The RhoA nuclear localization changes in replicative senescence:New evidence from in vitro human mesenchymal stem cells studies

All non-immortalized mesenchymal stem cells have a limited proliferative potential,that is,replicative senescence(RS)is an integral characteristic of the life of all mesenchymal stem cells(MSCs).It is known that one of the important signs of RS is a decrease of cell motility,and that violations of migration processes contribute to the deterioration of tissue regeneration.Therefore,the characterization of the properties of the cell line associated with RS is a prerequisite for the effective use of MSCs in restorative medicine.One of the key proteins regulating cell motility is the small GTPase RhoA.The main purpose of this work was to study the nuclear-cytoplasmic redistribution of the RhoA protein during RS in MSC lines recently obtained and characterized in our laboratory.The study found that a comparative analysis of the intracellular localization of RhoA in three cell lines(MSCWJ-1,FetMSC,DF2)showed a decrease in the nuclear localization of RhoA during RS.

Aminoguanidine delays the replicative senescence of human diploid fibroblasts

Background The accumulation of free radicals and advanced glycation end products （AGEs） in cell plays a very important role in replicative senescence. Aminoguanidine （AG） has potential antioxidant effects and decreases AGE levels. This study aimed to investigate its effect on replicative senescence in vitro. Methods The effects of aminoguanidine on morphology, replicative lifespan, cell growth and proliferation, AGEs, DNA damage, DNA repair ability and telomere length were observed in human fetal lung diploid fibroblasts （2BS）. Results Aminoguanidine maintained the non-senescent phenotype of 2BS cells even at late population doubling （PD） and increased cumulative population doublings by at least 17-21 PDs. Aminoguanidine also improved the potentials of growth and proliferation of 2BS cells as detected by the MTT assay. The AGE levels of late PD cells grown from early PD in DMEM containing aminiguanidine decreased significantly compared with those of late PD control cells and were similar to those of young control cells. In addition, the cells pretreated with aminoguanidine had a significant reduction in DNA strand breaks when they were exposed to 200 μmol/L H2O2 for 5 minutes which indicated that the compound had a strong potential to protect genomic DNA against oxidative stress. And most of the cells exposed to 100 μmol/L H2O2 had much shorter comet tails and smaller tail areas after incubation with aminoguanidine-supplemented DMEM, which indicated that the compound strongly improved the DNA repair abilities of 2BS cells. Moreover, PD55 cells grown from PD28 in 2 mmol/L or 4 mmol/L aminoguanidine retain telomere lengths of 7.94 kb or 8.12 kb, which was 0.83 kb or 1.11 kb longer than that of the control cells. Conclusion Aminoguanidine delays replicative senescence of 2BS cells and the senescence-delaying effect of aminoguanidine appear to be due to its many biological properties including its potential for proliferation improvement, its inhibitory effect of AGE formation, antioxidant effect, improvement of DNA repair ability and the slowdown of telomere shortening.

Two isomers of HDTIC isolated from Astragali Radix decrease the expression of p16 in 2BS cells

Background Astragafi Radix, the root of Astragalus membranceus （Fish） Bunge Var. mongholicus （Bge）, is a crude drug considered as one of the effective traditional Chinese anti-ageing material. The two isomers of 4-hydroxy-5-hydroxymethyl-[1,3]dioxolan-2,6＇-spirane-5＇,6＇,7＇,8＇-tetrahydro-indolizine-3＇-carbaldehyde （HDTIC）, HDTIC-1 and HDTIC-2, were first extracted from the herb in 2002. We demonstrated previously that 0.1 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2 strongly delay replicaUve senescence of human fetal lung diploid fibroblasts （2BS）. In this study, we chose them to investigate their effects on the expression of senescence-associated genes to explore the mechanism of how HDTIC delays replicative senescence. Methods The effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 were observed in vitro by RT-PCR and Western blot. The anti-oxidative activities of the compounds were also observed by phenotype alteration after treatment with antioxidants. Results There was an obvious expression of p16 in the control senescent cells. However, in the 2BS cells, after 56 population doublings （PDs） grown from PD28 in 0.1 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2, there was a weak mRNA expression of p16 and no protein expression of pl 6 was observed. The expression level of p21 increased with cell ageing Moreover, there was no difference between the expression level of p21 in the control cells and that in the same PD cells cultured with HDTIC compounds. The results also showed that 2BS cells exposed to 100 μmol/L H202 for 5 minutes retum to their non-senescent phenotype and continue to be confluent after incubating the damaged cells with HDTIC-1 （1.0 μmol/L ） or HDTIC-2 （10 μmol/L ） for I hour. Conclusions Expression of p16 by 2BS cells was strongly inhibited by HDTIC compounds, which could contribute to their delayed replicative senescence by the way of p16^INK4a/Rb/MAPK. The anti-oxidative activities of HDTIC-1 and HDTIC-2, described in this study for the first time, might be indirectly related to their inhibition of p16 expression.

Chondrogenic priming of human fetal synovium-derived stem cells in an adult stem cell matrix microenvironment

Cartilage defects are a challenge to treat clinically due to the avascular nature of cartilage.Low immunogenicity and extensive proliferation and multidifferentiation potential make fetal stem cells a promising source for regenerative medicine.In this study,we aimed to determine whether fetal synovium-derived stem cells(FSDSCs)exhibited replicative senescence and whether expansion on decellularized extracellular matrix(dECM)deposited by adult SDSCs(AECM)promoted FSDSCs’chondrogenic potential.FSDSCs from passage 2 and 9 were compared for chondrogenic potential,using Alcian blue staining for sulfated glycosaminoglycans(GAGs),biochemical analysis for DNA and GAG amounts,and real-time PCR for chondrogenic genes including ACAN and COL2A1.Passage 3 FSDSCs were expanded for one passage on plastic flasks(PL),AECM,or dECM deposited by fetal SDSCs(FECM).During expansion,cell proliferation was evaluated using flow cytometry for proliferation index,stem cell surface markers,and resistance to hydrogen peroxide.During chondrogenic induction,expanded FSDSCs were evaluated for tri-lineage differentiation capacity.We found that cell expansion enhanced FSDSCs’chondrogenic potential at least up to passage 9.Expansion on dECMs promoted FSDSCs’proliferative and survival capacity and adipogenic differentiation but not osteogenic capacity.AECM-primed FSDSCs exhibited an enhanced chondrogenic potential.

Effects of malondialdehyde on growth and proliferation of human bone marrow mesenchymal stem cells in vitro

Malondialdehyde(MDA)is a well known inducer of carbonyl stress in a variety of human cells,however,its effects on human bone marrow mesenchymal stem cells(hMSCs)have not been documented.In this study,the effects of MDA concentration on the growth rate and proliferation of hMSCs in vitro were assessed.Under high concentrations of MDA,the cell count was decreased and the population doubling time(PDT)was lengthened.Flow cytometry(FCM)demonstrated that MDA triggered cells to undergo apoptosis,in parallel with the findings in MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]assay which showed that it can also impair cellular viability.Surprisingly,FCM also determined that the percentage of hMSCs in G2/M-and S-phases also increased in a dose-dependent manner with respect to MDA concentration.These results strongly suggest that even though hMSCs were severely impaired by high concentrations of MDA,they were still able to send signals that resulted in accelerated cellular proliferation process.This study provided important insights on how carbonyl stress affects cell cycle and proliferation of hMSCs.