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P A Y W A L L E D S O U R C E : Experimental Gerontology
Highlights
• Senescence negatively impacts healthy aging and is responsible for many age-related diseases.
• Cellular reprogramming technology offers tremendous opportunities for disease modeling and regenerative medicine.
• Reprogramming could revert several aging hallmarks on the cellular level.
• Senescence has both cell-autonomous and non-cell autonomous effects on cellular reprogramming.
• Combining senescence elimination and reprogramming might hold great potential for tissue regeneration and rejuvenation.
Abstract
Aging is associated with diminished regenerative capacity and increased risk of chronic diseases. There is now compelling evidence suggests that aging process is reversible. Besides metabolic modification and systematic factors, both senescence elimination and cellular reprogramming showed beneficial effects on tissue regeneration and rejuvenation. Here we review recent studies on the interplay between cellular senescence and reprogramming. We discuss how both strategies could impact aging process and the possibility of combine them for more efficient regeneration and rejuvenation.
1. Introduction
Aging is associated with progressive functional degeneration of different tissues and a dramatically increased risk of many diseases. Moreover, it has been increasingly recognized that aging itself might be the underlying driving force for developing these diseases (Kennedy et al., 2014; Lopez-Otin et al., 2013). The last 30 years of extensive research, both in model organisms and humans, enlists nine tentative hallmarks of aging reflecting potential drivers of aging process (Lopez-Otin et al., 2013). Importantly, these studies highlight that the rate of aging is susceptive to modification (Campisi et al., 2019). Therefore, how to curb aging processes to improve the human healthspan is one of the most exciting challenges for biomedical research in the coming years.
Cellular senescence is one of the hallmarks of aging (Lopez-Otin et al., 2013), a form of stress response to various stimuli that leads to a permanent cell-cycle arrest. It is well established cells with markers of senescence accumulate in tissues of aged mammals, including rodents (Krishnamurthy et al., 2004; Bussian et al., 2018), primates (Herbig et al., 2006; Jeyapalan et al., 2007), and humans (Liu et al., 2009; Ressler et al., 2006; Melk et al., 2004; Dimri et al., 1995; Idda et al., 2020). Furthermore, recent studies demonstrated that senescence negatively impacts healthy aging (Baker et al., 2011; Baker et al., 2016; Xu et al., 2018). Noteworthy, removing senescent cells ameliorate a wide range of aging-associated disease conditions, including atherosclerosis (Childs et al., 2016), osteoarthritis (Jeon et al., 2017), liver steatosis (Ogrodnik et al., 2017), type 2 diabetes (Aguayo-Mazzucato et al., 2019), improve regeneration capacity of multiple tissues (Aguayo-Mazzucato et al., 2019; Bird et al., 2018), and extend both health and life span in mice (Baker et al., 2016; Xu et al., 2018). Therefore, recent studies have placed cellular senescence in the central stage of regeneration and aging (Gorgoulis et al., 2019; He and Sharpless, 2017).
Cellular reprogramming is the process of reverting terminal differentiated cells to the pluripotent state, which has tremendous potentials for regenerative medicine and aging research (Takahashi and Yamanaka, 2006; Robinton and Daley, 2012). Besides generating in vitro models to study aging and age-associated diseases, reprogramming has gained considerable attention recently for its rejuvenation potential (Mahmoudi et al., 2019; Ocampo et al., 2016a). In particular, reprogramming the cells from aged donner to pluripotency could erase several aging hallmarks in vitro (Mahmoudi and Brunet, 2012; Miller et al., 2013; Lapasset et al., 2011; Suhr et al., 2010; Mertens et al., 2015). Importantly, some rejuvenation effects preserve after re-differentiation (Miller et al., 2013; Lapasset et al., 2011; Mertens et al., 2015). Intriguingly, partial reprogramming, induced by short term OSKM expression, has been shown to enhance tissue regeneration in older mice and extend the life span of the progeroid mice (Ocampo et al., 2016b). Although the underlying mechanism remains largely unknown, the tremendous potentials merit further investigation.
Interestingly, recent studies revealed that senescence has both cell-autonomous and non-cell autonomous effects in reprogramming (Banito et al., 2009; Mosteiro et al., 2016; Chiche et al., 2017). At the same time, partial reprogramming could reduce certain senescent associated features on the cellular level (Ocampo et al., 2016b; Sarkar et al., 2020), suggesting that two processes are intimately connected. Both senescence and reprogramming have been extensively reviewed respectively elsewhere (Gorgoulis et al., 2019; Munoz-Espin and Serrano, 2014; Calcinotto et al., 2019; Srivastava and DeWitt, 2016; Takahashi and Yamanaka, 2016). Here, we summarize the current understanding of the interplay between senescence and reprogramming, with a particular focus on their potential implication in aging and regeneration. Examining the crosstalk between cellular senescence and reprogramming will further the mechanistic understanding of both processes and devise novel anti-aging and rejuvenation strategies exploiting the potential synergistic effect (Mahmoudi et al., 2019).
