has to be done before 40 or 50. He said it should be able to work whenever. I think he said this in his last ama on Reddit.

Posted by: Ham at July 10th, 2016 12:36 PM

Replacing whole tissues or organs make work better than replacing or rejuvenating individual cells. But I am not that pesimistic about aged environment because the parabiosis effect is a proven fact. Instead of full organism some artificial organelle can supply young factors after this mechanism is fully understood.

Posted by: Martin S. at July 10th, 2016 2:08 PM

@CANanonymity.

In your PS, you say "I suggest changing the strategy towards maintenance of redox".
I find this very interesting but fail to understand if you proposed this strategy in the rest of your PS.
If you didn't, please could you elaborate it a bit, including how to achieve this in living mammals, not only in cell culture experiments?

Thanks a lot!

Posted by: JP Le Rouzic at July 10th, 2016 4:09 PM

Hi Ham !

Ok, cool, I guess we could hope the efficiency of his therapies to be constant as we age. He said that the robust rejuvenation would make a chronologically-aged 60 or 70 year old rejuvunated
'to not be 60 biologically' up (thus extract 30 biological years to back to bio 30 years old) until his 90th chronological real-time birthday. Which means like giving a 30 year biological lease back in time.
When that human would be 90 years old (chronologically), he would be (again) 60 biologically (like he was when he did the therapy thirty years ago).
What I'm curious is to find out if this could be repeated that at, 90, he would be returned to '30 years old' biologically (freezing and rewinding time from biological 60 to 30 each time),
and so forth at 120, 150, 180 years chronological years..allowing eternal life from 'aging reversal 60 to 30 bio Loop-di-Loop' repeated rejuvenation.
In theory and with reason, it Should Repeat and Work, because when you reverse/revert back to 30 years biologically - you Are Reversing aging and thus - postpone death - yet again, for as many times this rewind loop 'back in biotime' rigg is repeated.
If you continuously and infinitely postpone death, which is exactly what LEV, through SENS, reaches, then that 90 year old could repeat these therapies forever - each 30 years - and live up to a 1000 years old if he wanted; equalling eternal life/immortality.

Is that really how it plays out ? Or, is it that reapplication of second, third, fourth, ...follow-up therapies become successivly not as efficient in effect - the effect 'loses' its potency. Meaning, even the body 'rigs' itsef to this kind of rigging
and it works...for a few time - not Forever. A while ago, I specified why, the damage that is accumulated, though infinitly small, would add-up after (a very long) while; thus, would affect the followup therapies that come later to rejuvenate yet again.
The damage is reduced/repaired each subsequent therapy...but, just like AdG's rusting car analogy, how long/many times can we repair it until we can't anymore because 'some tiny amounts of damage lingered each time' there was a rejuvenation/repair (we are to remember
that robust rejuvenation aims to 'reduce all types of damages to a comprehensive level (aka enough so it makes a difference and aging is thus partially reversed - the 'rig' is simply repeating it advitam eternam to get to LEV, but can we repeat it, when we can't remove damages in -utter- totality) ?

@Martin S.

Hi Martin!

I too hope that this could circumvent the problem, replacing the parts (organs with organoids/3D printed-scaffolded like organs) with fresh new young ones, would solve most things.
It seems more complicated though and some part are hard to replace (like the brain or skin tissue), you need them (your brain, its yours/
your identity/your memories/your You and no one else/where your soul lodges basically...how do you replace that and keep 'Your You Martin Identity' brain?)

@JP Le Rouzic

Hi JP!

Thanks a lot too! Je vais le réexpliquer sans problème.

The redox is basically the 'state' in which the cell is. There are two main states,
oxidized or reduced. With age, you guessed it, the cell accumulates more oxidizing
molecules and damages that alter its thiol pools (thiols are special sulfur antioxidants
such as the major antioxidant GSH (glutathione) which is present in nearly all parts (mitochondria, nucleus and cytosol, intra and extra-cellular),
they act in various ways, not just antioxidants but also as 'signalating' the various redox enzymes
(SOD, Catalase, GSH-S-Transferase, GPx (glutathione peroxidase), Glutaredoxins, Thioredoxins and Peroxiredoxins).
These systems are extremely fine-tuned and make sure the cell is continously ' in balance' between excessive reduction and excessive oxidation.
With age, the GSH pools are either reduced or unchanged, but even when unchanged they are offset by increased Oxidized GSH pools (called GSSG, oxidized glutathione). This alters the ratio up.
The cell state is measured as this ratio (GSH:GSSG) or as a cell millivoltage potential (mV, most healthy cells stay in the -300 mV, when they get damaged
and oxidized their mV rises up to 100 mV, at which point this collapses the mitochondrial membrane potential too, effectively killing the cells from inside from the damage).

I would suggest that the most important factors is maintaing the GSH levels (this can be changed by increasing the production through its major rate-limiting
producer Cysteine Gamma Ligase enzyme) or by altering the rate-limiting enzyme responsable for the GSSG side, GSH reductase.

