Aged‐senescent cells contribute to impaired heart regeneration

Aging leads to increased cellular senescence and is associated with decreased potency of tissue‐specific stem/progenitor cells. Here, we have done an extensive analysis of cardiac progenitor cells (CPCs) isolated from human subjects with cardiovascular disease, aged 32–86 years. In aged subjects (>70 years old), over half of CPCs are senescent (p16^INK4A, SA‐β‐gal, DNA damage γH2AX, telomere length, senescence‐associated secretory phenotype [SASP]), unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart. SASP factors secreted by senescent CPCs renders otherwise healthy CPCs to senescence. Elimination of senescent CPCs using senolytics abrogates the SASP and its debilitative effect in vitro. Global elimination of senescent cells in aged mice (INK‐ATTAC or wild‐type mice treated with D + Q senolytics) in vivo activates resident CPCs and increased the number of small Ki67‐, EdU‐positive cardiomyocytes. Therapeutic approaches that eliminate senescent cells may alleviate cardiac deterioration with aging and restore the regenerative capacity of the heart.     

Regulation of replicative senescence by insulin-like growth factor-binding protein 3 in human umbilical vein endothelial cells

Insulin/insulin-like growth factor (IGF) signaling pathways are among the most conserved processes in aging in organisms ranging from yeast to mammals. Previously, using cDNA microarray technology, we reported that expression of IGF-binding protein 3 (IGFBP3), one of the IGF-binding proteins, was increased with age in human dermal fibroblasts. In this study, the role of IGFBP3 on cellular senescence was studied in human umbilical vein endothelial cells (HUVEC). The expression levels of IGFBP3 mRNA and protein were increased in HUVECs with age. Knockdown of IGFBP3 in old cells with IGFBP3 short hairpin RNA (shRNA) retrovirus resulted in the partial reduction of a variety of senescent phenotypes, such as changes in cell morphology, and decreases in population doubling times and senescence-associated beta-galactosidase (SA-beta-gal) staining. Down-regulation of IGFBP3 rescued the growth arrest induced by p53 overexpression in young HUVECs. In contrast, up-regulation of IGFBP3 in young cells and prolonged IGFBP3 treatment accelerated cellular senescence, confirmed by cell proliferation and SA-beta-gal staining. The FOXO3a (forkhead box O3a) protein level was increased in old IGFBP3 shRNA cells. The treatment of young HUVECs with IGFBP3 repressed the levels of FOXO3a protein. Furthermore, calorie restriction reduced IGFBP3 protein levels, which were found to be increased with age in the rat liver and serum. These results suggest that IGFBP3 might play an important role in the cellular senescence of HUVECs as well as in vivo aging.     

Replicative senescence of human bone marrow and umbilical cord derived mesenchymal stem cells and their differentiation to adipocytes and osteoblasts

Mesenchymal stem cells (MSC) which have self-renewal and multiple differentiation potential in vitro play important roles in regenerative medicine and tissue engineering. However, long-term culture in vitro leads to senescence which results in the growth arrest and reduction of differentiation. In this study, MSC derived from human bone-marrow (BM-MSC) and umbilical cord (UC-MSC) were cultured in vitro lasted to senescence. Senescence and apoptosis detection showed that the senescent cells increased significantly but the increase of apoptosis was not significant in the long term culture. Senescence related genes p16, p21 and p53 increased gradually in BM-MSC. However, p16 and p53 reduced and then increased but with the gradual increase of p21 in UC-MSC. Adipogenic differentiation decreased whereas the propensity for osteogenic differentiation increased in senescent MSC. Real time RT-PCR demonstrated that both C/EBPα and PPARγ decreased in senescent BM-MSC. However, in UC-MSC, PPARγ decreased but C/EBPα increased in late phase compared to early phase. The study demonstrated p21 was important in the senescence of BM-MSC and UC-MSC. C/EBPα and PPARγ could regulate the balance of adipogenic differentiation in BM-MSC but only PPARγ not C/EBPα was involved in the adipogenic differentiation in UC-MSC.     

