A Youthful Touch: Reversal of Aging Hallmarks by Cell Reprogramming

Human life expectancy has increased by almost 10 years in the last century. The demographic comprising 65-year-olds and over, considered the elderly population, accounts for approximately 16.8% of the world’s inhabitants and is expected to double by 2050 [1]. However, changes to the health span (i.e., disease-free life expectancy) have not followed the same trajectory but stagnated. While life expectancy is expected to rise linearly in the next 4 decades, the number of age-related diseases is predicted to increase exponentially [2, 3]. Every year, two-thirds of deaths are linked to age-related pathologies, some of the most prevalent being cancer, arthritis, osteoporosis, diabetes, hypertension, cardiovascular disease, and different forms of neurodegeneration [4]. These diseases impact quality of life significantly, sometimes for years or even decades, but also cost the healthcare system billions of dollars. Ideally, however, lifespan and health span should overlap for as long as possible, and death should result from processes that preserve quality of life and exemplify healthy aging rather than from debilitating diseases [5, 6]. To make this possible, more research is necessary to understand the causes and underlying mechanisms of aging, to identify new strategies that treat or prevent age-related diseases, and to promote healthy aging.

Aging is a complex biological process that is characterized by gradual organismal decline and is not yet fully understood. Multiple non-mutually exclusive theories have been proposed to explain it. These include the free radical theory of aging, which attributes aging to accumulated cellular damage caused at large by reactive oxygen species (ROS), as proposed by Harran [7] in 1956; the evolutionary theory of aging, introduced by Hamilton [8] in 1966, which views aging as the accumulation of both random and deterministic deleterious effects; the programmed theory, which views aging as a genetic program directing senescence and death, initially suggested by Longo and colleagues [9] in 2005; and the hyperfunction theory, developed by Blagosklonny [10] in 2008, which considers aging a result of the hyperactivity of genes at specific developmental stages. Understanding such a complex process is challenging due to individual heterogeneity and the interrelated nature of many contributing factors considered in these theories [11]. Fortunately, the last decades have seen remarkable progress in understanding the biology of aging, leading to the identification of at least some of its key molecular and cellular markers. Proposed by López-Otín et al. [12] for the first time in 2013 and recently reviewed a decade later [13], the twelve most established hallmarks of aging are genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis. The identification and understanding – even if not yet complete – of these hallmarks is a first step toward developing strategies to reverse or slow down their progression.

Amidst the many antiaging approaches under investigation, one that has gained traction in recent years is the induction of cellular reprogramming through the overexpression of transcription factors related to stemness, in particular, the Nobel prize-winning cocktail formed by Oct3/4, Klf4, Sox2, and cMyc, also known as OKSM or Yamaka factors [14‒17]. While still a very young area of research, there is already considerable evidence that reprogramming adult cells to pluripotency, or even partially to intermediate states, reverses several molecular and cellular changes acquired during aging. In this article, we first provide a brief introduction to cellular reprogramming. We then review current knowledge on the mechanisms by which cell reprogramming acts on aging hallmarks. To finalize, we elaborate on the challenges ahead of developing in vivo cell reprogramming as a strategy to extend the health span.

Cellular reprogramming is a stepwise process that gradually reverses cell differentiation, ultimately guiding even fully differentiated, adult cells back to the pluripotent state if the reprogramming stimulus is sustained [18]. This reversal is possible because no genetic information is lost during differentiation. Instead, genes are expressed or silenced in a reversible manner, shaping the cell’s specific identity and function during various stages of growth and maturation. The genetic program of a cell can be reset via various techniques including somatic cell nuclear transfer, somatic cell fusion with pluripotent stem cells, or transcription factor-induced reprogramming. The latter was spotlighted by Takanashi and Yamanaka’s [19] pioneering work in 2006, in which they identified a combination of four transcription factors related to stemness (OKSM) that can reverse adult differentiated cells into pluripotent counterparts. Takanashi and Yamanaka’s [19] first work used retroviral vectors to deliver OKSM into mouse embryonic and adult fibroblasts, but later research has demonstrated that the same effect can be achieved using other delivery vectors, in different cell types, including those from human origin, and using slight variations of the reprogramming cocktail [20]. The earliest application of OKSM-induced pluripotency was to generate so-called induced pluripotent stem cells (iPSCs). These are virtually equivalent to embryonic stem cells in their differentiation potential but avoid the destruction of embryonic material and can be generated from specific patients [20]. iPSCs are nowadays routine research tools used to generate in vitro disease models for toxicology, pharmacology, and basic science studies. Moreover, there are hopes to use iPSC-derived cells as sources for cell therapies in the not-too-distant future.

