In Vivo Reprogramming of Cells to a Pluripotent, Partially Rejuvenated State Continues to Forge Ahead in the Lab

It has for some years now been possible to reprogram adult somatic cells into pluripotent stem cells that are functionally equivalent to embryonic stem cells. This is achieved by overexpressing some or all of the Yamanaka transcription factors, Oct4, Sox2, Klf4, and c-Myc (OSKM) proteins. One of the most interesting outcomes of this process is that cells so treated reverse epigenetic markers of aging to some degree, and repair their mitochondrial damage. Thus the research community has started to induce this same reprogramming in living animals to observe the results. If done haphazardly, the outcome is unrestrained cancer and tissue dysfunction, as one might expect. The surprise is that there are approaches that can lead to benefits with no such issues.

The discoveries of recent years in this part of the field have given rise to the company Turn.bio, who are attempting an implementation of transient partial reprogramming to rejuvenate cells throughout the body, as well as numerous research groups working on their own approaches to a basis for therapies capable of enhancing regeneration and function in old tissues. The work noted here is an example of the type, and is quite interesting for the further evidence that it is possible, given suitable methodologies, to deliver reprogramming factors to mice over a long period of time without causing noticeable harm.

Reversal of ageing- and injury-induced vision loss by Tet-dependent epigenetic reprogramming

In mammals, progressive DNA methylation changes serve as an epigenetic clock, but whether they are merely an effect or a driver of ageing is not known. In cell culture, the ectopic expression of the four Yamanaka transcription factors, namely Oct4, Sox2, Klf4, and c-Myc (OSKM), can reprogram somatic cells to become pluripotent stem cells, a process that erases most DNA methylation marks and leads to the loss of cellular identity. In vivo, ectopic, transgene-mediated expression of these four genes alleviates progeroid symptoms in a mouse model of Hutchison-Guilford Syndrome, indicating that OSKM might counteract normal ageing. Continual expression of all four factors, however, induces teratomas or causes death within days, ostensibly due to tissue dysplasia.

Ageing is generally considered a unidirectional process akin an increase in entropy, but living systems are open, not closed, and in some cases can fully reset biological age, examples being "immortal" cnidarians and the cloning of animals by nuclear transfer. Having previously found evidence for epigenetic noise as an underlying cause of ageing, we wondered whether mammalian cells might retain a faithful copy of epigenetic information from earlier in life, essentially a back-up copy of the original signal to allow for its reconstitution at the receiving end if information is lost or noise is introduced during transmission.

To test this hypothesis, we introduced the expression of three-gene OSK combination in fibroblasts from old mice and measured its effect on RNA levels of genes known to be altered with age, including H2A, H2B, LaminB1, and Chaf1b. We excluded the c-Myc gene from these experiments because it is an oncogene that reduces lifespan. OSK-treated old fibroblasts promoted youthful gene expression patterns, with no apparent loss of cellular identity or the induction of Nanog, an early embryonic transcription factor that can induce teratomas.

Next, we tested if a similar restoration was possible in mice. To deliver and control OSK expression in vivo, we engineered a tightly regulated adeno-associated viral (AAV) vector under the control of tetracycline response element (TRE) promoter. To test if ectopic OSK expression was toxic in vivo, we infected 5 month-old C57BL/6J mice with the vector delivered intravenously, then induced OSK expression by providing doxycycline in the drinking water. Surprisingly, continuous induction of OSK for over a year had no discernable negative effect on the mice, ostensibly because we avoided high-level expression in the intestine, thus avoiding the dysplasia and weight loss seen in other studies.

Post-mitotic neurons in the central nervous system are some of the first cells in the body to lose their ability to regenerate. Using the eye as a model tissue, we have shown that expression of OSK in mice resets youthful gene expression patterns and the DNA methylation age of retinal ganglion cells, promotes axon regeneration after optic nerve crush injury, and restores vision in a mouse model of glaucoma and in normal old mice. Thus we have shown that in vivo reprogramming of aged neurons can reverse DNA methylation age and allow them to regenerate and function as though they were young again.

The requirement of the DNA demethylases Tet1 and Tet2 for this process indicates that altered DNA methylation patterns may not just a measure of age but participants in ageing. How cells are able to mark and retain youthful DNA methylation patterns, then in late adulthood OSK can instruct the removal of deleterious marks is unknown. Youthful epigenetic modifications may be resistant to removal by the Tets by the presence of a specific protein or DNA modification that inhibits the reprogramming machinery. Even in the absence of this knowledge, these data indicate that the reversal of DNA methylation age and the restoration of a youthful epigenome could be an effective strategy, not just to restore vision, but to give complex tissues the ability to recover from injury and resist age-related decline.

Comments

Astounding research!

