Fight Aging!

Reprogramming of ordinary somatic cells into induced pluripotent stem cells (iPSCs) was initially thought to be a way to obtain all of the patient matched cells needed for tissue engineering or cell therapies. A great deal of work has gone towards realizing that goal over the past fifteen years or so; the research community isn't there yet, but meaningful progress has taken place. Of late, another line of work has emerged, in that it might be possible to use partial reprogramming as a basis for therapy, delivering reprogramming factors into animals and humans in order to improve tissue function, without turning large numbers of somatic cells into iPSCs and thus risking cancer or loss of tissue structure and function.

Reprogramming triggers some of the same mechanisms of rejuvenation that operate in the developing embryo, removing epigenetic marks characteristic of aged tissues, and restoring youthful mitochondrial function. It cannot do much for forms of damage such as mutations to nuclear DNA or buildup of resilient metabolic waste, but the present feeling is there is nonetheless enough of a potential benefit to make it worth developing this approach to treatments for aging. Some groups have shown that partial reprogramming - via transient expression of reprogramming factors - can reverse functional losses in cells from aged tissues without making those cells lose their differentiated type. But this is a complicated business. Tissues are made up of many cell types, all of which can need subtly different approaches to safe reprogramming.

Today's open access preprint is illustrative of the amount of work that lies ahead when it comes to the exploration of in vivo reprogramming. Different cell types behave quite differently, will require different recipes and approaches to reprogramming, different times of exposure, and so forth. It makes it very hard to envisage a near term therapy that operates much like present day gene therapies, meaning one vector and one cargo, as most tissues are comprised of many different cell types all mixed in together. On the other hand, the evidence to date, including that in the paper here, suggests that there are ways to create the desired rejuvenation of epigenetic patterns and mitochondrial function without the risk of somatic cells dedifferentiating into stem cells.

Partial reprogramming restores youthful gene expression through transient suppression of cell identity

Aging induces broad gene expression changes across diverse mammalian cell types, and these changes have been linked to many of the prominent hallmarks of aging. Cell reprogramming experiments have shown that young animals can develop from adult cells and aging features can be erased through complete reprogramming to pluripotency. Recent reports have further suggested that transient expression of the Yamanaka factors (SOKM) is sufficient to reverse features of aging and improve cell function. However, it was unclear whether these transient reprogramming interventions suppress somatic cell identities, activate late-stage pluripotency programs, or whether alternative reprogramming strategies could restore youthful gene expression.

Here, we investigated these questions using single cell measurements of gene expression to capture the phenotypic trajectory of transient reprogramming and evaluate the impact of alternative reprogramming methods. We found that transient reprogramming suppressed somatic cell identities and upregulated hallmark pluripotency programs, contrary to some previous reports but consistent with timecourse iPSC reprogramming experiments and lineage-tracing studies of transient SOKM expression. By inferring RNA velocity and applying numerical tools from dynamical systems, we also found that transiently reprogrammed cells transition back toward their original gene expression states after transit through an intermediate state. Our single cell profiles therefore revealed transient cell states that were likely masked in previous bulk measurements and support a model in which transient reprogramming suppresses somatic identities that are later reacquired through differentiation. Further experiments profiling single cell populations at multiple time-points during transient reprogramming will be necessary to confirm this hypothesis.

It remains unknown which of the Yamanaka Factors are required to restore youthful gene expression, or which subsets might exhibit distinct effects during transient reprogramming. Previous studies have explored only one set of factors at a time, preventing accurate comparisons to address these questions. Our pooled screens of all possible Yamanaka Factor subsets revealed that combinations of 3-4 Yamanaka Factors have remarkably similar effects, suggesting no single factor is required to restore youthful gene expression. Combinations of two Yamanaka Factors were also more similar to the full SOKM set than to control or single factor perturbations, and all reprogramming factor combinations reduced an aging gene expression score. Our screen demonstrates that no single pluripotency factor is required to mask features of aging and suggest oncogene-free reprogramming strategies may also restore youthful gene expression. Our multipotent reprogramming experiments in myogenic cells further support this suggestion, indicating that youthful gene expression may be restored even without activating the pluripotency factors.

Restoring youthful gene expression can improve tissue function, implying that transient reprogramming may be therapeutic. However, pluripotent reprogramming is well-known to be an oncogenic process, even when Myc is excluded from the reprogramming set. While it has been reported that transient reprogramming does not suppress somatic cell identities based on bulk measurements, our single cell results show that somatic cell identity is suppressed and late-stage pluripotency GRNs are activated in a transitional cell state in multiple cell types. This raises the possibility that even transient reprogramming may be oncogenic. Identifying alternative reprogramming strategies to restore youthful gene expression with lower neoplastic risk is therefore desirable. Toward this aim, we have shown that transient reprogramming with multiple subsets of the Yamanaka Factors induces highly similar transcriptional effects to the full set, and that a distinct multipotent reprogramming system can confer youthful expression. These results suggest the feasibility of disentangling the rejuvenative and pluripotency inducing effects of transient reprogramming and serve as a resource for further interrogation of transient reprogramming effects in aged cells.