2. Cellular senescence: the common cell fate within various contexts
Cellular senescence is a stable cell cycle arrest that occurs in diploid cells at the end of their replicative life span. In 1961, Hayflick and Moorhead demonstrated that human primary fibroblasts in culture could divide a limited number of times before irreversible cell cycle withdrawal (Hayflick and Moorhead, 1961). This process is known as the “Hayflick limit” or replicative senescence, which is caused by progressive shortening of telomeres upon each cell division. Besides, there is a wide range of stimuli that could induce senescence prematurely, including DNA damage, oncogenic stress, oxidative stress, protein misfolding, and genomic/epigenomic alterations, which eventually activate the p53/p21 and p16Ink4a/pRB pathways to establish and reinforce the persistent growth arrest (Munoz-Espin and Serrano, 2014; Hernandez-Segura et al., 2018). Moreover, there are many biological processes, such as tissue repair/regeneration (Demaria et al., 2014; Yun et al., 2015; Le Roux et al., 2015; Ritschka et al., 2017), and embryonic development (Munoz-Espin et al., 2013; Storer et al., 2013) rely on senescence.
Senescent cells are characterized collectively by several non-exclusive markers (Hernandez-Segura et al., 2018). Permanent cell cycle arrest is an essential feature of senescence. Senescent cells do not resume proliferation in response to mitogenic signals, which is different from quiescence, a state of reversible cell cycle arrest. At the same time, senescent cells frequently exhibit a persistent DNA damage response (DDR) and induction of antiapoptotic genes, which separate them from post-mitotic differentiated cells. The accumulation of mitochondria and lysosomal in the senescent cells allow the detection of the β-galactosidase activity in sub-optimal pH (senescence-associated β-galactosidase, SAβGal) (Dimri et al., 1995). Although senescent cells do not proliferate, they remain metabolically active and robustly express a senescence-associated secretory phenotype (SASP) (Coppe et al., 2010): secretion of many inflammatory cytokines, growth factors, and extracellular matrix metalloproteinases (MMPs). Therefore, up-regulation of the cyclin-dependent kinase inhibitors (CKIs), lack of proliferation, resistance to apoptosis, activation of DDR, SAβGal, and SASP factors are commonly used markers of senescence. Noteworthy, SASP factors play a crucial role in mediating senescence non-cell autonomous functions by attracting immune cells and altering tissue microenvironment (Munoz-Espin and Serrano, 2014). However, the temporospatial regulation of SASP is highly heterogeneous in a cell type and stress-dependent manner (Ito et al., 2017).
Work over the last decade expanded the involvement of cellular senescence to various biological and pathological processes, including embryonic development (Munoz-Espin et al., 2013; Storer et al., 2013), tissue repair /regeneration (Demaria et al., 2014; Yun et al., 2015; Ritschka et al., 2017), tumorigenesis (Calcinotto et al., 2019; Collado et al., 2007), and aging (Baker et al., 2016; Xu et al., 2018; Calcinotto et al., 2019). Of note, senescence can be either beneficial or detrimental for the organism depending on the cellular context. In the first scenario, transient and programmed senescence is initiated in the severely damaged or unwanted cells. Permanent withdrawal from proliferation is crucial for preventing the propagation of premalignant cells in the context of tumorigenesis. Then arrested cells secrete a mix of SASP factors, including cytokines, chemokines, growth factors, and metalloproteases. Besides communicating and recruiting the immune system (Yun et al., 2015), SASP could trigger the proliferation and differentiation of undamaged cells (Demaria et al., 2014), promote ECM deposition (Jun and Lau, 2010), optimize tissue remodeling (Demaria et al., 2014; Jun and Lau, 2010), and induce cellular plasticity and stemness of the neighboring cells (Ritschka et al., 2017). Eventually, removal of the senescent cells is essential to eliminate SASP and construct/reconstruct the tissue.