''Once oxidized, glutathione can be reduced back by glutathione reductase, using NADPH as an electron donor''

NAPDH is also another help (nicotinamide dinucleotide NAD+) as it controls and is used as electron donor to create GSH. But NAD itself alone is useless,
its GSH that protects the cell (feeding NAD in studies increased healthspan a bit but it does not seem to be capable to increase MLSP, but it increases fibroblast replicative lifespan but not the animal's chronological lifespan).
The three building amino acid blocks of GSH (a tripeptide protein) are Cysteine, Glutamate and Glycine. (Studies have
shown that NAC, Glycine and glutamate are all antioxidants,..but they fail at increasing lifespan, so there is a wrench. They fail at keeping the ratio
in a reduced state and the millivoltage deeperly negative. This can be explained, in part, because Cysteine Ligase and GSH reductase are enzymes
that 'age' too and don't respond well anymore no matter how much 'building blocks' you feed them - they are damaged too like any other enzymes with time.
THus the Redox becomes 'weak' and 'unresponsive', incapable of 'solving itself'...that is the hurdly - targetting the target to maintain the mV reduced and
the simplest way is by alterin this ratio GSH:GSSG. This ratio has dramatic effect on nearly everything that happens in the body.
I found out that, for example, when GSH drops or GSSG rises, telomerase is reduces up to 80% in activity...GSH and GSSG act in concert with the
proteasome (which itself is important to degrage the DNA junk). So there a million ways they communicate (like I said they are not just some antioxidant, they are
a multi-facet genetic regulation control of cell state).

One study showed that if critical enzymes fail (such as late in life) then it's game over, once you lose these important elements, you can't expect to
alter the ratio all that much. Since these enzymes regulate the ratio itself. Plus, why we have to do this young, is the simple fact that damage accrual
with high GSH:GSSG ratio is very low. If you start young, and keep your redox ratio high, you keep damage away and thus don'T accrue any with age.
That is how, extremely long lived mammals, have been capable of coping with oxidative stress and lived their very long lives.

Plus, one more thing, GSH (along with ascorbate) is critical in mitochondrial membranes. Mitochondrial membranes are the key element that drive aging.
They are 'multipliers' of damage because their phospholipids get oxidized and produced 'Peroxidation' of membranes and mitochondrial DNA nearby.
The fatty acids of the mitochondrial membranes phospholipids are the culprit. GSH is the first element that quenches this peroxidation-chain effect.
When mitochondrial Complex I and III produce ROS, they damaage the bilayer membrane phospholipid fatty acids (unsaturated fatty acids such as DHA, EPA and ARA Omega-3s and 6s)
which are higly peroxidizable-susceptible to ROS attack (creating Lipid Radicals and Lipid Hyperoxides (DHA is the major contributor to hydroperoxides).
Once peroxidized they let go a chain of peroxidizing events (forming reactive aldehydes, MDA malondialdehydes end products
and various ultra destroying end products such as Carbonyls, Methylglyoaxals, CML (CarboxyMethyl-Lysine), Pentosidine, Furosine, AGEs, Acrolein, Prostane, 4-HNE/4-HHE (4-Hydroxynonenal).
These molecules attack the GSH or inversely, GSH attacks them in a kamikaze/suicidal fashion 'sparing itself' to spare the cell DNA fragmentation and DSBs/SSBs (Double/Single strand breaks).
It turns becomes GSSG, oxidized. THen we have a problem that lingers...

Posted by: CANanonymity at July 10th, 2016 7:08 PM

Can we be sure that the fertilized isn't damaged, but then that damage is rapidly spread out among the daughter cells?

Posted by: Jim at July 11th, 2016 3:55 AM

Aging related inflammation can be lowered significantly when you remove senescent cells - we already know it's lowered in mice, we just have to test it in humans to see how good it works there. A big part of the aged environment is a lack of a proper immune system response.

Stem cell implantation does normally modulate inflammation to some degree - the way stem cell transplants are designed at the moment they rarely do anything else besides that regardless of the age of the recipient.

The problem with using Alzheimer's and Parkinson's as an argument when it comes to stem cell transplantation is kinda shallow, these diseases when they are diagnosed are typically the last stage of the disease so it's very hard to say what really causes them and no one knows for certain - but there is evidence these diseases might have some prion disease like qualities. They could start inside the cells but once there's synuclein or abeta outside the cells - typically when the diseases are detected unfortunately - it's already too late, implanting stem cells in that case would be as useful as implanting stem cells to treat AIDS.
The extracellular deposits of proteins will have to be cleared out somehow first for a stem cell therapy to work.
Anyway there are still stem cell therapies in development for PD and Alzheimer's regardless of the prior failures which show good results in animal testing. It's way to early to build an opinion on anything.

Posted by: Anonymoose at July 11th, 2016 4:34 AM

You say that but our researcher shows reduction of plaques using MSCs. We have a far more ambitious demonstration planned for stem cells shortly too at the MMTP.

Posted by: Steve Hill at July 11th, 2016 7:19 AM

I did say there are still stem cell therapies being developed for PD and AD.

I'm sure you can modulate the immune response in neurodegenerative diseases with stem cells and possibly even clear out the plaques to some degree, but then you'd have to identify why they were formed in the first place, because even if they are being cleared that just means they won't accumulate or that they will accumulate slower. We have to figure out why they're being produced in the first place.

And when it comes to implantation and what phenotype the cells acquire that's a different topic altogether.

Posted by: Anonymoose at July 11th, 2016 9:10 AM

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