Beyond the generation and applications of iPSCs, the last decade has also witnessed the utilization of OKSM factors to induce cell reprogramming directly in vivo. The first studies focused on demonstrating that the pluripotent state could be reacquired within the adult microenvironment. Yilmazer and colleagues [21] demonstrated that hepatocytes transfected with a single dose of plasmid DNA encoding OKSM transiently re-expressed endogenous pluripotency markers and together with de Lázaro et al. [20] confirmed functional pluripotency of in vivo reprogrammed liver cells. This was a critical finding to better understand the limits of cell plasticity in living organisms. However, the generation of tumors triggered by rapid proliferation and uncontrolled redifferentiation of pluripotent stem cells when these persisted in the tissue challenged the prospects for therapeutic interventions based on in vivo induced pluripotency [22, 23]. Yet, the stepwise nature of cell reprogramming makes it possible to stop the process before the cells reach a point of no return and commit to pluripotency, bypassing the risk of tumorigenesis, in a process broadly defined as partial reprogramming.

More specifically, partial reprogramming can entail dedifferentiation of adult cells to progenitor-like states, characterized by loss of at least some markers of the fully differentiated phenotype and, often, increased cell cycle activity which can be exploited to induce tissue regeneration [24, 25]. For example, Chen et al. [26] recently demonstrated that short expression of OKSM in mouse adult cardiomyocytes induced them to dedifferentiate into a proliferative state with fetal-like gene expression that contributed to myocardial regeneration after infarction. However, other reports of partial reprogramming have described instead the induction of a molecularly rejuvenated state, where cells maintain their identity and differentiation status but exhibit reversed aging markers and restored function [14, 26‒28]. For example, we recently showed that pulsatile OKSM overexpression in cardiomyocytes induces a significant decrease in their epigenetic age, without cell-cycle reentry, which has been seen to correlate with improved cardiac function in a mouse model of heart failure [16]. These different outcomes of reprogramming can be achieved by modulating the expression of OKSM, as represented in Figure 1. Overall, partial reprogramming has shown promising contributions to restoring injured, degenerated, and aged tissues in preclinical mouse models, although the underlying mechanisms have not been fully revealed [16, 26, 27, 29, 30]. Particularly, the option to induce rejuvenation without dedifferentiation is gaining a lot of attention given its anticipated safer profile and the overall increasing interest in antiaging and longevity research. Partial reprogramming has so far demonstrated effects in reversing some of the hallmarks of aging, decreasing epigenetic age, and extending longevity, which have been recently reviewed by other colleagues [31]. In this work, we take a deeper dive into the effects that induced reprogramming has on several of the hallmarks of aging and the underlying mechanisms that enable these effects.

The aging process is characterized by molecular and cellular alterations known as the hallmarks of aging [13]. To our knowledge, there is evidence that OKSM-induced cellular reprogramming can reset, or at least partially modify, nine out of these twelve hallmarks, some of which are highly interrelated. Further research may reveal still unknown effects on the remaining hallmarks. In this section, we review the hallmarks of aging that have been shown to be reversible through OKSM overexpression and discuss the mechanisms that underlie this transformative reset (Fig. 2).

Age-related genomic instability refers to the increased susceptibility to genetic alterations, including the accumulation of DNA damage, DNA mutations, telomere shortening, and chromosomal aberrations as organisms age [32]. In younger cells, robust DNA repair mechanisms efficiently rectify damage caused by environmental factors and cellular metabolism. These mechanisms include nucleotide excision repair, which rectifies helix-distorting lesions; base excision repair, responsible for repairing small, non-helix-distorting base lesions; mismatch repair, which corrects base mispairing post-replication; and double-strand break repair pathways, comprising nonhomologous end joining and homologous recombination, which repair the most lethal form of DNA damage [33, 34]. However, with aging, these repair systems become less effective, leading to the accumulation of DNA damage and mutations [32, 35]. Telomeres, the protective ends of chromosomes, also undergo significant changes as organisms age; they progressively shorten with each cell division, eventually reaching a critical length that triggers cellular senescence or apoptosis. Telomere shortening, while a hallmark of aging on its own, is a key driver of age-related genomic instability contributing to the overall decline in genomic integrity [36]. Additionally, older cells exhibit an increased rate of chromosomal abnormalities, such as aneuploidy, contributing further to genomic disarray [37]. In diseases such as progeria and other laminopathies, defects in the nuclear architecture, such as abnormalities in the nuclear lamina, have also been implicated in inducing genomic instability and driving accelerated aging [38, 39]. In turn, these changes in the genome are not merely passive markers but active contributors to the aging process, leading to a decline in cellular function, increased cellular senescence, and a heightened risk of age-related pathologies.