Senescence and apoptosis: dueling or complementary cell fates?

In response to a variety of stresses, mammalian cells undergo a persistent proliferative arrest known as cellular senescence. Many senescence‐inducing stressors are potentially oncogenic, strengthening the notion that senescence evolved alongside apoptosis to suppress tumorigenesis. In contrast to apoptosis, senescent cells are stably viable and have the potential to influence neighboring cells through secreted soluble factors, which are collectively known as the senescence‐associated secretory phenotype (SASP). However, the SASP has been associated with structural and functional tissue and organ deterioration and may even have tumor‐promoting effects, raising the interesting evolutionary question of why apoptosis failed to outcompete senescence as a superior cell fate option. Here, we discuss the advantages that the senescence program may have over apoptosis as a tumor protective mechanism, as well as non‐neoplastic functions that may have contributed to its evolution. We also review emerging evidence for the idea that senescent cells are present transiently early in life and are largely beneficial for development, regeneration and homeostasis, and only in advanced age do senescent cells accumulate to an organism's detriment.     

Deficiency in the DNA repair protein ERCC1 triggers a link between senescence and apoptosis in human fibroblasts and mouse skin

ERCC1 (excision repair cross complementing‐group 1) is a mammalian endonuclease that incises the damaged strand of DNA during nucleotide excision repair and interstrand cross‐link repair. Ercc1^?/Δ mice, carrying one null and one hypomorphic Ercc1 allele, have been widely used to study aging due to accelerated aging phenotypes in numerous organs and their shortened lifespan. Ercc1^?/Δ mice display combined features of human progeroid and cancer‐prone syndromes. Although several studies report cellular senescence and apoptosis associated with the premature aging of Ercc1^?/Δ mice, the link between these two processes and their physiological relevance in the phenotypes of Ercc1^?/Δ mice are incompletely understood. Here, we show that ERCC1 depletion, both in cultured human fibroblasts and the skin of Ercc1^?/Δ mice, initially induces cellular senescence and, importantly, increased expression of several SASP (senescence‐associated secretory phenotype) factors. Cellular senescence induced by ERCC1 deficiency was dependent on activity of the p53 tumor‐suppressor protein. In turn, TNFα secreted by senescent cells induced apoptosis, not only in neighboring ERCC1‐deficient nonsenescent cells, but also cell autonomously in the senescent cells themselves. In addition, expression of the stem cell markers p63 and Lgr6 was significantly decreased in Ercc1^?/Δ mouse skin, where the apoptotic cells are localized, compared to age‐matched wild‐type skin, possibly due to the apoptosis of stem cells. These data suggest that ERCC1‐depleted cells become susceptible to apoptosis via TNFα secreted from neighboring senescent cells. We speculate that parts of the premature aging phenotypes and shortened health‐ or lifespan may be due to stem cell depletion through apoptosis promoted by senescent cells.     

The role of growth factors in maintenance of stemness in bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are an active topic of research in regenerative medicine due to their ability to secrete a variety of growth factors and cytokines that promote healing of damaged tissues and organs. In addition, these secreted growth factors and cytokines have been shown to exert an autocrine effect by regulating MSC proliferation and differentiation. We found that expression of EGF, FGF-4 and HGF were down-regulated during serial passage of bone marrow-derived mesenchymal stem cells (BMSCs). Proliferation and differentiation potentials of BMSCs treated with these growth factors for 2 months were evaluated and compared to BMSCs treated with FGF-2, which increased proliferation of BMSCs. FGF-2 and -4 increased proliferation potentials at high levels, about 76- and 26-fold, respectively, for 2 months, while EGF and HGF increased proliferation of BMSCs by less than 2.8-fold. Interestingly, differentiation potential, especially adipogenesis, was maintained only by HGF treatment. Treatment with FGF-2 rapidly induced activation of AKT and later induced ERK activation. The basal level of phosphorylated ERK increased during serial passage of BMSCs treated with FGF-2. The expression of LC3-II, an autophagy marker, was gradually increased and the population of senescent cells was increased dramatically at passage 7 in non-treated controls. But FGF-2 and FGF-4 suppressed LC3-II expression and down-regulated senescent cells during long-term (i.e. 2 month) cultures. Taken together, depletion of growth factors during serial passage could induce autophagy, senescence and down-regulation of stemness (proliferation via FGF-2/-4 and differentiation via HGF) through suppression of AKT and ERK signaling.     

