Base excision repair but not DNA double‐strand break repair is impaired in aged human adipose‐derived stem cells

The decline in DNA repair capacity contributes to the age‐associated decrease in genome integrity in somatic cells of different species. However, due to the lack of clinical samples and appropriate tools for studying DNA repair, whether and how age‐associated changes in DNA repair result in a loss of genome integrity of human adult stem cells remains incompletely characterized. Here, we isolated 20 eyelid adipose‐derived stem cell (ADSC) lines from healthy individuals (young: 10 donors with ages ranging 17–25 years; old: 10 donors with ages ranging 50–59 years). Using these cell lines, we systematically compared the efficiency of base excision repair (BER) and two DNA double‐strand break (DSB) repair pathways—nonhomologous end joining (NHEJ) and homologous recombination (HR)—between the young and old groups. Surprisingly, we found that the efficiency of BER but not NHEJ or HR is impaired in aged human ADSCs, which is in contrast to previous findings that DSB repair declines with age in human fibroblasts. We also demonstrated that BER efficiency is negatively associated with tail moment, which reflects a loss of genome integrity in human ADSCs. Mechanistic studies indicated that at the protein level XRCC1, but not other BER factors, exhibited age‐associated decline. Overexpression of XRCC1 reversed the decline of BER efficiency and genome integrity, indicating that XRCC1 is a potential therapeutic target for stabilizing genomes in aged ADSCs.     

Mitochondrial-associated cell death mechanisms are reset to an embryonic-like state in aged donor-derived iPS cells harboring chromosomal aberrations

Somatic cells reprogrammed into induced pluripotent stem cells (iPSCs) acquire features of human embryonic stem cells (hESCs) and thus represent a promising source for cellular therapy of debilitating diseases, such as age-related disorders. However, reprogrammed cell lines have been found to harbor various genomic alterations. In addition, we recently discovered that the mitochondrial DNA of human fibroblasts also undergoes random mutational events upon reprogramming. Aged somatic cells might possess high susceptibility to nuclear and mitochondrial genome instability. Hence, concerns over the oncogenic potential of reprogrammed cells due to the lack of genomic integrity may hinder the applicability of iPSC-based therapies for age-associated conditions. Here, we investigated whether aged reprogrammed cells harboring chromosomal abnormalities show resistance to apoptotic cell death or mitochondrial-associated oxidative stress, both hallmarks of cancer transformation. Four iPSC lines were generated from dermal fibroblasts derived from an 84-year-old woman, representing the oldest human donor so far reprogrammed to pluripotency. Despite the presence of karyotype aberrations, all aged-iPSCs were able to differentiate into neurons, re-establish telomerase activity, and reconfigure mitochondrial ultra-structure and functionality to a hESC-like state. Importantly, aged-iPSCs exhibited high sensitivity to drug-induced apoptosis and low levels of oxidative stress and DNA damage, in a similar fashion as iPSCs derived from young donors and hESCs. Thus, the occurrence of chromosomal abnormalities within aged reprogrammed cells might not be sufficient to over-ride the cellular surveillance machinery and induce malignant transformation through the alteration of mitochondrial-associated cell death. Taken together, we unveiled that cellular reprogramming is capable of reversing aging-related features in somatic cells from a very old subject, despite the presence of genomic alterations. Nevertheless, we believe it will be essential to develop reprogramming protocols capable of safeguarding the integrity of the genome of aged somatic cells, before employing iPSC-based therapy for age-associated disorders.     

Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell‐based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC‐derived mesenchymal‐like progenitor population. We found the direct plating of undifferentiated iPSCs into high‐density micromass cultures in the presence of BMP‐2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP‐2 treatment of iPSC‐derived MSC‐like (iPSC–MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage‐specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell‐based approaches to repair joint cartilage damage. J. Cell. Biochem. 114: 480–490, 2013. © 2012 Wiley Periodicals, Inc.     