Cellular reprogramming can alleviate genomic instability and DNA damage by reinstating DNA repair mechanisms and normalizing defects in the nuclear architecture [27, 39]. The effect of reprogramming on telomeres, discussed in the next section, helps reduce chromosomal abnormalities such as aneuploidy, thus contributing to overall genomic stabilization [37, 40]. Lastly, the global epigenetic reset that takes place during cellular reprogramming also contributes to genomic stability. Importantly, at least some of these events take place from the initiation phase of cellular reprogramming, indicating that partial reprogramming could be sufficient to achieve these beneficial effects [27, 39, 41].

Telomere attrition entails shortening of the DNA sequences at the ends of chromosomes during each cell division. This shortening occurs because DNA polymerases, the enzymes that replicate DNA, cannot completely duplicate the end of the linear DNA molecule, leaving the very tip unreplicated, a problem known as the “end-replication problem.” Consequently, with each round of cell division, a small portion of telomeric DNA is lost [42, 43]. Over time, the telomeres reach a critically short length, leading to genomic instability and signaling the cell to cease division and enter senescence or to undergo programmed cell death (apoptosis). In cultured human fibroblasts, the critical telomere length has been estimated as 5–10 kilobases [44, 45]. Similarly, in human leukocytes, telomere critical lengths typically range from 5 to 15 kilobases and serve as biomarkers for aging and related diseases, with shorter lengths linked to higher morbidity and mortality [46, 47]. However, this number is not universally fixed and varies across species, cell types, and likely other individual cellular factors. Telomerase, a ribonucleoprotein, can prevent these changes by adding repetitive nucleotide sequences to the ends of chromosomes and, therefore, elongating telomeres [48, 49]. However, most mammalian somatic cells do not express high levels of this enzyme [40].

In iPSCs, telomere length is extended compared to parental cells thanks to the upregulation of telomerase during reprogramming [17, 37, 48]. By resetting telomere length, reprogrammed cells can undergo potentially unlimited divisions without entering a state of senescence or apoptosis. However, current evidence suggests that the reactivation of telomerase takes place once the point of no return has been reached, and cells are irreversibly committed to pluripotency [17, 48]. This, together with the fact that telomere attrition offers protection against malignancy by limiting proliferation, suggests that it could be complicated to benefit from the erasure of this aging hallmark without incurring significant risks.

Epigenetic alterations linked to aging include changes in DNA methylation patterns, aberrant histone modifications, and chromatin remodeling. As organisms age, there is a global loss of DNA methylation at CpG islands. This demethylation can inadvertently activate previously silenced genes, some of which might be implicated in oncogenic pathways. Simultaneously, there is hypermethylation at other sites, potentially silencing genes integral to the maintenance of a healthy aging process [32, 50]. Histone modifications, which affect how tightly DNA is coiled around histones and thus its accessibility for transcription, also change with age [32, 51]. Histone methylation, including H3K9me3 and H3K27me3, contributes to more condensed, less transcriptionally active chromatin (heterochromatin), while histone demethylation, involving the removal of methyl groups, can influence chromatin relaxation and gene expression changes in aged cells [17, 27, 52]. Moreover, aging influences other crucial posttranslational modifications of histones, such as acetylation and ubiquitination [32, 51]. Histone acetylation, generally associated with an open chromatin configuration and active gene expression, tends to decline with age, further limiting the ability of cells to adapt their transcriptional plasticity [53‒55]. Ubiquitination of histones, which can signal either chromatin opening or compaction depending on the specific modification, also exhibits age-related changes, further complicating the epigenetic regulation landscape in aging cells [51, 56]. The net result of these epigenetic alterations is a disruption in the fine-tuned balance of gene expression maintained in younger cells, contributing to the decline in cellular function, increased susceptibility to age-related diseases, and the overall aging phenotype [4, 32].

