52 replies to this topic

[Turnbuckle]

Epigenetic drift in stem cells is certainly possible, and is in fact crucial in SC aging according to the following-

 

Summarizing, our data suggest that DNA methylation plays a crucial role in adult stem cell aging, as DNA methylation increases with donor age in Ad-MSCs. We could show that 5-Azacytidine, a small molecule inhibitor for DNMTs, not only passively but also actively promotes DNA demethylation in Ad-MSCs from aged donors. This balanced the expression levels of pluripotency genes and improved the osteogenic differentiation potential, thus rejuvenating aged Ad-MSCs.

 https://www.ncbi.nlm...les/PMC3946260/

 

 

But as long as there are non-hypermethylated SCs in a niche, the fraction thereof will be increased by self-renewal. Rescue of SCs turned inactive by hypermethylation would be greatly advantageous when no more are available. This would be especially useful in small niches, such at follicle roots. Substances like 5-Azacytine discussed in the above paper are highly toxic, however, unlike alpha ketoglutaric acid,

 

 

[Edit_XYZ]

Epigenetic drift in stem cells is certainly possible, and is in fact crucial in SC aging according to the following-

 

 

But as long as there are non-hypermethylated SCs in a niche, the fraction thereof will be increased by self-renewal. Rescue of SCs turned inactive by hypermethylation would be greatly advantageous when no more are available. This would be especially useful in small niches, such at follicle roots. Substances like 5-Azacytine discussed in the above paper are highly toxic, however, unlike alpha ketoglutaric acid,

As I understand it, stem cells act very coordinated, very regimented, in their niches.

I do not think they act like bacteria in a culture, with non-hypermethylated, active stem cells multiplying and outcompeting inactive stem cells. As long as inactive stem cells exist in the niche, they are not being replaced.

 

My reasoning is also supported on the fact that there is a process of aging, a cumulative disfunction of the body with age. If active stem cells were continuously replacing inactive stem cells in their niches, the aging process would not exist in the organism; at least, aging would not be caused by lack of stem cell activity.

[Turnbuckle]

As I understand it, stem cells act very coordinated, very regimented, in their niches.

I do not think they act like bacteria in a culture, with non-hypermethylated, active stem cells multiplying and outcompeting inactive stem cells. As long as inactive stem cells exist in the niche, they are not being replaced.

 

My reasoning is also supported on the fact that there is a process of aging, a cumulative disfunction of the body with age. If active stem cells were continuously replacing inactive stem cells in their niches, the aging process would not exist in the organism; at least, aging would not be caused by lack of stem cell activity.

 

 If an SC becomes non-functional because it lost its ability to replicate due to hypermethylation, then it is not available for proliferation or differentiation. If it was, then the process of de novo methylation would fix it. Functional SC's do not replace dysfunctional SCs, so far as I know, but if the numbers of functional SCs are proliferated by the correct techniques, the SC niche can be topped off.

 

The life-long persistence of stem cells in the body makes them particularly susceptible to the accumulation of cellular damage, which ultimately can lead to cell death, senescence or loss of regenerative function. Indeed, stem cells in many tissues have been found to undergo profound changes with age, exhibiting blunted responsiveness to tissue injury, dysregulation of proliferative activities and declining functional capacities. These changes translate into reduced effectiveness of cell replacement and tissue regeneration in aged organisms.

https://www.ncbi.nlm...rative function.

 

 

Many researchers are looking at ways to fix dysfunctional SCs, but why do that when you can proliferate the good ones? This does not happen naturally, but it can be forced.

Edited by Turnbuckle, 01 August 2022 - 11:25 AM.

[Edit_XYZ]

 If an SC becomes non-functional because it lost its ability to replicate due to hypermethylation, then it is not available for proliferation or differentiation. If it was, then the process of de novo methylation would fix it. Functional SC's do not replace dysfunctional SCs, so far as I know, but if the numbers of functional SCs are proliferated by the correct techniques, the SC niche can be topped off.

 

 

Many researchers are looking at ways to fix dysfunctional SCs, but why do that when you can proliferate the good ones? This does not happen naturally, but it can be forced.