In contrast, senescent cells accumulate in tissues during physiological aging (Calcinotto et al., 2019) and in age-associated pathologies (Baker and Petersen, 2018; Childs et al., 2017). There are several non-mutually exclusive possibilities of why senescent cells accumulate with age. Firstly, the senescent cell production rate might be increased owing to more aged cells containing a higher level of damage. Secondly, SASP mediated paracrine senescence (Acosta et al., 2013) could further accelerate this process. Thirdly, the aging immune system may be less efficient in removing senescent cells. Alternatively, senescent cells may evolve in a way to directly impair the immune surveillance in the aged tissues. Noteworthy, a recent study used both experimental and mathematical models to address this question. The authors used datasets from previous longitudinal studies to identify the most suitable model describing the dynamics of the senescent cells, which was further validated by senescence induction in mice of different ages. The model suggests that the turnover rate of senescent cells decreases with age due to increased production and reduced removal (Karin et al., 2019). Surprisingly, senescent cells actively inhibit their removal. Although the mechanisms by which senescent cells use to disrupt their removal is unknown, this study proposed a provocative scheme with exciting implications in senolytic approaches.
Recent studies provided vital clues on how senescence promotes age-related tissue dysfunction in both cell-autonomous and non-cell autonomous manner. First of all, a permanent cell cycle arrest directly impairs tissue regeneration. Senescent adult stem cells (ASCs), such as hematopoietic stem cells and muscle stem cells, fail to re-enter the cell cycle and resume proliferation after exiting from the quiescence (Sousa-Victor et al., 2014; Chang et al., 2016). Moreover, a persistent proliferative arrest could disrupt the cell turnover resulting in permanent cell loss in tissues that do not have ASCs. Besides, senescent cells can lose many cellular functions. Senescent vascular cells reduced endothelial tight junction and lessen barrier integrity in vitro, which might contribute to the age-related blood-brain barrier disruption (Yamazaki et al., 2016). Senescent chondrocytes fail to secrete various ECM factors that are important for articular cartilage maintenance (Calcinotto et al., 2019; McCulloch et al., 2017), while senescent pancreatic β cells lose the cell identity and cannot produce insulin (Aguayo-Mazzucato et al., 2019).
Although it remains to be demonstrated in vivo, SASP could promote a dysfunctional stem cell niche (Chang et al., 2016) and induce paracrine senescence in the neighboring cells to exacerbate the diminished stem cell functionality. Moreover, SASP factors could induce tissue degradation (Jeon et al., 2017; Farr et al., 2017), interfere with differentiation process (Farr et al., 2017), and stimulate tissue fibrosis (Ferreira-Gonzalez et al., 2018). Finally, SASP may contribute to the proinflammatory microenvironment in many age-related pathologies (Jeon et al., 2017; Baker and Petersen, 2018; Childs et al., 2017; Palmer et al., 2019). Work over the last decade demonstrated that selective elimination of senescent cells, either genetically or by senolytic drugs, ameliorates various age-related pathologies in mice (Bussian et al., 2018; Childs et al., 2016; Jeon et al., 2017; Baker and Petersen, 2018; Childs et al., 2017; Palmer et al., 2019), and significantly extends both the health and life span (Baker et al., 2016; Xu et al., 2018; Chang et al., 2016; Zhu et al., 2015).
Why does the acute and chronic senescence have the opposite impact on tissue regeneration? There are two potential explanations. Cell autonomously, young ASCs are spared from acute senescence, due to either higher resistance to damage or protective stem cell niche. Therefore, their regenerative functions are preserved. On the contrary, senescence most likely occurs to all the cell types during aging. The irreversible cell cycle arrest prevents the activation of ASCs, which are indispensable for tissue regeneration. Non-cell autonomously, the SASP components might be different between acute and chronic senescence. The SASP is possibly more proinflammatory with age, which might further inhibit the functions of ASCs and impair regeneration.
In summary, there are many questions remain to be elucidated, including how senescence is induced during embryonic development and tissue repair, why senescent cells accumulate in various disease conditions, and why senescence elimination ameliorates organism aging. In vivo senescence program is highly heterogeneous, containing different cell types and SASP composition, which might have a significant impact on senescence phenotype and functions. Therefore, further investigation of in vivo senescence in different contexts will determine the shared and distinct features among various types of senescence, which might allow specifically target the detrimental effects of senescence.
3. Cellular reprogramming: plasticity beyond stem cells
Cell identity is established in the course of lineage differentiation during development, maintained by epigenetic memories and defined by a broad range of molecular and functional properties, which is generally stable for the terminally differentiated cells (Morris, 2019). Previously, it was thought that differentiation is an irreversible process. In the 1950s, Briggs and King began to test the developmental potential of differentiated cells in frogs. They transferred the nucleus from one cell to an enucleated cell (oocytes, in this case), a method known as nuclear transfer. Later, using the same methodology but a different frog species, Xenopus laevis, Gurdon successfully cloned sexually mature frogs using donor nuclei from cells at various developmental stages (Gurdon et al., 1958) and fully differentiated intestinal cells (Gurdon, 1962). Gurdon's seminal work provided the first evidence that certain factors in the oocyte cytoplasm can erase the cellular identity encoded in the nucleus of a somatic cell, strongly supported the principle of nuclear equivalence and laid the foundation for the future development of reprogramming cell identity.
