Rejuvenation takes place very early in embryonic development. The germline cells that go into the creation of an embryo are well protected and maintained in comparison to the average somatic cell in the adult body. Nonetheless, there is an accumulation of age-related epigenetic changes and molecular damage. Cells purge themselves of as much of this change and damage as possible, in order to ensure that the young are born with young somatic cells and tissues. This is primarily a resetting of epigenetic controls over gene expression, decorations on the structure of the genome that control shape and access to specific genes by the molecular machinery responsible for producing proteins from genetic blueprints.
A cell is a state machine, largely governed in operation by the matter of which proteins are produced, and in what quantities. Not completely governed: some damage, such as mutations to nuclear DNA, is irreversible. Some molecular waste cannot be managed even by cells in a youthful epigenetic state, and will degrade normal function. In a collection of replicating cells, that waste can be diluted via cell division, or even passed off entirely to a sacrificial daughter cell in a process of asymmetric division. So long as no one cell or small number of cells are vital, even serious mutation can be evaded by replication, provided that mutated cells are rejected. This is how single celled life, such as bacteria, can continue indefinitely. Further, a few lower organisms, such as the hydra, essentially a tiny bundle of stem cells in which every structure is replaceable, use this strategy in order to achieve individual immortality. Higher animals, with complex central nervous systems that include many non-replicating cells that cannot be sacrificed, cannot use this strategy, and so suffer from degenerative aging.
Embryonic rejuvenation is a process that can be understood, induced, and manipulated. The creation of induced pluripotent stem cells from normal adult somatic cells via reprogramming is one example of what becomes possible given sufficient knowledge and technical aptitude. This combines, in the same way as occurs in the early embryo, both an epigenetic reset and loss of somatic cell state, such as the shape and function of a skin cell or a brain cell, producing dedifferentiation into a pluripotent stem cell state. Researchers are presently looking beyond experiments in cell cultures towards the application of reprogramming in living animals. An epigenetic reset is a desirable outcome for somatic tissues throughout the aged body, likely able to reverse to some degree many age-related issues, such as loss of mitochondrial function. Dedifferentiation of somatic cells in an adult individual, on the other hand, is a roadblock and a challenge. It will lead to cancer where it occurs to a lesser degree, and it will cause pathology and death if prevalent. Differentiated cell state is vital to normal tissue function.
Thus an important question currently under investigation is whether or not these two aspects of reprogramming are inseparable. Is there an approach to reprogramming that will produce maximal epigenetic rejuvenation with minimal dedifferentiation? If so, that could prove to the the basis for a very useful approach to the treatment of aging. It likely cannot help much in the case of stochastic nuclear DNA damage leading to somatic mosaicism, and it cannot help with the accumulation of some forms of persistent molecular waste in long-lived cells, but it could nonetheless be beneficial enough to be interesting.
A recent addition to the anti-ageing strategies being developed comes from cellular reprogramming approaches. Induced pluripotency studies provided evidence that age-related cellular phenotypes such as mitochondrial morphology, function and number, as well as nuclear envelope integrity, are not irreversible. However, developmental cellular reprogramming turns a cell to a pluripotent state, where it has the potential to generate any somatic cell type. This process is not appropriate for an anti-ageing therapy in vivo because it requires not only the loss of the original cellular identity, but also the re-establishment of self-renewal capabilities. Therefore, induction of pluripotency or the direct injection of pluripotent cells in vivo, invariably lead to cancer in mice. For a cellular reprogramming-based intervention to be considered rejuvenative (turning an old cell into a younger cell), we need to uncouple its effects from dedifferentiation (loss of somatic cell identity).
Cellular reprogramming has demonstrated potential not only in regenerative medicine, but also in the ageing field through the amelioration of both physiological and cellular ageing hallmarks. While partial reprogramming might be used as a catch-all term to describe this type of rejuvenation, it does not reflect the fact that the described interrupted cellular reprogramming techniques are applied with the aim of (epigenetic) rejuvenation as opposed to inducing pluripotency (loss of cell identity). Reprogramming-induced rejuvenation (RIR) is a better term, capturing the nature of the utilised process and final aim of the interventions.
RIR has shown promise as a treatment to safely reverse ageing whilst retaining the ability to revert to or maintain original cell identity, both in vivo and in vitro. However, the precise nature of RIR still needs to be fully understood before it can be safely implemented as an anti-ageing treatment. For example, tracking any traces of pluripotency in partially reprogrammed cells (particularly in vivo) is a necessary precaution to minimise long-term cancer risk. Additionally, can rejuvenated partially reprogrammed cells be cultured long-term? The rejuvenated phenotype of some OSKM-treated cells lasts at least four weeks, but does this phenotype remain stable or eventually start to deteriorate at a rate faster than normal ageing?
Other important RIR safety concerns include how the reprogramming factors are introduced in vivo. Retroviruses are commonly used to integrate reprogramming factors into the genome. However, this method bears risks, such as insertional mutagenesis, residual expression and re-activation of reprogramming factors, and retrotransposon activation, all of which could increase cancer risk in vivo. Non-integrative delivery methods, such as transient transfection, non-integrating viral vectors, and RNA transfection are safer alternatives. For example, researchers have successfully used mRNA transfection to non-integratively conduct RIR. Another safe alternative is chemical-based reprogramming, which involves direct conversion of a somatic cell to a pluripotent state by use of small molecules and growth factors. It is conceivable that, in the future, chemical-based reprogramming could be adapted to achieve rejuvenation, however, this reprogramming approach currently only works for mice.
While RIR applied to skeletal muscle stem cells appears effective in improving regenerative capacity and muscle function in immunocompromised mice, further analysis is required regarding the somatic mosaicism of partially reprogrammed stem cells. Somatic variants at a stem or early progenitor cell level in turn can cause lineage bias, reduced stem cell function, and increased risk of developing haematologic cancer (e.g. age-related clonal haematopoesis). This can lead to the development of pre-malignant cells, which have a higher propensity to transform to a malignant state, the effect of which could be attenuated or exacerbated by RIR.
It also remains to be further explored whether and how RIR would work on post-mitotic terminally differentiated cells, such as neurons, cardiomyocytes, or adipocytes, but also other non-dividing cells such as quiescent or senescent cells. Pilot work has been done in the latter two states, demonstrating that a rejuvenated phenotype is achievable after restoration of cell division. These results may point to a scenario where proliferation is an essential requirement for rejuvenation. Indeed, induced pluripotency of postnatal neurons was only possible after forced cell proliferation via p53 expression. Coincidentally, the natural rejuvenation event in the early mouse embryo spans over stages of very active cell proliferation.
Overall, RIR is currently the best prospect to achieve epigenetic rejuvenation. Further studies are required to fully determine its limitations and efficacy.