Loss of SIRT2 leads to axonal degeneration and locomotor disability associated with redox and energy imbalance

Sirtuin 2 (SIRT2) is a member of a family of NAD⁺‐dependent histone deacetylases (HDAC) that play diverse roles in cellular metabolism and especially for aging process. SIRT2 is located in the nucleus, cytoplasm, and mitochondria, is highly expressed in the central nervous system (CNS), and has been reported to regulate a variety of processes including oxidative stress, genome integrity, and myelination. However, little is known about the role of SIRT2 in the nervous system specifically during aging. Here, we show that middle‐aged, 13‐month‐old mice lacking SIRT2 exhibit locomotor dysfunction due to axonal degeneration, which was not present in young SIRT2 mice. In addition, these Sirt2^?/? mice exhibit mitochondrial depletion resulting in energy failure, and redox dyshomeostasis. Our results provide a novel link between SIRT2 and physiological aging impacting the axonal compartment of the central nervous system, while supporting a major role for SIRT2 in orchestrating its metabolic regulation. This underscores the value of SIRT2 as a therapeutic target in the most prevalent neurodegenerative diseases that undergo with axonal degeneration associated with redox and energetic dyshomeostasis.     

Insulin‐like growth factor‐1 regulates the SIRT1‐p53 pathway in cellular senescence

Cellular senescence, which is known to halt proliferation of aged and stressed cells, plays a key role against cancer development and is also closely associated with organismal aging. While increased insulin‐like growth factor (IGF) signaling induces cell proliferation, survival and cancer progression, disrupted IGF signaling is known to enhance longevity concomitantly with delay in aging processes. The molecular mechanisms involved in the regulation of aging by IGF signaling and whether IGF regulates cellular senescence are still poorly understood. In this study, we demonstrate that IGF‐1 exerts a dual function in promoting cell proliferation as well as cellular senescence. While acute IGF‐1 exposure promotes cell proliferation and is opposed by p53, prolonged IGF‐1 treatment induces premature cellular senescence in a p53‐dependent manner. We show that prolonged IGF‐1 treatment inhibits SIRT1 deacetylase activity, resulting in increased p53 acetylation as well as p53 stabilization and activation, thus leading to premature cellular senescence. In addition, either expression of SIRT1 or inhibition of p53 prevented IGF‐1‐induced premature cellular senescence. Together, these findings suggest that p53 acts as a molecular switch in monitoring IGF‐1‐induced proliferation and premature senescence, and suggest a possible molecular connection involving IGF‐1‐SIRT1‐p53 signaling in cellular senescence and aging.     

Inducing pluripotency in vitro: recent advances and highlights in induced pluripotent stem cells generation and pluripotency reprogramming

Induced pluripotent stem cells (iPSCs) are considered patient‐specific counterparts of embryonic stem cells as they originate from somatic cells after forced expression of pluripotency reprogramming factors Oct4, Sox2, Klf4 and c‐Myc. iPSCs offer unprecedented opportunity for personalized cell therapies in regenerative medicine. In recent years, iPSC technology has undergone substantial improvement to overcome slow and inefficient reprogramming protocols, and to ensure clinical‐grade iPSCs and their functional derivatives. Recent developments in iPSC technology include better reprogramming methods employing novel delivery systems such as non‐integrating viral and non‐viral vectors, and characterization of alternative reprogramming factors. Concurrently, small chemical molecules (inhibitors of specific signalling or epigenetic regulators) have become crucial to iPSC reprogramming; they have the ability to replace putative reprogramming factors and boost reprogramming processes. Moreover, common dietary supplements, such as vitamin C and antioxidants, when introduced into reprogramming media, have been found to improve genomic and epigenomic profiles of iPSCs. In this article, we review the most recent advances in the iPSC field and potent application of iPSCs, in terms of cell therapy and tissue engineering.     

Aging impairs double‐strand break repair by homologous recombination in Drosophila germ cells

Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double‐strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error‐free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I‐SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR‐white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals.     

Senescence promotes in vivo reprogramming through p16INK4a and IL‐6

Cellular senescence is a damage response aimed to orchestrate tissue repair. We have recently reported that cellular senescence, through the paracrine release of interleukin‐6 (IL6) and other soluble factors, strongly favors cellular reprogramming by Oct4, Sox2, Klf4, and c‐Myc (OSKM) in nonsenescent cells. Indeed, activation of OSKM in mouse tissues triggers senescence in some cells and reprogramming in other cells, both processes occurring concomitantly and in close proximity. In this system, Ink4a/Arf‐null tissues cannot undergo senescence, fail to produce IL6, and cannot reprogram efficiently; whereas p53‐null tissues undergo extensive damage and senescence, produce high levels of IL6, and reprogram efficiently. Here, we have further explored the genetic determinants of in vivo reprogramming. We report that Ink4a, but not Arf, is necessary for OSKM‐induced senescence and, thereby, for the paracrine stimulation of reprogramming. However, in the absence of p53, IL6 production and reprogramming become independent of Ink4a, as revealed by the analysis of Ink4a/Arf/p53 deficient mice. In the case of the cell cycle inhibitor p21, its protein levels are highly elevated upon OSKM activation in a p53‐independent manner, and we show that p21‐null tissues present increased levels of senescence, IL6, and reprogramming. We also report that Il6‐mutant tissues are impaired in undergoing reprogramming, thus reinforcing the critical role of IL6 in reprogramming. Finally, young female mice present lower efficiency of in vivo reprogramming compared to male mice, and this gender difference disappears with aging, both observations being consistent with the known anti‐inflammatory effect of estrogens. The current findings regarding the interplay between senescence and reprogramming may conceivably apply to other contexts of tissue damage.     