Almost 50 years after Gurdon's experiments, Yamanaka demonstrated that a small set of transcription factors, Sox2, Klf4, Oct4 and c-Myc (OSKM) are sufficient to convert somatic cells into the pluripotent state, known as induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). Currently, there are several routes to reprogram cell identity. For example, cells can be firstly reverted to the pluripotent state, followed by differentiation to desired identities. Alternatively, one cell type can be directly converted to another cell identity by expressing specific factors (Jopling et al., 2011; Aydin and Mazzoni, 2019). The method, also known as lineage reprogramming, bypasses embryonic states altogether and eliminates the tumorigenic risk of undifferentiated pluripotent cells, an important safety issue for therapeutic applications (Jopling et al., 2011). Noteworthy, cell identity can also be manipulated in vivo via forced expression of the same transcription factors combination as their in vitro counterparts (Srivastava and DeWitt, 2016). Several reprogrammable mouse models are engineered to express Yamanaka factors (OSKM) upon doxycycline treatment to induce reprogramming in vivo, which are evaluated by the Nanog expression (a pluripotency marker) and teratoma formation (Ocampo et al., 2016b; Abad et al., 2013; Ohnishi et al., 2014). Importantly, these studies indicate the tissue microenvironment can support full reprogramming, which raised the possibility to modulate cell fate in situ to promote tissue regeneration. Nowadays, direct lineage reprogramming of undamaged cells into the desired cell type using defined factors in situ is an emerging alternative to improve self-repair and tissue regeneration (Jessen et al., 2015; Sanchez Alvarado and Yamanaka, 2014).
The ground-breaking advances in cellular reprogramming led to a paradigm shift in the biomedical research, with exciting implications for advances in disease modeling and regenerative medicine (Takahashi and Yamanaka, 2016; Passier et al., 2016). Studies from the last decade have identified many combinations of factors to facilitate cell fate conversion, including transcription factors, small molecules, and microRNAs, which firmly demonstrated the remarkable plasticity of the differentiated cells both in vitro and in vivo (Xu et al., 2015). Thanks to the advance in single cell biology, cellular reprogramming has become a tractable system to study the mechanisms of cell fate conversion (Takahashi and Yamanaka, 2016). The next challenge in the field is to understand how cellular plasticity is regulated in vivo, which will provide essential insights for improving tissue regeneration in a controlled manner.
4. Partial reprogramming: an emerging rejuvenation strategy
Cellular reprogramming offers many exciting opportunities for aging research. Generation of iPSCs from Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) patients provided powerful in vitro models to unravel the molecular mechanisms of premature and physiological aging and to facilitate the drug development for these devastating rare diseases (Zhang et al., 2011; Liu et al., 2011; Zhang et al., 2015). Besides, many age-related pathologies that are associated with losing functional cells could benefit from iPSCs-based regenerative therapies. Moreover, reprogramming aged cells may gain pivotal insight into epigenetic rejuvenation (Rando and Chang, 2012). Lastly, in vivo lineage-reprogramming based tissue repair in situ would be particularly important in the aged organism with diminished regenerative capacity.
Recent research highlighted that reprogramming could be a rejuvenation process (Mahmoudi et al., 2019; Mahmoudi and Brunet, 2012). Previously, iPSCs derived from aged donors revert several age-associated features, including elongated telomere length due to the reactivation of telomerase during reprogramming (Marion et al., 2009a), improved mitochondrial quality and function, and reset of heterochromatin marks and genes expression signatures (Miller et al., 2013; Lapasset et al., 2011; Studer et al., 2015). Importantly, fibroblasts and neurons differentiated from old donors iPSCs preserved the youthful state, including the transcriptomic profile and proliferative capacity (in the case of fibroblasts) (Miller et al., 2013; Lapasset et al., 2011; Studer et al., 2015). Interestingly, neurons generated from old donor fibroblasts via transdifferentiation (bypassing the pluripotency stage) retained the age-associated signature of the donor cells (Mertens et al., 2015). Therefore, rejuvenation might occur specifically during reprogramming to pluripotency process, where extensive proliferation is required (Buganim et al., 2013).
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Edited by Engadin, 29 July 2020 - 08:16 PM.