Senescent cells spread the word: non‐cell autonomous propagation of cellular senescence

Nat Cell Biol advance online publication, June162013; doi:10.1038/ncb2784Senescence has long been considered a cell autonomous arrest programme restricting the propagation of damaged cells in tissues. Now there is accumulating evidence that senescent cells can communicate with their environment. In a recent report by Gil and colleagues (Acosta et al, 2013), it now seems senescence can be transmitted in a paracrine fashion in several in vitro and in vivo contexts. In addition to broadening our understanding of the biology of senescence, these new findings may have interesting implications for tissue homeostasis and future cancer therapies.Senescence is a form of stress-induced cell cycle arrest that restricts the proliferative capacity of damaged and/or potentially harmful cells (Rodier and Campisi, 2011), thereby promoting tissue homeostasis and tumour suppression. While the senescence-associated cell cycle arrest involves the well-studied Rb and p53 pathways, senescent cells also possess the less understood ability to secrete growth factors, cytokines and chemokines into their environment. This process, collectively known as the senescence-associated secretory phenotype (SASP; Rodier and Campisi, 2011), was originally used to mark senescent cells, but is now known to enforce cell cycle arrest, modify the microenvironment and trigger immune surveillance of senescent cells (Xue et al, 2007; Krizhanovsky et al, 2008; Rodier and Campisi, 2011).Adding to our understanding of this process, a recent report by Gil and colleagues showed that the SASP can also mediate paracrine transmission of cellular senescence (Acosta et al, 2013). By co-culturing cells undergoing oncogene-induced senescence (OIS) with normal cells, the authors showed that the senescence phenotype could be transmitted to surrounding cells via the soluble SASP proteins. Coupling quantitative proteomics with small-molecule inhibitor screens, they identified key players mediating the paracrine transmission of senescence, including TGFB, VEGF and CCL2 pathways. A search for upstream regulators of SASP pointed at IL-1 signalling and the inflammasome, molecules that operate cell autonomously to control SASP production and non-cell autonomously to spread the senescent phenotype via the SASP (Figure 1). Complementing the in vitro senescence findings, experiments using mouse and human models of OIS demonstrated evidence for paracrine senescence transmission in vivo.Open in a separate windowFigure 1Cell autonomous and non-cell autonomous effects of cellular senescence. Stress stimuli such as activation of oncogenes and DNA damage can trigger normal mitotic cells to go into senescence. This involves inflammosome-mediated activation of IL-1 signalling, which initiates the SASP response. The SASP acts cell autonomously (autocrine) to reinforce the senescent phenotype via cytokines such as IL-6. The SASP also acts non-cell autonomously (paracrine) to influence the cells in the surrounding environment. For example, SASP components such as VEGF, TGFB and CCL2 can trigger bystander senescence on neighbouring cells. Paradoxically, the SASP can also exert pro-mitogenic stimulation of neighbouring cells via cytokines like IL-6, which appear to play dual roles depending on the context. Furthermore, the SASP can act on the immune system via pro-inflammatory cytokines, leading to immune cell recruitment and subsequent targeting and clearance of senescent cells. Alternatively, the SASP can trigger upregulation of p16 and p21 levels on neighbouring immune cells, the functional consequences of which are not yet so clear.The ability of senescent cells to propagate their phenotype is consistent with previous studies identifying IGFBP7 as a paracrine senescence regulator (Wajapeyee et al, 2008) and provides important insights into senescence biology. It is conceivable to think that the induction of paracrine cell cycle arrest could expand the senescence footprint of the pre-neoplastic lesion to the surrounding epithelium. This could potentially serve to amplify the tissue damage signal, recruit more immune cells and ensure more efficient clearance of damaged cells. In parallel, the induction of paracrine senescence in other cell types within the tissue, for example tumour-associated fibroblasts, could repress their reported paracrine tumour-promoting effects (Krtolica et al, 2001).Despite the biological implications, a number of questions remain. Why, for instance, is paracrine senescence triggered in some cells surrounding pre-neoplastic lesions but not in others? Similarly, what is the functional significance of paracrine senescence induction in the surrounding immune cells? Intriguingly, recent evidence implies that p16 can also be induced in tumour-infiltrating immune cells (Burd et al, 2013). It will be important to determine whether the paracrine p16 induction in immune cells leads to the same consequences as in non-immune cells and whether the induction of a potential arrest programme compromises the ability of the immune cell to clear senescent cells.Beyond the biological implications, the key regulators of paracrine senescence have potential to be manipulated therapeutically. It is commonly believed, for instance, that senescent cells accumulate in aging tissues and disrupt tissue architecture and function (Rodier and Campisi, 2011). In this context, antagonists of paracrine senescence might limit the spread of senescence and prove beneficial for some age-associated disorders. In the context of cancer, both chemotherapeutic drugs and radiation are known to induce senescence in tumour cells (Schmitt, 2007; Prise and O''Sullivan, 2009). The use of agents agonizing paracrine senescence as adjunctive therapy could potentially increase the effectiveness of chemo- and radiotherapy by triggering a bystander response.Nonetheless, it is critical to keep in mind that the SASP may not always relay an arrest-inducing message onto the surrounding cells. Indeed, the SASP component IL-6 has been shown to elicit a pro-mitogenic response in a paracrine fashion (Kuilman et al, 2008). Similarly, the SASP has been shown to be pro- and anti-tumorigenic depending on the microenvironment (Krtolica et al, 2001; Xue et al, 2007; Lujambio et al, 2013; Figure 1). Collectively, these findings suggest that the ultimate outcome of senescence within a tissue is highly dependent on the context. But what then determines this context? One decisive factor could be whether or not the senescence signal engages in sufficient modulation of the immune system to provoke clearance. In cases where the senescent cells in a tissue are not cleared, the pro-mitogenic arm of the SASP signal could persist long enough to have an overall pro-tumorigenic effect. It will thus be important to understand all the flavours of SASP to modulate it safely for therapeutic purposes.     

干细胞衰老与衰老微环境调控的研究进展

干细胞衰老会损害机体组织的稳态,衰老的干细胞丧失修复能力从而引发衰老相关疾病。衰老微环境是促进机体衰老的重要因素之一。衰老相关分泌表型(SASP)是构成衰老微环境的主要成分,影响干细胞的组织修复能力,进而推动机体衰老进程。细胞外囊泡(EVs)被认为在衰老微环境中发挥重要作用,衰老细胞分泌的EVs通过运载mi RNAs等非编码RNA及SASP在内的多种活性分子参与调控衰老微环境,本文就干细胞衰老的诱发因素以及衰老微环境的研究进展进行综述,以期为干细胞的临床应用提供实验基础和理论基础。     

Pharmacological CDK4/6 inhibition reveals a p53‐dependent senescent state with restricted toxicity

Cellular senescence is a state of stable growth arrest and a desired outcome of tumor suppressive interventions. Treatment with many anti‐cancer drugs can cause premature senescence of non‐malignant cells. These therapy‐induced senescent cells can have pro‐tumorigenic and pro‐disease functions via activation of an inflammatory secretory phenotype (SASP). Inhibitors of cyclin‐dependent kinases 4/6 (CDK4/6i) have recently proven to restrain tumor growth by activating a senescence‐like program in cancer cells. However, the physiological consequence of exposing the whole organism to pharmacological CDK4/6i remains poorly characterized. Here, we show that exposure to CDK4/6i induces non‐malignant cells to enter a premature state of senescence dependent on p53. We observe in mice and breast cancer patients that the CDK4/6i‐induced senescent program activates only a partial SASP enriched in p53 targets but lacking pro‐inflammatory and NF‐κB‐driven components. We find that CDK4/6i‐induced senescent cells do not acquire pro‐tumorigenic and detrimental properties but retain the ability to promote paracrine senescence and undergo clearance. Our results demonstrate that SASP composition is exquisitely stress‐dependent and a predictor for the biological functions of different senescence subsets.     

Proteomic Analysis Reveals a Role for Bcl2-associated Athanogene 3 and Major Vault Protein in Resistance to Apoptosis in Senescent Cells by Regulating ERK1/2 Activation

Senescence is a prominent solid tumor response to therapy in which cells avoid apoptosis and instead enter into prolonged cell cycle arrest. We applied a quantitative proteomics screen to identify signals that lead to therapy-induced senescence and discovered that Bcl2-associated athanogene 3 (Bag3) is up-regulated after adriamycin treatment in MCF7 cells. Bag3 is a member of the BAG family of co-chaperones that interacts with Hsp70. Bag3 also regulates major cell-signaling pathways. Mass spectrometry analysis of the Bag3 Complex revealed a novel interaction between Bag3 and Major Vault Protein (MVP). Silencing of Bag3 or MVP shifts the cellular response to adriamycin to favor apoptosis. We demonstrate that Bag3 and MVP contribute to apoptosis resistance in therapy-induced senescence by increasing the level of activation of extracellular signal-regulated kinase1/2 (ERK1/2). Silencing of either Bag3 or MVP decreased ERK1/2 activation and promoted apoptosis in adriamycin-treated cells. An increase in nuclear accumulation of MVP is observed during therapy-induced senescence and the shift in MVP subcellular localization is Bag3-dependent. We propose a model in which Bag3 binds to MVP and facilitates MVP accumulation in the nucleus, which sustains ERK1/2 activation. We confirmed that silencing of Bag3 or MVP shifts the response toward apoptosis and regulates ERK1/2 activation