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.
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.