I see - you are arguing that you can use various medicines to 'encourage' active, correctly methylated SCs to proliferate, and keep the stem cell niches functional when, without such intervention, they would stop working.

I have become aware of the thread in which you present your protocol for just this:

https://www.longecit...newal-with-c60/

It's a lot to read. BTW, congratulations for the huge methylation age reversal you have achieved.

 

I do have a few questions:

Does your protocol have any effect on VSELs (Very Small Embryonic-Like Stem Cells)? As you know, their number dwindles with age.

 

Do you have any idea about what happens with the inactive SCs, if you 'encourage' the active, correctly methylated SCs to proliferate in the niche? Do they undergo apoptosis? If such a mechanism confirmed for stem cells?

 

About the proliferation and differentiation of SCs - you wrote about how SCs de novo methylate during these processes. I would appreciate it if you could point to studies/other information sources with more detail on this.

Edited by Edit_XYZ, 01 August 2022 - 12:17 PM.

[Turnbuckle]

I see - you are arguing that you can use various medicines to 'encourage' active, correctly methylated SCs to proliferate, and keep the stem cell niches functional when, without such intervention, they would stop working.

I have become aware of the thread in which you present your protocol for just this:

https://www.longecit...newal-with-c60/

It's a lot to read. BTW, congratulations for the huge methylation age reversal you have achieved.

 

I do have a few questions:

Does your protocol have any effect on VSELs (Very Small Embryonic-Like Stem Cells)? As you know, their number dwindles with age.

 

Do you have any idea about what happens with the inactive SCs, if you 'encourage' the active, correctly methylated SCs to proliferate in the niche? Do they undergo apoptosis? If such a mechanism confirmed for stem cells?

 

 

 

If VSELs actually exist, then I would expect them to respond. I'm using retail epigenetic tests to determine efficacy of the treatments and the tests only measure a limited number of easily obtained cells from a subject. Presumably the replacement rate goes up when epigenetic age goes down, as somatic cells derived from SCs will have a near zero epigenetic age and lower the average. Since all SC's are controlled by mito switches, all are likely affected by the treatment. As for stem cell niche homeostasis, that is unclear. However, I have worked under the assumption that homeostatic processes are at play that trim back SCs if there are too many of them. And thus I usually combine a proliferation/fusion treatment with one or more differentiation/fission treatments back-to-back to take advantage of the excess as soon as possible.

[Learner056]

Well, where the rubber meets the road, Mr Sinclair is the "it", he has that needed sense of urgency and vision. Now you can argue all that you want that you are not even close to his foot-nail, or that he too far superior to you in accomplishments, that you feel too small because of it, or that he is too good in marketing, or too greedy unlike yourself, but the matter of fact is that he has "explained concepts" to ignorant like myself at zero dollars.  That said there are also plenty of genuine reasons to be skeptical of him, but one needs to stay honest, give credit where due.  The few research publications that carry his name I looked at, I seldom see that depth, that level of detail, and data.  His name is usually the last and I feel that he positions people working underneath to take a leading role in research - that is a commendable human trait.  His stack, comprised of Res, NMN, ALA, PQQ, Quercetin, these are all indeed powerful compounds.  Now I do think one needs to exercise prudence while using these compounds, I do think that Mr Sinclair needs to explain A LOT LOT more concepts, but then I also think that I should be king of Namibia (its just another story that they haven't anointed me yet).  

[Learner056]

This is immaculate. I was trying to understand these precise relationships.  Some people experience a loss of Melanocyte SC's on their scalp, don't you think fission/fusion cycling done correctly can be utilized in that context?, as aging I feel is strongly tissue specific.  I think the biggest hurdle is that some people have certain longevity mechanisms activated (as a consequence of DNA damage etc) and as a result their epigenetic changes prevent the systemic circulation of certain compounds (say BCAAs).  So while there overall aging rate may be small but still they are experiencing tissue specific manifestations.  I suspect in such cases, topical route overcomes these enterocyte specific limitations, and a fission/fusion treatment (complemented through a localized intervention i.e. topical route) provide them the material needed for MSC re-awakening or production?

Since all SC's are controlled by mito switches,  Assumption that homeostatic processes are at play that trim back SCs if there are too many of them. And thus I usually combine a proliferation/fusion treatment with one or more differentiation/fission treatments back-to-back to take advantage of the excess as soon as possible.