Posted by: Thomas Balsløv at August 3rd, 2019 2:03 AM

Ah from David Sinclair, his idea is that the epigenetic information get's messed up in later life an all you have to do is reprogram the cells to an earlier state, not exactly the SENS approach but if he can pull it off good for him ... to be honest it sounds to good to be true.

Posted by: Tom at August 3rd, 2019 6:48 AM

Yes, this is important work that adds to the long data history of reprogramming going back to the 1952 Briggs and King experiments

And it has its place in the rejuvenation basket because even if the ability was here to eliminate all forms of cellular damage tomorrow restoring biologic fitness, the epigenetic states of your trillions of cells, as pertaining to both biological robustness and resilience, would still technically be that of an older organism, opening up many tough questions

The issue that still remains in 2019 surrounding these approaches is a translational one that must be addressed as one thinks about more complex tissues and the way biological aging occurs at the tissue level in humans - not like what we see in the lab experiments above and throughout the pre-clinical literature, but in the way cells in various tissue micro-environments, begin to "explore" new transcriptional regulatory states based on their epigenetic status, and the respective communities of cellular competition are established

When one goes into the regenerative biology literature, you find that all organisms capable of complex reprogramming for regeneration and rejuvenation purposes undergo BOTH 1) cellular reprogramming (to re-establish the "embryogenic" potential of their cells / genomes / gene regulatory networks, AND 2) appropriate "morphodynamic" remodeling of their tissues (to re-establish the hierarchical architecture of those cells in the correct configuration within tissues / organs / limbs / body segments) - not "salt-and-pepper" randomness

This morphodynamic aspect will be critical for realizing the potential of cellular reprogramming in humans

Posted by: Ira S. Pastor at August 3rd, 2019 8:15 AM

Hi there! Just a 2 cents.

''How cells are able to mark and retain youthful DNA methylation patterns, then in late adulthood OSK can instruct the removal of deleterious marks is unknown.''

Cells that retain youthful DNA methylation patterns have (highly) active methylation going on, that is the major reason why. With age, global demethylation is the main driver of epiaging in epigenome. Hypermethylation happens with age, only, in specific areas (mainly the inflammatory ones/cancer/oncogenic ones/hypermethylation in CpG-rich DNA), the rest (the Large rest/which encompasses anti-inflammation and anti-oxidative/repair/redox mechanisms/hypermethylation in CpG-poor DNA) is becoming demethylated. In a sense that, inflammatory genes are unsilenced with age, while anti-inflammatory ones are silenced; and that's when things go bad, health worsens and you see clear advancement of epiclock age too.

I am not surprised to read that optical nerve neuron OSKM-delivery epigenetic reversal reverses blindness and glaucoma of age disease (type II diabetes causes glaucoma/cataract/destruction of mitoPUFAs in crystalin, my father (72) had glaucoma from his t2d but he reduced it with metformin, he stopped taking metformin and for a while it was ok...but then high glucose levels again, they kept rising - again (since he eats well/sweets/deserts and still had t2d (he told me, he doesn't care at this point anymore/at his age, he wants to keep fit enough but not restrict the joys of eating/quality of life - even if it makes a bit shorter lifespan, he prefers quality of it, and eating whaterver he wants including sugary cakes because has sweet tooth)...sugar/browning oxidation, ocular ceroid/lipofuscin/A2E/AGEs/ALEs/Maillard-Amadori, etc), so he is back on it).

Just like what happens with axolotl/zebrafish/salamader who 'regrow' a cut limb (through cell dedifferentiation by epigenetic methylation changes) or babies who completely 'rebuild' tissue from an injury to a 'scarless' one (while in children/adults, scar formation happens from macro-granulation to fill the scar hole). It means that epigenetic mechanism not only control aging, they control damages, repair of damages and the morphology of ECM and entire tissues; in all organs. When you reverse the epiclock it is a signal to reconstruct to what it 'once was', meaning true reversal to a young state/morphostate. During the reversal/rejuvenation, damages are repaired, residues/depots are excreted and the Entire Tissue is Rebuilt to a morphologically young construction - Exactly, as it was back then; thus, truely young. The epigenome controls the reprogramming/program, it is like a 'code' that tells the body to reverse itself in entirety - a wipe out - just like 'overwriting code', if you overwrite it, it's like a new clean slate; the old code is gone forever. The 'new young' code tells the body what it needs to reconstruct the body to a young morphologic form/construct. That means repair/removing all damages/excrete the crap/junk (lyso/proteo), rebuild the chaperones/proteins and increase telomeres/make strong methylome (silenced), stop chronic inflammation of age, reduce mito ROS and reinstate correct redox milieu.

Just a 2 cents.

Posted by: CANanonymity at August 3rd, 2019 12:11 PM

important work

Posted by: huling at August 4th, 2019 1:43 AM

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