Epigenetic Reprogramming With a Methylation Inhibitor

[pegasus2]

I looked into DNMT1 inhibitors some time ago to demethylate HFSCs, but ultimately I did not try it due to this study showing epidermal deletion of DNMT1 induces severe hair loss in mice, and this one showing that increased methylation of the AR in occipital HF may protect them from balding. However, I came across this study on reproductive follicles that made me revisit the idea.

DNA methylation mediated RSPO2 to promote follicular development in mammals

Inhibition of DNA methylation or DNMT1 knockdown could decrease the methylation level of CpG island (CGI) in RSPO2 promoter and upregulate the expression level of RSPO2 in porcine GCs

RPSO2 is a master regulator of Wnt signaling, it primes stem cells for renewal and organ regeneration. When its expression is high the HF stays in anagen

RSPO2 knockdown promoted the apoptosis of GCs, blocked the development of follicles, and delayed the onset of puberty with decreasing the expression level of Wnt signaling pathway-related genes (LGR4 and CTNNB1) in vivo. Taken together, the hypomethylation of −758/−749 and −563/−553 regions in RSPO2 promoter facilitated the occupancy of E2F1 and enhanced the transcription of RSPO2, which further promoted the proliferation and E2 secretion of GCs, inhibited the apoptosis of GCs, and ultimately ameliorated the development of follicles through Wnt signaling pathway. This study will provide useful information for further exploration on DNA-methylation-mediated RSPO2 pathway during follicular development.

CTNNB1 is B-catenin and E2 is 17b-estradiol.

Furthermore:

To determine the effect of RSPO2 on the E2 secretion of GCs, the expression of genes related to estrogen signaling pathway were detected after overexpression and knockdown of RSPO2. qRT-PCR (Fig. 4A) and WB (Fig. 4B) results indicated that RSPO2 overexpression significantly promoted the mRNA and protein levels expression of CYP19A1 and HSD17B1, while RSPO2 silencing significantly inhibited the mRNA and protein expression levels of HSD17B1 as well as the protein expression level of CYP19A1. Moreover, RSPO2 overexpression significantly promoted the secretion of E2 in GCs, while RSPO2 knockdown displayed the opposite effects (Fig. 4C). Similarly, RSPO2-siRNA5 also significantly inhibited the mRNA expression of CYP19A1 and HSD17B1 (Fig. 4D) and the secretion of E2 in GCs. (Fig. 4E). In summary, RSPO2 promoted the secretion of E2 in porcine ovarian GCs.

Of note here is that HSD17B1 is an estrogenic enzyme. It converts E1 into the more potent E2 and it also weakens androgenic activity by breaking down T into weaker androgens.

Influence of estrogens on the androgen metabolism in different subunits of human hair follicles ​

RSPO in two studies is the second most highly correlated gene with Androgenetic Alopecia after the androgen receptor. Its induction has been shown to induce precocious telogen-anagen transition and to prevent catagen, so anything that upregulates RSPO is worth close inspection.

The following studies explore the use of DNMT1 inhibitors in cochlea hair follicle regeneration:

Epigenetic DNA Demethylation Causes Inner Ear Stem Cell Differentiation into Hair Cell-Like Cells​

5-aza was applied to mouse utricle sensory epithelia-derived progenitor cells (MUCs) to investigate whether 5-aza stimulated MUCs to become sensory hair cells. After treatment, MUCs increased expression of hair cell genes and proteins. The DNA methylation level (indicated by percentage of 5-methylcytosine) showed a 28.57% decrease after treatment, which causes significantly repressed DNMT1 protein expression and DNMT activity. Additionally, FM1-43 permeation assays indicated that the permeability of 5-aza-treated MUCs was similar to that of sensory hair cells, which may result from mechanotransduction channels. This study not only demonstrates a possible epigenetic approach to induce tissue specific stem/progenitor cells to become sensory hair cell-like cells, but also provides a cell model to epigenetically modulate stem cell fate determination.