 

[Phoebus]

DS stole $8M from big pharma with his fake resveratrol research that could never be replicated, so yeah very few are at his level of theivery. But good for him, mad respect to anyone who can steal that much money and still be respected scientist. 

[Learner056]

I am not understanding this concept, pleeeease help this grandma quick (before it passes away one night in sleep, so time is the essence here, you all petulant children: 

 

I associate: Fission=Division=Proliferation  vs Fusion=Differentiation=Commitment

But Turnbuckle is opposite, I think he is associating Proliferation=Fusion  and Differentation=Fission?   How can that be right.

I usually combine a proliferation/fusion treatment with one or more differentiation/fission treatments back-to-back to take advantage of the excess as soon as possible.

 

[Turnbuckle]

I am not understanding this concept, pleeeease help this grandma quick (before it passes away one night in sleep, so time is the essence here, you all petulant children: 

 

I associate: Fission=Division=Proliferation  vs Fusion=Differentiation=Commitment

But Turnbuckle is opposite, I think he is associating Proliferation=Fusion  and Differentation=Fission?   How can that be right.

 

 

Your use of equals signs is incorrect.

 

Mito fusion + a UCP2 blocker (C60) results in SC proliferation.

Mito fission + a senolytic agent results in the apoptosis of senescent cells.

Paracrine signaling during apoptosis signals SCs to replace the eliminated cells.

 

Also, you seem to be confused about mitochondria fission/fusion vs stem cell division, which can be either differentiation or proliferation.

 

Mitochondrial morphology (fission and fusion) is used to control cell fate. Stem cell division may be asymmetric (differentiation into a somatic and a stem cell) or symmetric (proliferation into two stem cells). The latter is also called self renewal, which occurs only about 20% of the time unless you force the SC mitochondria to a fusion state.

 

The process of reducing epigenetic age first requires proliferation of SCs using mito fusion along with the restoration of SC ATP production with a UCP2 blocker, then on subsequent days, mito fission to encourage the apoptosis of senescent cells. Senolytics can help promote apoptosis.

 

To summarize:

Mito morphology can be fission, fusion, or mixed. Fusion is needed for SC proliferation, while fission is needed for the apoptosis of senescent cells.

Stem cells can differentiate or proliferate.

C60 is a putative UCP2 blocker. UCP2 is a protein that forms pores in the mitochondria of SCs. It's purpose is to leak protons across the inner mito membrane that would otherwise be used by ATP synthase to make ATP. Blocking those pores thus restores ATP production and wakes up SCs.

Edited by Turnbuckle, 23 August 2022 - 11:30 PM.

[Learner056]

So while I am deeply impressed by your theory Turnbuckle, but I am also a born skeptic.  I understand our universe is not perfect, but what you present, is there some reasonable level of consensus?  I opened two papers and I read something different, so appreciate your wisdom here:

 

Mito Dynamics: Fission and Fusion in fate determination of MSCs, Lin Ren et al, 2020.  They contend that: "Active mitochondrial fission, adapted to glycolytic dependence on energy production, is critical for the self-renewal and pluripotency of MSCs",

2019: Mitochondrial fission is essential for maintaining stemness in MSCs, and inhibiting fission leads to a reduction in the expression of stemness markers and multidirectional differentiation potential (Feng et al., 2019)

While this may not be applicable but it does illuminate:  2016: "During somatic cell reprogramming to iPSCs, cells undergo mitochondrial reconstruction, usually from a mature network toward immature mitochondria, and a metabolic shift, usually from OXPHOS toward glycolysis. Drp1-dependent mitochondrial fission is also necessary for the acquisition of cellular pluripotency during the early stage of embryonic fibroblast induction to form iPSCs" (Prieto et al., 2016)

A one important concept the aforementioned paper is introducing: "However, over fission induced by Mff overexpression impairs the pluripotency of ESCs and iPSCs, suggesting that active fission should be within a certain threshold to better maintain stem cell function"  

 

 

Mitochondrial morphology (fission and fusion) is used to control cell fate. Stem cell division may be asymmetric (differentiation into a somatic and a stem cell) or symmetric (proliferation into two stem cells). The latter is also called self renewal, which occurs only about 20% of the time unless you force the SC mitochondria to a fusion state.