During cell reprogramming, widespread epigenetic remodeling occurs, which includes the erasure of DNA methylation marks that have accumulated with age [57, 58]. This has been shown in partially reprogrammed cells, in which many of these age-associated methylation marks are reset, rejuvenating the cell’s epigenetic landscape to mirror a more youthful-like state [16, 59]. Epigenetic clocks, sophisticated models that predict biological age based on methylation patterns, have validated that reprogrammed cells exhibit a reversal in their epigenetic age, demonstrating the effectiveness of this approach and providing a valuable tool for aging research [60]. In terms of histone modifications, partial reprogramming induces a global reduction in H3K9me3 levels, which is typically associated with transcriptional repression and found in regions of heterochromatin. This contributes to the loosening of tightly packed heterochromatin and facilitates a more open chromatin structure conducive to gene activation. Reprogramming also decreases H3K27me3, linked to the silencing of developmental genes in stem cells [52], which is essential for deactivating lineage-specific genes and activating pluripotency-associated genes [17, 27, 61]. Ultimately, through these mechanisms, reprogramming offers the potential to recalibrate gene expression patterns, making them more similar to those observed in younger cells [62, 63].

Proteostasis is a crucial process that regulates the production, maintenance, and degradation of proteins within cells to ensure cellular homeostasis and health [64]. As organisms age, the efficiency of the proteostasis network diminishes, leading to the impairment in the mechanisms responsible for protein synthesis, folding, trafficking, and degradation [65]. Three critical components of this system, the ubiquitin-proteasome system (UPS), the autophagy-lysosomal pathway, and the chaperone system, are compromised with age. The UPS, responsible for degrading damaged or misfolded proteins, shows reduced activity leading to the accumulation of aberrant proteins [66]. Similarly, autophagy, the process by which cells remove damaged organelles and proteins, becomes less effective, further contributing to the buildup of protein aggregates [67]. This accumulation of misfolded and aggregated proteins is a characteristic feature of many age-related diseases, such as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis [64, 66]. Additionally, the decline in the cellular chaperone system, which assists in proper protein folding, exacerbates the problem by allowing more misfolded proteins to accumulate [65]. The combined effects of these deteriorations lead to cellular dysfunction, increased cellular stress, and the manifestation of age-related pathologies.

The mechanisms through which reprogramming may improve proteostasis are not fully elucidated, but a few key events have already been described in this process. Buckley et al. [68] demonstrated that specific components of the UPS, such as the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7, are integral in modulating the stability and levels of key pluripotency factors like c-Myc, influencing the efficiency of reprogramming murine embryonic fibroblasts to iPSCs. While it remains to be confirmed whether the UPS is directly restored through partial reprogramming, others have shown that this process enhances proteolytic activity in aged human fibroblasts and endothelial cells, as evidenced by increased formation of autophagosomes and elevated chymotrypsin-like proteasome activity [69]. Together, these changes contribute to a broader restoration of the proteostasis network and enhance the cell’s protein quality control mechanisms, ensuring the survival and proper function of newly differentiated cells [68, 69]. Thanks to all these events, reprogramming can reduce the levels of protein aggregates characteristic of various neurodegenerative diseases [70, 71]. Lastly, the rejuvenated cellular environment resulting from reprogramming might bolster proteostasis indirectly. This can be achieved by enhancing mitochondrial function and curtailing oxidative stress, both factors that can detrimentally affect protein integrity [65, 69].

Autophagy is the cellular process of enveloping and degrading cytoplasmic material in vesicles called autophagosomes. Autophagy homeostasis declines with age, thus impairing the removal of cytoplasmic materials, dysfunctional organelles, and pathogens, leading to a buildup of cellular debris and increased inflammation that contribute to accelerating aging [72]. This decline is multifactorial. Age-related changes in the expression of autophagy-related genes including ATG5 and ATG7, a decrease in the efficiency of the autophagic machinery, and alterations in cellular signaling pathways are all contributing factors [73, 74]. One of the main regulatory pathways of autophagy involves AMP-activated protein kinase (AMPK) as a positive regulator and the mechanistic target of rapamycin (mTOR) as a negative regulator. Changes in the functionality of these regulatory proteins are also implicated in the age-related decline of autophagy. For instance, as cells age, declines in the activation of AMPK and increases in mTOR activity can occur, both of which lead to reduced autophagy [75]. Aging is also associated with an accumulation of oxidative stress and damage, which further impacts autophagic pathways negatively. For example, oxidative stress can harm components essential for autophagy, such as autophagy-related proteins and lysosomes, and mitochondrial DNA (mtDNA), leading to mitochondrial dysfunction and, subsequently, impairing the autophagic turnover of these critical energy-producing organelles [76].