in a panel of diverse breast cancer cell lines. This study highlights Bag3-MVP as an important complex that regulates a potent prosurvival signaling pathway and contributes to chemotherapy resistance in breast cancer.Cellular senescence plays an important role in determining the response of tumors to cancer therapy (1). Senescence is regulated by the p53 and p16-pRB tumor suppressor pathways and characterized by irreversible cell cycle arrest and expression of the lysosomal protein, senescence associated beta galactosidase (SA-β-gal)¹. Additional characteristics of senescent cells include the presence of senescence-associated heterochromatic foci, and a senescence associated secretory phenotype (SASP) (2). Because of the SASP of senescent cells, therapy-induced senescence (TIS) may be harmful in cancer and the quantitative elimination of senescent cells could prove to be therapeutically beneficial. A recent study demonstrated that pharmacologically targeting the metabolic pathways of TIS in vivo prompted tumor regression and improved treatment outcomes (3).A characteristic of senescent cells is their ability to resist apoptosis although the responsible mechanism is poorly understood. Impairment of apoptosis in senescent cells is associated with a poor outcome in cancer (4). Manipulation of the apoptotic machinery may serve as a therapeutic means of eliminating senescent cells with harmful SASP. It has been proposed that in senescent cells, p53 may preferentially activate genes that arrest proliferation, rather than those that facilitate apoptosis. Alternatively, resistance to apoptosis may be caused by altered expression of proteins that inhibit, promote, or mediate apoptotic cell death, such as Bcl2.Bcl2 associated athanogene 3 (Bag3) is a member of the BAG family of chaperones that interacts with the ATPase domain of heat shock protein-70 (Hsp70). In addition to its BAG domain, Bag3 contains a WW domain and a proline-rich (PXXP) repeat, which mediates binding to partners other than Hsp70. Bag3 is expressed in response to cellular stress under the induction of HSF1 and is known to suppress apoptosis and regulate autophagy (5–6). Suppression of apoptosis may be partially explained by the ability of Bag3 to protect Bcl2 family members against proteasomal degradation (7). In normal cells, Bag3 is constitutively expressed in only a few cell types, including cardiomyocytes (8). Bag3 is overexpressed in leukemia and several solid tumors where it has been reported to sustain cell survival, induce resistance to therapy, and promote metastasis. The pleiotropic functions of Bag3 may reflect its ability to assemble scaffolding complexes, which participate in multiple signal transduction pathways (9).In this study, we describe a role for Bag3 in regulating cancer chemotherapy induced senescence in breast cancer cell. Using a quantitative SILAC approach, we show that Bag3 is up-regulated in TIS. Mass spectrometry analysis reveals that Bag3 binds to the Major Vault Protein (MVP) complex, a protein complex strongly associated with chemotherapy resistance. We also show that Bag3 and MVP contribute to apoptosis resistance by regulating ERK1/2 signaling in senescent MCF7 and ZR751 cells.     

Secretome analysis of ionizing radiation-induced senescent cancer cells reveals that secreted RKIP plays a critical role in neighboring cell migration

Cellular senescence is a physiological program of irreversible growth arrest that is considered to play an important role in tumor suppression. Recent studies demonstrated that senescent cells secrete multiple growth regulatory proteins that could alter the behavior of neighboring cells. In this study, we investigated the effect of secretory proteins from ionizing radiation (IR) induced senescent tumor cells on normal and tumor cells. Conditioned medium (CM) from IR-induced senescent MCF7 cells significantly increased cell proliferation, invasion, migration, and wound healing activity in MCF7 cells and HUVECs. Comparative proteomics analysis revealed 24 differentially secreted protein spots including Raf kinase inhibitor protein (RKIP), α-Enolase, AKAP9, and MARK4, and the findings were confirmed by Western blot analysis of IR-induced senescent cancer cells. We found that RKIP was secreted via the classical pathway, and the transfection of small interfering RNA against RKIP suppressed CM-induced migration in MCF7 cells. Treatment with recombinant human RKIP increased the migratory activity of MCF7 cells. Taken together, our results demonstrate that the senescence-associated secretory protein RKIP could be the principal target to prevent the potential effects of the secretome from IR-induced senescent tumor cells on neighboring cell migration.     

自噬与间充质干细胞衰老的关系研究进展

间充质干细胞(mesenchymal stem cells,MSCs)具备多向分化、免疫调控和靶向迁移的能力,在再生医学领域一直备受关注。但是,随着供体年龄的增长和体外培养时间的延长,MSCs通常表现出衰老特征。MSCs衰老以及功能衰退被认为是机体衰老和相关退行性疾病发展的重要诱发因素,同时也制约着MSCs在再生医学领域中的应用。自噬是溶酶体依赖途径介导细胞内物质的降解和再循环过程,是真核细胞的非核(细胞质)部分得以更新的有效途径,对维持细胞稳态至关重要,是调节MSCs衰老的潜在调控靶标。对MSCs衰老的表型特征、功能变化和分子机制,以及自噬与衰老之间的关系进行综述,为促进MSCs临床应用提供理论基础。     

Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7

Expression of an oncogene in a primary cell can, paradoxically, block proliferation by inducing senescence or apoptosis through pathways that remain to be elucidated. Here we perform genome-wide RNA-interference screening to identify 17 genes required for an activated BRAF oncogene (BRAFV600E) to block proliferation of human primary fibroblasts and melanocytes. Surprisingly, we find a secreted protein, IGFBP7, has a central role in BRAFV600E-mediated senescence and apoptosis. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 (rIGFBP7) induces apoptosis in BRAFV600E-positive human melanoma cell lines, and systemically administered rIGFBP7 markedly suppresses growth of BRAFV600E-positive tumors in xenografted mice. Immunohistochemical analysis of human skin, nevi, and melanoma samples implicates loss of IGFBP7 expression as a critical step in melanoma genesis.     

Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages

Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker^® staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration.     

Proteomic analysis of the impact of static culturing on the expansion of rat bone marrow mesenchymal stem cells

The clinical potential of mesenchymal stem cells (MSC) in tissue engineering and regenerative medicine is due to their self-renewal, proliferation and multi-lineage differentiation potential. Clinical use requires large cell numbers; which can, theoretically, be generated by ex vivo expansion of plastic adherent, MSC subpopulation, of bone marrow cells (BMC). Effects of serial culture on MSC phenotype were investigated using non-gel based quantitative proteomic methodology for static monolayer cultures of rat BMC. In total, 382 proteins were relatively quantified (≥ 2 peptides). Nine proteins were up-regulated and seven down-regulated at passage 4 relative to passage 2 (p ≤ 0.05). We propose that serial culture impacts on MSC expansion (observed decline in colony forming potential and colony size) is through a combination of osteogenic differentiation and ageing/senescence and propose six novel protein biomarkers as candidates for quality control purposes in bioprocessing.     

Senescent cells and macrophages: key players for regeneration?

Over the last decade, our understanding of the physiological role of senescent cells has drastically evolved, from merely indicators of cellular stress and ageing to having a central role in regeneration and repair. Increasingly, studies have identified senescent cells and the senescence-associated secretory phenotype (SASP) as being critical in the regenerative process following injury; however, the timing and context at which the senescence programme is activated can lead to distinct outcomes. For example, a transient induction of senescent cells followed by rapid clearance at the early stages following injury promotes repair, while the long-term accumulation of senescent cells impairs tissue function and can lead to organ failure. A key role of the SASP is the recruitment of immune cells to the site of injury and the subsequent elimination of senescent cells. Among these cell types are macrophages, which have well-documented regulatory roles in all stages of regeneration and repair. However, while the role of senescent cells and macrophages in this process is starting to be explored, the specific interactions between these cell types and how these are important in the different stages of injury/reparative response still require further investigation. In this review, we consider the current literature regarding the interaction of these cell types, how their cooperation is important for regeneration and repair, and what questions remain to be answered to advance the field.     

Mesenchymal stem cell‐derived extracellular vesicles reduce senescence and extend health span in mouse models of aging

Aging drives progressive loss of the ability of tissues to recover from stress, partly through loss of somatic stem cell function and increased senescent burden. We demonstrate that bone marrow‐derived mesenchymal stem cells (BM‐MSCs) rapidly senescence and become dysfunctional in culture. Injection of BM‐MSCs from young mice prolonged life span and health span, and conditioned media (CM) from young BM‐MSCs rescued the function of aged stem cells and senescent fibroblasts. Extracellular vesicles (EVs) from young BM‐MSC CM extended life span of Ercc1 ^−/− mice similarly to injection of young BM‐MSCs. Finally, treatment with EVs from MSCs generated from human ES cells reduced senescence in culture and in vivo, and improved health span. Thus, MSC EVs represent an effective and safe approach for conferring the therapeutic effects of adult stem cells, avoiding the risks of tumor development and donor cell rejection. These results demonstrate that MSC‐derived EVs are highly effective senotherapeutics, slowing the progression of aging, and diseases driven by cellular senescence.     

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.