 

The process of reducing epigenetic age first requires proliferation of SCs using mito fusion along with the restoration of SC ATP production with a UCP2 blocker, then on subsequent days, mito fission to encourage the apoptosis of senescent cells. Senolytics can help promote apoptosis.

 

Attached Files

•  fcell-08-580070-g002.jpg   106.95KB   0 downloads

Edited by Learner056, 28 August 2022 - 12:35 AM.

[Rib Jig]

this thread implies people are self-medicating alleged "aging reversal" supplements...?

any nonagenarians out there looking & acting like they're under 50 years old?
any centenarians out there looking & acting like they're under 60 years old?
(looking & acting 10 or 20 years younger is a natural occurrence for some, proves nothing IMO) 

authoritative first-hand evidence please,

otherwise I'll stick to my cryonics scheme...       

Edited by Rib Jig, 28 August 2022 - 12:31 AM.

[Turnbuckle]

So while I deeply impressed by your theory Turnbuckle, but I am also a born skeptic.  I understand our universe is not perfect, but what you present, is there some reasonable level of consensus?  I opened two papers and I read something different, so appreciate your wisdom here: 

 

Mito Dynamics: Fission and Fusion in fate determination of MSCs, Lin Ren et al, 2020.  They contend that: "Active mitochondrial fission, adapted to glycolytic dependence on energy production, is critical for the self-renewal and pluripotency of MSCs",

2019: Mitochondrial fission is essential for maintaining stemness in MSCs, and inhibiting fission leads to a reduction in the expression of stemness markers and multidirectional differentiation potential (Feng et al., 2019)

While this may not be applicable to our scenario but still:  2016: "During somatic cell reprogramming to iPSCs, cells undergo mitochondrial reconstruction, usually from a mature network toward immature mitochondria, and a metabolic shift, usually from OXPHOS toward glycolysis. Drp1-dependent mitochondrial fission is also necessary for the acquisition of cellular pluripotency during the early stage of embryonic fibroblast induction to form iPSCs" (Prieto et al., 2016)

A one important concept the aforementioned paper is introducing: "However, over fission induced by Mff overexpression impairs the pluripotency of ESCs and iPSCs, suggesting that active fission should be within a certain threshold to better maintain stem cell function"  I assume this concept would be applicable to MSCs / adult stem cells in our bodies as well?

 

 

Let's get on the same page with the terminology, as not everyone uses the same definitions.

 

Herein proliferation is symmetric division, or self-renewal. One SC becomes two SCs. 

Differentiation is asymmetric division. One SC becomes one SC and one somatic cell. 

Stemness is a long-term fundamental property of SCs. Without stemness, SCs will not be able to proliferate or differentiate. 

 

The first paper you referenced is talking about fission being required for the maintenance of stemness in culture, not for proliferation in the sense I used above. Both fission and fusion are necessary for maintaining the health of all cells, not just SCs, and thus mitochondria are highly active, constantly fusing and fissioning.

 

Mitochondrial fusion is used herein to bias SCs to proliferation (one SC becoming two), while mito fission biases SCs for differentiation. See the following paper --

 

In this model, elongated [fused] mitochondria in NSCs maintain low ROS levels and promote self-renewal, while a transition of mitochondria to a more fragmented [fission] state results in a modest increase in ROS levels, thereby inducing the expression of genes that inhibit self-renewal (Botch) and promote commitment and differentiation 

https://www.cell.com...20?showall=true

 

 

As an aside, I don't believe they are correct about lower ROS being the operative factor, for if it was, good antioxidants would make a substantial impact on viable SC levels and thus longevity, but they don't. This applies to C60 as well. It is an excellent antioxidant, but used without a fusion agent, if fails to extend lifespans. In my view a clue can be found in the first paper when they write about the "asymmetrical segregation of mitochondria" during differentiation. An asymmetry in the SC is needed for asymmetric division.

 

For example, it has been suggested that young mitochondria go to the new SC, while the old ones go to the new somatic cell. 