Generation of new hair cells by DNA methyltransferase (Dnmt) inhibitor 5-azacytidine in a chemically-deafened mouse model​

Regeneration of mature mammalian inner ear hair cells remains to be a challenge. This study aims to evaluate the ability of DNA methyltransferase (Dnmt) inhibitor 5-azacytidine (5-aza) to generate outer hair cells (OHCs) in a chemically-deafened adult mouse model. 5-aza was administrated into the mouse inner ear via the round window. Immunofluorescence was used to examine the expression of hair cell specific proteins following 5-aza treatment. The results showed that in the chemically-deafened mouse cochlea, new OHCs were found post 5-aza treatment, whereas OHCs were completely lost in saline-treated mice. New hair cells expressed multiple hair cell markers included Myosin VIIa, Pou4f3 and Myosin VI. Newly-generated hair cells presented in three cochlear turns and were able to survive for at least six weeks. The effects of new hair cells generation by 5-aza were concentration dependent. Quantitative PCR study indicates that 5-aza may function through Dnmt1 inhibition. The results of this report suggest that the Dnmt inhibitor 5-aza may promote hair cell regeneration in a chemically-deafened mouse model.

This one shows that 5-aza upregulates SOX2 which is necessary for HF morphogenesis.

The DNA methylation inhibitor RG108 protects against noise-induced hearing loss

This study suggests that inhibition of DNMT1 ameliorates noise-induced hearing loss and indicates that DNMT1 may be a promising therapeutic target.

Genome-Wide Demethylation by 5-aza-2’-Deoxycytidine Alters the Cell Fate of Stem/Progenitor Cells​

DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine (5-aza-CdR) is able to cause DNA demethylation in the genome and induce the expression of silenced genes. Whether DNA demethylation can affect the gene expression of stem/progenitor cells has not been understood. Mouse utricle epithelia-derived progenitor cells (MUCs), which possess stem cell features as previously described, exhibit a potential DNA methylation status in the genome. In this study, MUCs were treated with 5-aza-CdR to determine whether DNMT inhibitor is able to induce the differentiation of MUCs. With 5-aza-CdR treatment for 72 hr, MUCs expressed epithelial genes including Cdh1, Krt8, Krt18, and Dsp. Further, hair cell genes Myo7a and Myo6 increased their expressions in response to 5-aza-CdR treatment. The decrease in the global methylated DNA values after 5-aza-CdR treatment indicated a significant DNA demethylation in the genome of MUCs, which may contribute to remarkably increased expression of epithelial genes and hair cell genes. The progenitor MUCs then turned into an epithelial-like hair cell fate with the expression of both epithelial and hair cell genes. This study suggests that stem cell differentiation can be stimulated by DNA demethylation, which may open avenues for studying stem cell fate induction using epigenetic approaches.

This leads us to a patent filed by George Cotsarelis for the use of a DNMT inhibitor for WIHN. When applied topically for 2 consecutive days 5-aza-2’-deoxycytidine increased hair follicle generation by 8-fold. There was nothing else used, no minoxidil, just two applications of this DNMT1 inhibitor.

Currently, no therapy exists to regenerate normal skin that includes hair follicles and sweat glands from a wound or scar and hence, there is an existing need for such a therapy. [0003]The present invention addresses this need by providing DNA methyl transferase (DNMT) inhibitors, e.g., 5-aza-2′-deoxycytidine or 5-azacytidine. When inhibitors of DNMT activity (i.e., blocks DNA methylation) are administered following wound closure, enhanced hair follicle regeneration occurs, suggesting that DNA methyl transferase modulates the regeneration program in a strict temporal manner. Also demonstrated is that administration of 5-aza-2′-deoxycytidine or other related DNA methyltransferase inhibitors may promote skin regeneration in healing cutaneous wounds.

When applied to the wound site immediately following scab detachment for 2 days only, Aza-dC greatly enhanced hair follicle regeneration in healed wounds (from 3 to 8 fold) in a dose-dependent manner. (FIG. 4). Therefore, up-regulation of certain genes, via inhibition of DNA methylation, at the times when hair follicles begin to form plays a critical role in the hair follicle regeneration. This stimulatory effect on regeneration was diminished when the topical treatment was extended to 5 days (from SD0 to SD4), suggesting that the expression levels of some of these genes need to be temporally controlled to support hair follicle formation"

Curiously, systemic administration of the compound had the opposite effect, and applying the topical compound for 5 consecutive days instead of 2 reduced its effectiveness. This could be due to a temporal window during regenration after which DNMT1 inhibition must be stopped for the next step in regeneration to take place, or it could be due to cytotoxicity. If the latter is the case then RG108, which does not display cytotoxicity, may be better suited for this.

