The free radical theory of aging proposes that will cumulative damage by free oxygen radicals is the fundamental reason for cellular and tissue aging. In this study, we all showed that MSC isolated from donors of comparable age had distinct in vitro expansion potential which usually mirrored underlying differences in mitochondrial oxidative status.
MSC were divided into two groupings, weak and vigorous, based on their fitness in lifestyle. More in-depth analysis demonstrated differences between the mitochondrial functionality indicators of the MSC: changes in mitochondrial activity, mtDNA copies, superoxide radical (O 2 – ) production, mitochondrial membrane potential (Δ ψ m), mitonuclear protein imbalance, and expression associated with transcription factors involved in mitochondrial biogenesis. At early pathways, vigorous MSC had lower Δ ψ m plus O 2 – production, and expressed lower levels of mitochondrial biogenesis genetics, such as POLG2 and TFAM. All these parameters together described a higher level of cell fitness, further indicated by considerably longer telomeres, reduced loss of CD90 + cells, faster division rate, and longer stability in culture. At late passages, vigorous MSC sits firmly their metabolic activity at lower levels: fewer mitochondria reflected by fewer mtDNA copies, and consequently decreased creation of O 2 – . Conversely, weak MSC significantly increased their metabolic action with culture time, by increasing the number of mitochondria plus O 2 – , in addition to by a rising gene expression of POLG2 and PGC-1α. Although no difference in the progression of replicative senescence was observed between the two MSC groups, these information indicate an energetic dysfunction which affects the long-term life-span of cells in culture, and shows the restrictions of the standard test of replicative senescence when this kind of tests are used as a substitute for cell quality assessment.
Aging of mitochondria is inspired by several maintenance and turnover biological processes. Higher energetic rates result in accumulation of reactive oxygen types (ROS) which are detrimental for cell components; for instance, mtDNA is very susceptible to oxidative damage [ 13 ]. At increased levels, the same is true for high energy consuming tissues, like the heart and brain, ultimately leading to the formation associated with damage in tissues and organs [ 33 ]. However, low levels of ROS are well known to trigger an adaptive hormetic response, generating a stress resistant reaction plus increased cell longevity [ 34 ]. Successful aging is related to functional mitochondrial dynamics, where centenarians were proven to have high rates of mitochondrial autophagy [ 35 ]. At the molecular level, mitochondrial protein turnover has been connected with extension of longevity by activating the mitochondrial unfolded protein response (UPR mt ). Mitochondrial misfolded proteins activate chaperones and proteases in order to re-establish the proteostasis balance, promoting a stronger response to tension by the cells [ 36 ]. Although a dysfunctional ubiquitin-proteasome system and high rate of protein oxidative harm are among the main causes, UPR mt activation is also elicited by the reduction in the gene expression of mitochondrial ribosomal protein S5 (MRPS5), that is involved in translation of mtDNA-encoded proteins. In fact , 50% downregulation of MRPS5 expression in mice extended their life-span by ~250 days and, in C. elegans , RNA inhibition of the ähnlich MRPS5 prolonged lifespan by 50% [ 37 ]. Furthermore, in our experimental setting, methylene blue (MB) downregulated MRPS5 gene expression by ~40%; however , we could not notice any peculiar UPR mt service in MSC. Thus, downregulation of MRPS5 alone cannot explain the increased MSC lifespan. Additionally , MB decreased gene expression of MRPS5 in the weak MSC with out influencing their expansion potential.
MB is a synthetic dye with several interesting functions: it is readily available, inexpensive, and approved for medical make use of. Since its first application as an anti-malarial drug, these days MB is used in the treatment of methemoglobinemia [ 38 ], cyanide poisoning [ 39 ], and ifosfamide-induced encephalopathy [ 40 ]. Furthermore, MB enhances mitochondrial oxidative phosphorylation and decreases anabolism in glioblastomas, leading to a decrease in cancer cellular proliferation [ 41 ]. In August 2016, 86 medical trials involved MB use, ranging from treatment of pain in order to bipolar disorder (clinicaltrials. gov). The MB mechanism of actions relies on its low redox potential (close to that associated with oxygen) which allows MB to act as an alternative electron acceptor inside the mitochondria. Furthermore, MB has an antioxidant effect, scavenging bad particals from the proximity of the enzymes where O two – are produced [ 23 ]. We also observed an increase in MSC metabolic activity in cultures with MB, coupled with reduced ROS production. Recent observations suggest that an increase in the cell metabolism is one of the effects of caloric restriction [ 42 ], the only identified intervention that extends the lifespan of several microorganisms.
We speculate that MEGABYTES promotes the life extension of MSC in a similar way, by marketing increases in cell metabolic rate and mitochondrial membrane possible, and lowering superoxide production. In parallel, we suggest that MB preserves functional mitochondria for a longer time due to reduced biogenesis and increased expression of the mitochondrial superoxide dismutase. Likewise, high metabolic rates promoted a lifespan extension within mice, based on the “ uncoupling to survive” theory [ 43 ]. In mitochondria, protons are transferred to the intramembrane space by the electron transport chain, eventually promoting ADP phosphorylation. However , not all protons contribute to ATP formation, seeping through the inner membrane. This process, known also as mitochondrial uncoupling, supports high cellular respiration and low ROS and ATP production. We also observed that strenuous MSC were characterized by the lowest mitochondrial membrane potential. In the recent study, Sukumar et al. showed that a reduced mitochondrial membrane potential is a marker of hematopoietic come cells [ 44 ]. Likewise, we propose that MSC along with lower mitochondrial membrane potential possess enhanced stemness, which may explain the longer in vitro lifespan. In contrast, within weak MSC, MB could not induce any lifespan expansion, although the effects of MB at the molecular level were logical. We speculate that mitochondria of these cells were towards a more advanced state of dysfunction which could not be rescued simply by MB; hence, MB only has a preventative role.
Limitations of the study were the particular limited number of donors per group and the absence of further tests, such as mitochondrial proton conductance or accumulation associated with mutations in mtDNA. We included only elderly contributor in the project because MSC isolated from older people possess lower in vitro expansion potential compared to younger donors [ 10 ], and so an increase in cell culture lifespan will be more beneficial for them from a cell therapy prospective. Moreover, to avoid undesired alterations to the endoplasmic reticulum and mitochondrial properties promoted by the addition of bFGF to the lifestyle media [ 45 ], we controlled for this confounding impact by maintaining a constant bFGF concentration among all groupings.