 

Old mitochondria are preferentially segregated to the differentiating daughter, whereas stem cells inherited fewer older mitochondria during the asymmetric division of human mammary stem-like cells, suggesting that stem cells might be protected from senescence by eliminating old, nonfunctional mitochondria (Fig. 4 D; Katajisto et al., 2015).

https://www.ncbi.nlm...les/PMC6219723/

 

 

According to that paper, segregating mitochondria might be a benefit, but in my view, it is actually the fate determinant. By preventing segregation of mitochondria by fusing them, that would dampen any asymmetry, and without asymmetry you get proliferation.

Edited by Turnbuckle, 28 August 2022 - 01:13 AM.

[Learner056]

I marvel at your clarity (and eloquence with which you explained) that I was unable to decipher it earlier.  I can be wrong, but my inability to distinguish this context, may even be one underlying reason why sometimes researchers get so far apart.  But back to this beautiful learning of mito:  

 

Fission: asymmetric division/differentiation/

Fusion: symmetric division/stem cell renewal/proliferation

1. Would it be right, that in Fission: while stem cells are biased for differentiation, it means higher rate of somatic cell proliferation (e.g. epithelial cells)?  And importantly to further say, that fibrosis/scar tissue will be associated to excess of fission?

 

2. Being only focused on the science aspect here, is ejaculation as a process (part of the male anatomy obviously), involved in excess mito fusion or with fission by chance?

 

3. C60 being a putative UCP2 blocker.  I sometimes read that C60 is only good for over 65, though I have not seen it explained as a rationale?  for e.g there is some fear that C60 has a dependence on the G6PD (glucose 6 phosphate dehydrogenase) pathway or maybe that it shuts that pathway down - but still I don't understand why it would be a concern in that enzyme deficient state, if any should "alleviate" that deficiency

 

 

Let's get on the same page with the terminology, as not everyone uses the same definitions.

 

Edited by Learner056, 29 August 2022 - 10:03 PM.

[Turnbuckle]

I marvel at your clarity (and eloquence with which you explained) that I was unable to decipher it earlier.  I can be wrong, but my inability to distinguish this context, may even be one underlying reason why sometimes researchers get so far apart.  But back to this beautiful learning of mito:  

 

Fission: asymmetric division/differentiation/

Fusion: symmetric division/stem cell renewal/proliferation

1. Would it be right, that in Fission: while stem cells are biased for differentiation, it means higher rate of somatic cell proliferation (e.g. epithelial cells)?  And importantly to further say, that fibrosis/scar tissue will be associated to excess of fission?

 

With epithelial cells (and many other cell types), most of the differentiation is by transit amplifying cells, which are intermediate between stem cells and somatic cells. (See this paper.) Likely epigenetic aging is occurring there.  Some fading of new scar tissue can occur with this protocol, but the older the scar, the less likely it will fade. I don't know if there is an association between scar tissue and mito morphology.

 

2. Being only focused on the science aspect here, is ejaculation as a process, involved with excess mito fusion or with fission?

 

I have no idea. 

 

3. C60 being a putative UCP2 blocker.  I sometimes read that C60 is only good for over 65, though I have not seen it explained as a rationale?  for e.g there is some fear that C60 have a dependence on the G6PD (glucose 6 phosphate dehydrogenase) pathway or maybe that it shuts it down - a concern for those with G6PD deficiency?

 

I have suggested that this C60 protocol be used with caution by those not of the geriatric crowd (who have a lot less to lose). Certainly, if you use C60 as many are doing without considering fusion, you will definitely deplete your SC niches and see a fading of the initial positive results. As for an association with G6PD, I've not heard of that.

 

[Learner056]

I did not follow this, can you lay down the process steps that would amount to C60 depleting SCs (without considering fusion)?  Many thanks.

 

 

Edited by Learner056, 29 August 2022 - 10:54 PM.

[Turnbuckle]

I did not follow this, can you lay down the process steps that would amount to C60 depleting SCs (without considering fusion)?  Many thanks.

 

 

See the thumbnail plot at the bottom of this post. Viable SC numbers are on a decaying exponential curve throughout life. If you just stimulate them without regard to the balance of differentiation to proliferation, you can get a temporary improvement that may last a year or two, but essentially you are robbing Peter to pay Paul, as the SC decline will go faster in the future as you use them up today. But if you use fusion + stimulation to drive SCs into proliferation, you can refill the niches and push that curve to the left.

 

Normally SCs differentiate 80% of the time and proliferate 20%, which is not enough proliferation to prevent depletion. Using fusion will bias that split to proliferation instead of differentiation, and restore SC niches.

Edited by Turnbuckle, 29 August 2022 - 11:57 PM.

[johnhemming]

I have wondered what the relationship of HIF 1 alpha is to the proliferation of stem cells

https://www.ncbi.nlm...les/PMC6457711/

 

 

[Learner056]

Mechanistically, C60 by itself only blocks MMP (through UCP2) thereby increasing ATP.   You stated that Stearic Acid (or equivalent) amplifies Fusion.  But isn't it that mito in a fused state (Fusion) implicitly also increases ATP?.  So C60 has some intrinsic characteristic of stimulating stem cells or is through increased ATP?  This part I got it, that Fusion biases for Symmetric/Self renewal. 

 

Normally (approximations): SC's differentiate 80% and Proliferate 20% (you mean here 20% symmetric proliferation, or even this 20% is assymetric).   

 

The above numbers are in a mixed Fission/Fusion state (where one is not deliberately intending anything), or this would be when e.g. person following your protocol of being in predominant Fusion state?

 

Fusion state helps increase Symmetric/Self-renewal replication.  Roughly how much increase would that be?   

 

Apologies for these so many questions:  I read that too much Fission or too much Fusion can be detrimental as they contribute towards mito dysfunction over time.  What are your thoughts on that?.  Would there be some 'intuitive' way (by chance) to maybe even 'feel' or know when Fission state might be getting too high or Fusion state running too high? By chance even some typical blood marker that approximates it?

 

See the thumbnail plot at the bottom of this post. Viable SC numbers are on a decaying exponential curve throughout life. If you just stimulate them without regard to the balance of differentiation to proliferation, you can get a temporary improvement that may last a year or two, but essentially you are robbing Peter to pay Paul, as the SC decline will go faster in the future as you use them up today. But if you use fusion + stimulation to drive SCs into proliferation, you can refill the niches and push that curve to the left.

 

Normally SCs differentiate 80% of the time and proliferate 20%, which is not enough proliferation to prevent depletion. Using fusion will bias that split to proliferation instead of differentiation, and restore SC niches.

 

Edited by Learner056, 31 August 2022 - 07:35 PM.

[Rib Jig]

cellular senescence / zombie cell build-up:
https://www.yahoo.co...-050436011.html

[Gern]

My own epigenetic age dropped by 28 years, and I am, presumably, a mammal.

Is it possible the David Sinclair is aware of your protocol? He mentioned people who have turned back their epigenetic age by more than 20 years, and it made me wonder.

[Turnbuckle]

Is it possible the David Sinclair is aware of your protocol? He mentioned people who have turned back their epigenetic age by more than 20 years, and it made me wonder.

 

 

I haven't seen that, but I rather doubt it. He seems to swear by calorie restriction and MNM.

[Learner056]

I have been intrigued by Hypoxia.  I get the feeling that Hypoxia may decrease the anabolic/ATP/mTOR signaling barrier for SC to come out of deeper quiescent states on one hand, while on the other hand it has certain limitations such as neuronal/myogenic differentiation of cells (below).  I don't understand well currently but trying - my youngest son's issues where his cells do not fully differentiate, if they are somehow tied to his psychiatric issue, like for one under stress, his body can change/maneuver its shape and size.  I notice during his episodes, he unknowingly induces hypoxia under stress (and even during joy), he does not realize it at the time ... it worries a mother, so trying to learn this as quickly I can. 

 

Hypoxia blocks neuronal and myogenic differentiation in a Notch-dependent manner.    Ref:  :text=The%20Notch%20signaling%20pathway%20is,self%20renewal%20and%20multi-potency' class='bbc_url' title='External link' rel='nofollow external'>https://www.ncbi.nlm...d multi-potency.

 

I have wondered what the relationship of HIF 1 alpha is to the proliferation of stem cells

https://www.ncbi.nlm...les/PMC6457711/