Topical Aza-dC treatment enhanced hair follicle regeneration in healed wounds. (A) 10 μL of Aza-dC (dissolved in DMSO) was applied topically to the wound site, once a day, for 2 consecutive days at the time of scab detachment (SD0 & SD1). Aza-dC treatment at all doses tested greatly enhanced hair follicle (HF) neogenesis compared to the vehicle control, with the strongest enhancement seen with the highest tested dose (10 μg). (A) 10 μL of Aza-dC (dissolved in DMSO) was applied topically to the wound site, once a day, for 5 consecutive days at the time of scab detachment (SD0 to SD1). While the two lower doses (1 & 5 μg) showed little effect on the regeneration, the highest dose (10 μg) still enhanced hair follicle regeneration.

I believe this could complement the CHIR99021+VPA reprogramming experiment that some people are trying. This is all very serious stuff though that is risky to play around with. Cellular reprogramming is unpredictable, untested, and dangerous. We don't understand this stuff very well yet and the chance of causing cancer is real. This is all theoretical for now.

A. shows hair regeneration when applied for 2 days after scab detatchment. B. shows hair regeneration when applied for 5 days after scab detachment. This is even more effective than Cotsarelis' wounding study with SAG.

 

[Hairful]

This is all very serious stuff though that is risky to play around with. Cellular reprogramming is unpredictable, untested, and dangerous. We don't understand this stuff very well yet and the chance of causing cancer is real. This is all theoretical for now.

Yes, our knowledge needs to progress a lot further before we can even think about trying this stuff. And at that point, we would have cure for a lot more serious diseases than hairloss.

If there was only one gene for baldness, perhaps cispr/gene therapy could’ve been a viable option now but there’s so many, that’s out the window too .

 

[pegasus2]

Yes, our knowledge needs to progress a lot further before we can even think about trying this stuff. And at that point, we would have cure for a lot more serious diseases than hairloss.

If there was only one gene for baldness, perhaps cispr/gene therapy could’ve been a viable option now but there’s so many, that’s out the window too .

You mean it needs to progress a lot further before anyone else can think about trying this stuff. I will be trying it soon. A DNMT1 inhibitor alone is not that crazy. There have been clinical trials with 5-aza and two others are FDA approved. The really crazy part comes in combining this with CHIR99021 and VPA, which is what I'm going to do.

 

[Feramon1]

It may sound naive, but why not smooth out the side effects of really working Androgenetic Alopecia drugs with another drugs? Or do it in 3 steps? I mean create a longer chain for the end goal.

For example we mitigate the side effects of antibiotics with probiotics or immune preparations and it works. I remember a guy on this forum who reduced the side effects of finasteride with Tangat Ali and it seemed to work for him.

You mean it needs to progress a lot further before anyone else can think about trying this stuff. I will be trying it soon. A DNMT1 inhibitor alone is not that crazy. There have been clinical trials with 5-aza and two others are FDA approved. The really crazy part comes in combining this with CHIR99021 and VPA, which is what I'm going to do.

Man, we still need you.

 

[pegasus2]

It may sound naive, but why not smooth out the side effects of really working Androgenetic Alopecia drugs with another drugs? Or do it in 3 steps? I mean create a longer chain for the end goal.

You mean counter estradiol with proviron? Obviously that's a bad idea. Maybe you have a good suggestion for countering the only drugs that work? I messed up when I did my protocol because I was too arrogant in thinking I was too high T for anything to happen to me. Had I kept dutasteride to .5mg and not used estradiol and spironolactone I think I would've been fine. ASC+RU is better than castration and no sides. Add to that estriol which I think is as good as estradiol or nearly as good, but without the sides, and you are getting close. Still need to demethylate to reactivate the genes that are switched off permanently by AR

For example we mitigate the side effects of antibiotics with probiotics or immune preparations and it works. I remember a guy on this forum who reduced the side effects of finasteride with Tangat Ali and it seemed to work for him.


Man, we still need you.

 

[Feramon1]

You mean counter estradiol with proviron? Obviously that's a bad idea. Maybe you have a good suggestion for countering the only drugs that work? I messed up when I did my protocol because I was too arrogant in thinking I was too high T for anything to happen to me. Had I kept dutasteride to .5mg and not used estradiol and spironolactone I think I would've been fine. ASC+RU is better than castration and no sides. Add to that estriol which I think is as good as estradiol or nearly as good, but without the sides, and you are getting close. Still need to demethylate to reactivate the genes that are switched off permanently by AR

Right, but this is just an example. I'm actually trying to make this a goal instead of the default dgt, but I can't say I've had any real success. There are no specific tips yet, but I have some thoughts that I want to test in practice and if there are results, I will create a separate branch. At the moment I am limited in funds, some things are too expensive for me to experiment.