A high‐fat diet reverses metabolic disorders and premature aging by modulating insulin and IGF1 signaling in SIRT6 knockout mice

Mammalian sirtuin 6 (SIRT6) is involved in the regulation of many essential processes, especially metabolic homeostasis. SIRT6 knockout mice undergo premature aging and die at age ~4 weeks. Severe glycometabolic disorders have been found in SIRT6 knockout mice, and whether a dietary intervention can rescue SIRT6 knockout mice remains unknown. In our study, we found that at the same calorie intake, a high‐fat diet dramatically increased the lifespan of SIRT6 knockout mice to 26 weeks (males) and 37 weeks (females), reversed multi‐organ atrophy, and reduced body weight, hypoglycemia, and premature aging. Furthermore, the high‐fat diet partially but significantly normalized the global gene expression profile in SIRT6 knockout mice. Regarding the mechanism, excessive glucose uptake and glycolysis induced by the SIRT6 deficiency were attenuated in skeletal muscle through inhibition of insulin and IGF1 signaling by the high‐fat diet. Similarly, fatty acids but not ketone bodies inhibited glucose uptake, glycolysis, and senescence in SIRT6 knockout fibroblasts, whereas PI3K inhibition antagonized the effects of a high‐fatty‐acid medium in vitro. Overall, the high‐fat diet dramatically reverses numerous consequences of SIRT6 deficiency through modulation of insulin and IGF1 signaling, providing a new basis for elucidation of SIRT6 and fatty‐acid functions and supporting novel therapeutic approaches against metabolic disorders and aging‐related diseases.     

Metformin and tenovin‐6 synergistically induces apoptosis through LKB1‐independent SIRT1 down‐regulation in non‐small cell lung cancer cells

Sirtuin 1 (SIRT1) is known to play a role in a variety of tumorigenesis processes by deacetylating histone and non‐histone proteins; however, antitumour effects by suppressing SIRT1 activity in non‐small cell lung cancer (NSCLC) remain unclear. This study was designed to scrutinize clinicopathological significance of SIRT1 in NSCLC and investigate effects of metformin on SIRT1 inhibition. This study also evaluated new possibilities of drug combination using a SIRT1 inhibitor, tenovin‐6, in NSCLC cell lines. It was found that SIRT1 was overexpressed in 300 (62%) of 485 formalin‐fixed paraffin‐embedded NSCLC tissues. Its overexpression was significantly associated with reduced overall survival and poor recurrence‐free survival after adjusted for histology and pathologic stage. Thus, suppression of SIRT1 expression may be a reasonable therapeutic strategy for NSCLC. Metformin in combination with tenovin‐6 was found to be more effective in inhibiting cell growth than either agent alone in NSCLC cell lines with different liver kinase B1 (LKB1) status. In addition, metformin and tenovin‐6 synergistically suppressed SIRT1 expression in NSCLC cells regardless of LKB1 status. The marked reduction in SIRT1 expression by combination of metformin and tenovin‐6 increased acetylation of p53 at lysine 382 and enhanced p53 stability in LKB1‐deficient A549 cells. The combination suppressed SIRT1 promoter activity more effectively than either agent alone by up‐regulating hypermethylation in cancer 1 (HIC1) binding at SIRT1 promoter. Also, suppressed SIRT1 expression by the combination synergistically induced caspase‐3‐dependent apoptosis. The study concluded that metformin with tenovin‐6 may enhance antitumour effects through LKB1‐independent SIRT1 down‐regulation in NSCLC cells.     