Emerging evidence, however limited and understudied, indicates that reprogramming can enhance macroautophagy, reinstating a more youthful autophagic capability. Reprogramming increases the expression of autophagy-related genes, enhancing the formation of autophagosomes and ensuring more efficient degradation and recycling of cellular materials [69, 73, 77]. Autophagy is relevant and essential in the early phases of cell reprogramming during the generation of iPSCs [74, 77], which offers hopes that its beneficial effects can be attained without the need for complete reprogramming. Indeed, Sarkar et al. [69] found that transient reprogramming (without reacquisition of pluripotency) of fibroblasts and endothelial cells from old patients (60–90 years old) significantly increased autophagosome formation. Overall, a restored autophagy system could provide protective effects against age-related pathologies. For instance, improved autophagy better counteracts the buildup of toxic aggregates in neurodegenerative diseases, reduces inflammation, and maintains cellular homeostasis [72, 78].

Mitochondria, the powerhouses of the cell, undergo functional decline with aging which leads to compromised cellular bioenergetics, increased production of ROS, and potential inflammation and cell death due to permeabilization of mitochondrial membranes. Aging-induced mitochondrial dysfunction arises from several factors such as accumulation of mtDNA mutations, destabilization of respiratory complexes that facilitate energy production, reduced organelle turnover, and imbalances in the dynamics governing mitochondrial fusion and fission [79‒81]. These factors not only impair the primary function of mitochondria but also increase their propensity to release factors that can induce inflammation or trigger cell death [79, 82].

Reprogramming can rejuvenate the state of cellular mitochondria by reducing accumulated mtDNA mutations and via a reset of mitochondrial dynamics and quality control processes including mitophagy, the targeted degradation of dysfunctional mitochondria [37, 81, 83, 84]. This rejuvenation ensures that cells have a renewed energy production capacity, reduced ROS generation, and increased resilience against mitochondrial-induced cellular stress [15, 27, 80, 81, 84, 85]. Evidence has been shown that even partial reprogramming affects mitochondrial health. Partial reprogramming of human aged fibroblasts and endothelial cells showed increased mitochondrial activity and decreased mitochondrial ROS accumulation, although the changes were small [69]. The same experiments evidenced a decrease in mitochondrial mass that could be attributed to enhanced autophagy. Using in vivo mouse models, Ocampo et al. [27] also noted a decrease in mitochondrial ROS upon partial reprogramming and Chondronasiou et al. [15] observed a decrease in age-related transcriptional signatures related to mitochondrial processes.

Aging is accompanied by significant changes in intercellular communication, leading to a decline in tissue function and homeostasis. A marked increase in pro-inflammatory signaling is evidenced by elevated levels of cytokines such as IL-6 and TNF-alpha, disrupting the delicate balance of cellular interactions and promoting chronic inflammation. Hormonal communication, vital for regulating metabolism, reproduction, and stress responses, also becomes imbalanced with age [86, 87]. Furthermore, neurotransmission in the nervous system experiences alterations in neurotransmitter levels and receptor sensitivities, contributing to cognitive decline and neurodegenerative diseases. The extracellular matrix, essential for providing structural support and biochemical and mechanical signaling, undergoes changes and degradation that further impair cell-to-cell communication and tissue integrity [4, 88, 89]. Overall, these changes in intercellular communication are critical contributors to the aging process, affecting a wide range of organismal functions, from immune responses to tissue repair and regeneration.

Cellular reprogramming has been shown to reverse some altered intercellular communication processes associated with aging through various mechanisms. The expression of some extracellular matrix genes has been reported to undergo significant changes during the process of somatic cell reprogramming to pluripotency [90], and partial reprogramming has been shown to decease fibrosis in several mouse tissue injury [16, 91] models that could otherwise also affect cellular communication [92]. Additionally, cellular reprogramming reduces pro-inflammatory signaling secreted by senescent cells, as discussed in the next section, that otherwise can induce secondary senescence [90].

Senescence is a state of permanent cell cycle arrest, no longer responsive to growth factors or other proliferative stimuli. Initially identified as a natural barrier to uncontrolled cell proliferation, senescence is now understood as a complex phenomenon in response to various stress factors such as DNA damage, telomere shortening, and oxidative stress, which impacts aging at multiple levels [88]. Senescent cells secrete various inflammatory and tissue-remodeling factors, collectively known as SASP, which alter the tissue microenvironment. This in turn can drive chronic inflammation and degenerative changes, playing a pivotal role in the progression of age-related diseases [88, 93, 94]. The evolving understanding of cellular senescence reflects its dual nature: a defense mechanism in youth, turning into a driver of aging and pathology in later life.

Independent research in a mouse model of progeria and in cultured endothelial cells isolated from aged human subjects has shown that partial reprogramming can induce downregulation of key senescence markers such as the expression of p16, p21, and p53 and the activity of senescence-associated beta-galactosidase (SA-β-gal) enzyme [27, 69]. Partially reprogrammed endothelial cells also showed a decrease in proinflammatory senescence secretory phenotype cytokines [69]. Indeed, reprogramming has been shown to effectively reduce the SASP by modulating the expression of specific miRNAs and lncRNAs, among other mechanisms [62, 90], and the observation that these changes take place even when only partial reprogramming is induced reaffirms that many of the rejuvenating effects of cellular reprogramming do not require the reacquisition of pluripotency [14, 27, 59]. The capabilities of reprogramming to reinstate cell division in senescent cells are, however, so far less clear. Lapasset and colleagues [37] generated iPSCs from human fibroblasts induced into replicative senescence, demonstrating that they can not only be forced back into the cell cycle but also into pluripotency. However, increased numbers of proliferating cells have been observed among both partially reprogrammed aged human cells (chondrocytes from patients diagnosed with late stage osteoarthritis) [69] and partially reprogrammed mouse tissues from progeroid mice (stomach, kidney, skin) [30], but it remains to be confirmed whether those dividing cells were previously senescent.

Stem cell exhaustion involves the progressive decline in the number and function of tissue resident stem cells, leading to impaired tissue regeneration and maintenance. As we age, stem cells in various tissues, such as hematopoietic stem cells in the bone marrow and mesenchymal stem cells in the bone and muscle, exhibit reduced proliferative capacity and regenerative potential [32, 50]. This decline is attributed to intrinsic factors like DNA damage, telomere shortening, and epigenetic changes, as well as extrinsic factors including changes in the stem cell niche and systemic environment [32, 95, 96]. The cumulative effect of these changes is a decrease in the body’s ability to repair and maintain tissues, contributing significantly to aging and the development of age-related diseases [94, 97].

Cell reprogramming has been shown to restore youthful characteristics in aged stem cells through direct and paracrine mechanisms [69, 98]. Specifically, Sarkar et al. [69] observed that partial reprogramming of aged mouse- and human-derived skeletal muscle stem cells enhanced their proliferative capacity without altering their capacity to enter myogenic differentiation (measured by the expression of the myogenic marker MyoD) and enhanced their capacity to generate new muscle in vitro and in vivo. Using reprogrammable transgenic mice, Ocampo and colleagues [27] noted a significant increase in the number of hair follicle stem cells in skin and satellite cells in the muscle upon in vivo partial reprogramming. Most recently, Wang and colleagues [30] described a paracrine mechanism by which the induction of partial reprogramming in muscle fibers has beneficial effects in the stem cell niche which resulted in enhanced activation of satellite cells and muscle regeneration. However, their observations that direct reprogramming of satellite cells did not reproduce the same beneficial effects in the tissue contrast with the previous findings from Sarkar and Ocampo and guarantee further investigation.

The last few years have seen a significant increase in research groups and studies investigating in vivo partial reprogramming in the context of tissue regeneration and rejuvenation. However, there are still standing questions on both the potential benefits and the possible risks of this approach. Extending our fundamental knowledge about the process of reprogramming and of the factors that may impact it in a living organism, as well as finding safe ways to bypass anticipated caveats, will be necessary to translate the observations of the last few years into a potential rejuvenation intervention. Here, we highlight key gaps in knowledge, challenges, and limitations associated with this revolutionary approach for rejuvenating aging systems.

While several reports have demonstrated rejuvenating effects resulting from in vivo partial reprogramming in preclinical mouse models, very few of them have confirmed the fate of reprogrammed cells and whether they are directly or indirectly responsible for such effects. Both direct and paracrine mechanisms have been reported, and these may also be dependent on the extent of reprogramming (e.g., dedifferentiation vs. rejuvenation only) and the cell type that is reprogrammed [29, 30]. In addition, most studies analyzed biological age and rejuvenation shortly after the end of the OKSM reprogramming stimuli [16]. Encouragingly, a recent study demonstrated that short OKSM upregulation early in life has long-lasting effects throughout the lifespan of mice [99]. However, more studies, preferably in longer lived models, are required to confirm these findings and also to rule out the potential appearance of undesired effects long-term.

One of the most critical challenges in utilizing cellular reprogramming for healthy aging lies in precisely controlling the extent of reprogramming. The objective is to revert cells to a more youthful state without erasing their specialized functions or inducing an embryonic-like pluripotent state, which could lead to uncontrolled cell growth and tumorigenesis or loss of function in differentiated tissues [100]. Striking this balance is complex. Moreover, it is well established that different cell types require different levels of OKSM upregulation to undergo reprogramming, complicating this further. For example, it has been challenging to induce reprogramming in the skeletal or cardiac muscles using reprogrammable mouse models in which OKSM are expressed ubiquitously because cells in the gut become fully reprogrammed and tumorigenic much faster than muscle fibers [16, 101]. Cell specificity will likely be required for safe reprogramming.

The presence of cMyc, a known oncogene, as part of the reprogramming cocktail, contributes further to the risk of malignancy and will likely complicate regulatory approval. Limited efforts have been made to avoid cMyc in the induction of reprogramming [21, 29]. Yilmazer et al. [21] demonstrated upregulation of reprogramming markers in the absence of tumorigenesis upon transfection of mouse hepatocytes with OKS in vivo but did not investigate the reprogramming effect achieved further. More recently, Lu et al. [29] overexpressed OSK in mouse retinal ganglion cells using doxycycline-inducible adeno-associated viral vectors and demonstrated an increase in axon regeneration and reversed vision loss in in vivo models of glaucoma and aging without loss of cellular identity or tumor formation. Further research is necessary to confirm whether efficient cell reprogramming can be achieved without c-Myc in other tissues and to address if downstream druggable targets can be identified to avoid the OKSM cocktail altogether.

The effective delivery of reprogramming factors to specific cells or tissues in a safe, controlled, and efficient manner remains a formidable challenge. The best tools to control OKSM expression to date are cell type-specific reprogrammable mouse models in which the expression of reprogramming factors is controlled by cell-specific promoters and tet-ON doxycycline-inducible systems [16, 24, 30, 102]. However, these lack any potential for clinical translation. Most attempts at viral and nonviral delivery so far have suffered from either insufficient levels of gene expression rendering the efficiency of reprogramming suboptimal, or, on the contrary, from the appearance of tumors due to prolonged OKSM expression [103, 104]. The most sophisticated viral vector for cell reprogramming remains a doxycycline-inducible AAV encoding OKS developed by the Sinclair lab [29]. While this system allows switching the reprogramming machinery on and off, it could still be affected by general AAV shortcomings including immunogenicity and limited to tissues and cells for which AAVs show specific tropism. Synthetic nanoparticles could be an alternative with improved safety profiles. However, to our knowledge, no candidates have been tested for in vivo delivery of OKSM yet and in vitro examples have relied on commercially available nanoparticle transfection reagents with known cytotoxicity that precludes their use in vivo [69, 105]. Further research in the formulation of appropriate delivery vectors will be imperative to realize the demonstrated biological benefits of cell reprogramming.

Cellular reprogramming is a promising strategy to address the challenges of age-related diseases that are on the rise due to the significant increase in the elderly population. A definitive advantage is that partial reprogramming can reverse many aging hallmarks in concert without inducing loss of cell identity or reacquisition of pluripotency. However, years or even decades of research will be necessary to further our understanding of reprogramming mechanisms and outcomes, to engineer translatable approaches to induce partial reprogramming, and to eliminate tumorigenic risks and off-target effects.

The authors have no conflicts of interest to declare.

E.M. wishes to thank the NYU Tandon School of Engineering for an SoE Graduate Fellowship and the A.G. Leventis Foundation for an educational grant. I.dL. acknowledges start-up funds from NYU School of Engineering.

E.M.: conceptualization, writing – original draft, and visualization. I.dL.: conceptualization, writing – original draft, writing – review and editing, and supervision.