The catch is that side effects are always associated with cancer or feminization, I'm afraid this trend will continue and we will have to aim at eliminating these side effects, and not finding a cure without them. Obviously, everything we have today is found by accident or as a side effect. It turns out that when they try to treat one, it turns out that it affects the other, while when they are looking for something specific, they do not find it, a paradox. That's what I mean, i think change the angle, change the question.

I messed up when I did my protocol because I was too arrogant in thinking I was too high T for anything to happen to me. Had I kept dutasteride to .5mg and not used estradiol and spironolactone I think I would've been fine.

I read your regimen, it was quite aggressive, but also not bad results. I guess you rolled back?

 

[pegasus2]

5-Aza-2′-Deoxycytidine and Valproic Acid in Combination with CHIR99021 and A83-01 Induce Pluripotency Genes Expression in Human Adult Somatic Cells

A generation of induced pluripotent stem cells (iPSC) by ectopic expression of OCT4, SOX2, KLF4, and c-MYC has established promising opportunities for stem cell research, drug discovery, and disease modeling. While this forced genetic expression represents an advantage, there will always be an...

www.mdpi.com

 

[Hairful]

You mean it needs to progress a lot further before anyone else can think about trying this stuff. I will be trying it soon. A DNMT1 inhibitor alone is not that crazy. There have been clinical trials with 5-aza and two others are FDA approved. The really crazy part comes in combining this with CHIR99021 and VPA, which is what I'm going to do.

You’re a brave man

 

[RagnarLothbrok]

Don't try it man, this is crossing the line.
Why would you risk your own life like that, wouldn't it be similar ethics to buy a f*****g pig or monkey and do it to him first lol

I absolutely agree this is 100% the true and only road for curing baldness (epigenetic programming) and sounds very exciting to see experiments with it but I hope it doesn't mean you harm yourself too much

 

[pegasus2]

Don't try it man, this is crossing the line.
Why would you risk your own life like that, wouldn't it be similar ethics to buy a f*****g pig or monkey and do it to him first lol

I absolutely agree this is 100% the true and only road for curing baldness (epigenetic programming) and sounds very exciting to see experiments with it but I hope it doesn't mean you harm yourself too much

Demethylation agents are used in humans safely, and CHIR/VPA has been tested on a handful of people with a single dose safely. They just haven't been combined on a human, and CHIR/VPA hasn't been used for a prolonged period of time. However, I did use CHIR with another weaker HDAC inhibitor without side effects. I'm planning to use these for only 2 to 3 consecutive days, perhaps one or two times a month

 

[indie85]

Demethylation agents are used in humans safely, and CHIR/VPA has been tested on a handful of people with a single dose safely. They just haven't been combined on a human, and CHIR/VPA hasn't been used for a prolonged period of time. However, I did use CHIR with another weaker HDAC inhibitor without side effects. I'm planning to use these for only 2 to 3 consecutive days, perhaps one or two times a month

The trouble is no meaningful safety data can be obtained from 1 case study even if works..

 

[Redgate]

The trouble is no meaningful safety data can be obtained from 1 case study even if works..

why is that trouble? he wants to use it for himself, not provide clinical data. read the studies and decide for yourself

 

[indie85]

why is that trouble? he wants to use it for himself, not provide clinical data. read the studies and decide for yourself

As others less aware of/unable to understand the theoretical risks may be tempted to try this regimen. Of course it's a free world..

 

[pegasus2]

As others less aware of/unable to understand the theoretical risks may be tempted to try this regimen. Of course it's a free world..

I don't think anyone else is going to try this. It potentially upregulates 3 of the 4 Yamanaka factors, excluding c-MYC. However, SOX2 is the only one highly upregulated.

Edit: It's a small phase I trial, but it's more than one case study https://journals.lww.com/otology-ne...eech_intelligibility_in_subjects_with.14.aspx

This isn't a group buy thread. This is just for discussion.

 

[RagnarLothbrok]

Well if you go for it at least document it

Btw I used to read your hairloss recovery thread back then, what is your hair loss status nowdays since last update if you dont mind sharing? (did you mantain or even improve? or worsened a little maybe)

 

[pegasus2]

Elevated TWIST1 expression in myelodysplastic syndromes/acute myeloid leukemia reduces efficacy of hypomethylating therapy with decitabine | Haematologica

www.haematologica.org

Twist1 mediates decitabine resistance in AML.