Comparative interactomes of SIRT6 and SIRT7: Implication of functional links to aging

Sirtuins are NAD⁺‐dependent deacetylases that regulate a range of cellular processes. Although diverse functions of sirtuins have been proposed, those functions of SIRT6 and SIRT7 that are mediated by their interacting proteins remain elusive. In the present study, we identified SIRT6‐ and SIRT7‐interacting proteins, and compared their interactomes to investigate functional links. Our interactomes revealed 136 interacting proteins for SIRT6 and 233 for SIRT7 while confirming seven and 111 proteins identified previously for SIRT6 and SIRT7, respectively. Comparison of SIRT6 and SIRT7 interactomes under the same experimental conditions disclosed 111 shared proteins, implying related functional links. The interaction networks of interactomes indicated biological processes associated with DNA repair, chromatin assembly, and aging. Interactions of two highly acetylated proteins, nucleophosmin (NPM1) and nucleolin, with SIRT6 and SIRT7 were confirmed by co‐immunoprecipitation. NPM1 was found to be deacetylated by both SIRT6 and SIRT7. In senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased levels of SIRT6 and SIRT7, suggesting that the acetylation of NPM1 could be regulated by SIRT6 and SIRT7 in the aging process. Our comparative interactomic study of SIRT6 and SIRT7 implies important functional links to aging by their associations with interacting proteins. All MS data have been deposited in the ProteomeXchange with identifiers PXD000159 and PXD000850 ( http://proteomecentral.proteomexchange.org/dataset/PXD000159 , http://proteomecentral.proteomexchange.org/dataset/PXD000850 ).     

SIRT6 rescues the age related decline in base excision repair in a PARP1-dependent manner

In principle, a decline in base excision repair (BER) efficiency with age should lead to genomic instability and ultimately contribute to the onset of the aging phenotype. Although multiple studies have indicated a negative link between aging and BER, the change of BER efficiency with age in humans has not been systematically analyzed. Here, with foreskin fibroblasts isolated from 19 donors between 20 and 64 y of age, we report a significant decline of BER efficiency with age using a newly developed GFP reactivation assay. We further observed a very strong negative correlation between age and the expression levels of SIRT6, a factor which is known to maintain genomic integrity by improving DNA double strand break (DSB) repair. Our mechanistic study suggests that, similar to the regulatory role that SIRT6 plays in DNA DSB repair, SIRT6 regulates BER in a PARP1-depdendent manner. Moreover, overexpression of SIRT6 rescues the decline of BER in aged fibroblasts. In summary, our results uncovered the regulatory mechanisms of BER by SIRT6, suggesting that SIRT6 reactivation in aging tissues may help delay the process of aging through improving BER.     

SIRT6 rescues the age related decline in base excision repair in a PARP1-dependent manner

In principle, a decline in base excision repair (BER) efficiency with age should lead to genomic instability and ultimately contribute to the onset of the aging phenotype. Although multiple studies have indicated a negative link between aging and BER, the change of BER efficiency with age in humans has not been systematically analyzed. Here, with foreskin fibroblasts isolated from 19 donors between 20 and 64 y of age, we report a significant decline of BER efficiency with age using a newly developed GFP reactivation assay. We further observed a very strong negative correlation between age and the expression levels of SIRT6, a factor which is known to maintain genomic integrity by improving DNA double strand break (DSB) repair. Our mechanistic study suggests that, similar to the regulatory role that SIRT6 plays in DNA DSB repair, SIRT6 regulates BER in a PARP1-depdendent manner. Moreover, overexpression of SIRT6 rescues the decline of BER in aged fibroblasts. In summary, our results uncovered the regulatory mechanisms of BER by SIRT6, suggesting that SIRT6 reactivation in aging tissues may help delay the process of aging through improving BER.     

Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression

The utility of induced pluripotent stem cells (iPSCs) as models to study diseases and as sources for cell therapy depends on the integrity of their genomes. Despite recent publications of DNA sequence variations in the iPSCs, the true scope of such changes for the entire genome is not clear. Here we report the whole-genome sequencing of three human iPSC lines derived from two cell types of an adult donor by episomal vectors. The vector sequence was undetectable in the deeply sequenced iPSC lines. We identified 1,058-1,808 heterozygous single-nucleotide variants (SNVs), but no copy-number variants, in each iPSC line. Six to twelve of these SNVs were within coding regions in each iPSC line, but ~50% of them are synonymous changes and the remaining are not selectively enriched for known genes associated with cancers. Our data thus suggest that episome-mediated reprogramming is not inherently mutagenic during integration-free iPSC induction.     

Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells

The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50–70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer.