Autophagy: An evolutionarily conserved process in the maintenance of stem cells and aging.

Autophagy is an evolutionarily conserved process that degrades and recycles defective organelles, toxic proteins, and various other aggregates on the cytoplasmic surface by sequestering them into autophagosomes which, then, fuse with lysosomes which degrade them. If these aggregates are not cleared, they accumulate and damage the cell resulting in cellular senescence and aging. Stem cells, with their capacity to differentiate, are crucial for tissue homeostasis. In addition to differentiation, the stemness of stem cells must be preserved. Recent studies in stem cells show the importance of autophagy in evading cellular senescence. In this review, we describe the conservative nature of the autophagy process, carried out throughout evolution. In particular, we highlight the role of autophagy in various evolutionarily diverse species and how it evolved to maintain tissue homeostasis and regulate aging and cellular senescence in stem cells.

The tubulin deacetylase sirtuin-2 regulates neuronal differentiation through the ERK/CREB signaling pathway.

Modification of microtubule (MT) dynamics is important for diverse aspects of cellular function including differentiation, cargo trafficking, migration, and adhesion. MTs also play a crucial role in the progression of neuronal development. The MT deacetylase Sirtuin 2 (Sirt2) and histone deacetylase 6 (HDAC6) regulate MT dynamics by deacetylating alpha-tubulin (α-tubulin). In this study, we investigated the role of MT deacetylation in the progression of neuronal differentiation. For this, we examined acetylated α-tubulin levels during the differentiation of stem cells into neurons. Acetylated α-tubulin levels were significantly altered during differentiation, and these changes were abolished following treatment with 10 µM AGK2 (Sirt2 inhibitor) or 3 µM tubastatin A (HDAC6 inhibitor). However, neural-specific protein expression (Nestin, NF-M, and MAP-2) was reduced in AGK2-treated hBM-MSCs (AGK-MSCs), but not in tubastatin A-treated hBM-MSCs (Tubastatin A-MSCs). Inhibition of Sirt2 led to a decrease in ERK phosphorylation (p-ERK) level, but HDAC6 inhibition had no such effect. Similar results were obtained for CREB phosphorylation (p-CREB). The results suggest that Sirt2 plays a crucial role in neuronal differentiation via the ERK-CREB signaling pathway.

Metformin promotes neuronal differentiation and neurite outgrowth through AMPK activation in human bone marrow-mesenchymal stem cells.

Metformin is an AMP-activated kinase (AMPK) activator that plays a role in glucose energy metabolism and cell protection. It is widely used to treat several diseases, including type 2 diabetes, cardiovascular diseases, cancer, and metabolic diseases. In this study, we investigated whether AMPK activation upon treatment with metformin may promote neurite outgrowth during the progression of neuronal differentiation in human bone marrow-mesenchymal stem cells (hBM-MSCs). Differentiation of metformin-treated MSCs (Met-MSCs to Met-diMSCs) in the neuronal induction media resulted in an increase in the number of differentiated cells in a metformin concentration dependent manner. The differentiation rate reached its maximum at 3 H after the initial treatment with neuronal induction media. At 3 H of induction, the neurite length increased significantly in Met-diMSCs as compared with control cells without metformin treatment (diMSCs). diMSCs showed a significant increase in the expression of neuronal-specific marker genes; however, the expression of dendrite-specific markers MAP-2 and Tuj-1 was significantly increased in Met-diMSCs as compared to diMSCs, as confirmed by immunoblotting. This effect was abolished upon treatment with the AMPK inhibitor, compound C, as evident by quantitative PCR, immunoblotting, and immunocytochemical staining. Thus, metformin treatment promotes neuronal differentiation and neurite outgrowth in hBM-MSCs through AMPK activation.

Functional Restoration of Amyotrophic Lateral Sclerosis Patient-Derived Mesenchymal Stromal Cells Through Inhibition of DNA Methyltransferase.

Alteration of DNA methylation is highly associated with aging and neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). Remedying these aberrant methylation patterns may serve to improve these diseases. Previously, we reported that human bone marrow mesenchymal stromal cells isolated from ALS patients (ALS-MSCs) have functionally decreased stem cell potency, and excessively express DNA methyltransferases (DNMTs). In this study, we examined the correlation between excessive DNMT expression and functional decline in ALS-MSCs. The DNMT inhibitor RG108 was used for this. RG108-treated ALS-MSCs exhibit increased expression of the anti-senescence genes TERT, VEGF, and ANG, and decreased expression of the senescence-related genes ATM and p21. The activity of SA-ß-galactosidase and the expression of senescence proteins p53 and p16 were reduced in RG108-treated ALS-MSCs. The abilities of cell migration and protection against oxidative damage were improved in the treated ALS-MSCs. In neuronal differentiation experiments, the treated MSCs more effectively differentiated into neuron-like cells. These results suggest that ALS-MSC function can be restored by inhibiting excessively expressed DNMTs, an approach that may ultimately provide better efficacy in stem cell therapy.

Accumulation of apoptosis-insensitive human bone marrow-mesenchymal stromal cells after long-term expansion.

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long-term-cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long-term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence-associated ß-gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15-19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7-10 passages). This resistance occurred via caspase-9 and caspase-3 rather than via caspase-8. The senescent cells are gradually accumulated during long-term expansion. The oxidative stress-sensitive proteins ataxia-telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl-2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late-passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long-term-cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long-term expansion. Copyright © 2016 John Wiley & Sons, Ltd.

Endogenous ROS levels are increased in replicative senescence in human bone marrow mesenchymal stromal cells.

Cellular senescence is characterized by functional decline induced by cumulative damage to DNA, proteins, lipids, and carbohydrates. Previous studies have reported that replicative senescence is caused by excessive amounts of reactive oxygen species (ROS) produced as a result of aerobic energy metabolism. In this study, we established human bone marrow mesenchymal stromal cells (hBM-MSCs) in replicative senescence after culture over a long term to investigate the relationship between ROS levels and stem cell potential and to determine whether differentiation potential can be restored by antioxidant treatment. Intracellular ROS levels were increased in hBM-MSCs; this was accompanied by a decrease in the expression of the antioxidant enzymes catalase and superoxide dismutase (SOD)1 and 2 and of phosphorylated forkhead box O1 (p-FOXO1) as well as an increase in the expression of p53 and p16, along with a reduction in differentiation potential. When the antioxidant ascorbic acid was used to eliminate excess ROS, the levels of antioxidant enzymes (catalase, SOD1 and 2, p-FOXO1, and p53) were partly restored. Moreover, differentiation into adipocytes and osteocytes was higher in hBM-MSCs treated with ascorbic acid than in the untreated control cells. These results suggest that the decline in differentiation potential caused by increased endogenous ROS production during in vitro expansion can be reversed by treatment with antioxidants such as ascorbic acid.

Anti-senescence effects of DNA methyltransferase inhibitor RG108 in human bone marrow mesenchymal stromal cells.

Alteration of DNA methylation is highly associated with ageing and ageing-related diseases. Remedy of the altered methylation pattern may provide beneficial efficacy in these diseases. In this study, we used a DNA methyltransferase inhibitor, RG108, to investigate the senescence effects in human bone marrow mesenchymal stromal cells (hBM-MSCs). First, we determined the optimized dose and time of RG108 treatment in hBM-MSCs to be 5 µM for 48 H, respectively. Under these conditions, the anti-senescence genes TERT, bFGF, VEGF, and ANG were increased, whereas the senescence-related genes ATM, p21, and p53 were decreased. The number of ß-galactosidase-positive cells was significantly decreased in RG108-treated MSCs, whereas the rates of MSC migration and cellular protection were increased. We have shown that RG108 significantly induces the expression of TERT by blocking methylation at the TERT promoter region. Thus, these data indicate that an optimized dose of RG108 may improve the cell migration, protection, cellular senescence, which may provide a better efficacy of these cells in stem cell therapy.

Trichostatin A modulates intracellular reactive oxygen species through SOD2 and FOXO1 in human bone marrow-mesenchymal stem cells.

Engraft cells are often exposed to oxidative stress and inflammation; therefore, any factor that can provide the stem cells resistance to these stresses may yield better efficacy in stem cell therapy. Studies indicate that histone deacetylase (HDACs) inhibitors alleviate damage induced by oxidative stress. In this study, we investigated whether regulation of reactive oxygen species (ROS) occurs through the HDAC inhibitor trichostatin A (TSA) in human bone marrow-mesenchymal stem cells (hBM-MSCs). Intracellular ROS levels increased following exposure to hydrogen peroxide (H2 O2 ), and were suppressed by TSA treatment. Levels of the antioxidant enzyme superoxide dismutase 2 (SOD2) increased following treatment with 200 nM TSA and to a lesser level at 1-5 µM TSA. Cell protective effects against oxidative stress were significantly increased in TSA-MSCs after treatment with low doses of TSA (50-500 nM) and decreased with high doses of TSA (5-10 µM). Consistent results were obtained with immunoblot analysis for caspase3. Investigation of Forkhead box O1 (FOXO1), superoxide dismutase 2 (SOD2), and p53 levels to determine intracellular signaling by TSA in oxidative stress-induced MSCs demonstrated that expression of phosphorylated-FOXO1 and phosphorylated-SOD2 decreased in H2 O2 -treated MSCs while levels of p53 increased. These effects were reversed by the treatment of 200 nM TSA. These results suggest that the main function of ROS modulation by TSA is activated through SOD2 and FOXO1. Thus, optimal treatment with TSA may protect hBM-MSCs against oxidative stress.

Proteomic analysis reveals KRIT1 as a modulator for the antioxidant effects of valproic acid in human bone-marrow mesenchymal stromal cells.

Valproic acid (VPA) protects human bone marrow-mesenchymal stromal cells (hBM-MSCs) against oxidative stress and improves their migratory ability through increasing the secretion of trophic factors. This suggests that VPA may be an excellent candidate for improving stem cell function. However, the molecular mechanisms of VPA in BM-MSCs are not known. In this study, we used a proteomic approach to investigate VPA-associated targets under oxidative stress conditions. Krev/Rap1 interaction Trapped-1 (KRIT1), a modulator for the homeostasis of intracellular reactive oxygen species (ROS), was identified as a target protein by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analyses. The up-regulation of KRIT1 and its target proteins (SOD2 and FoxO1) with VPA treatment of hBM-MSCs was revealed by qPCR and immunoblot analysis. Damage from oxidative stress was reduced in VPA-pretreated BM-MSCs, which was also confirmed by qPCR and immunoblot analysis. In addition, increased in intracellular ROS by H2O2 were also reduced by VPA pretreatment in BM-MSCs. This suggests that VPA reduces intracellular ROS level by the modulation of KRIT1 and its correlated proteins, FoxO1, SOD2, and cyclin D1. Thus, this study is the first to provide evidence that VPA modulates KRIT1 and intracellular ROS in BM-MSCs.

Synthesis and evaluation of 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives as glycogen synthase kinase-3 (GSK-3) inhibitors.

New potent glycogen synthase kinase-3 (GSK-3) inhibitors, 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives, were designed by modeling, synthesized and evaluated in vitro. Compound 17c showed good potency in enzyme and cell-based assays (IC50=111 nM, EC50=1.78 µM). Moreover, it has demonstrated desirable water solubility, PK profile, and moderate brain penetration.

Human angiogenin presents neuroprotective and migration effects in neuroblastoma cells.

Human angiogenin (ANG) has been highlighted as an angiogenic factor which supports primary and metastatic tumor growth. Recent genetic studies have shown that ANG is presented as a susceptibility gene for amyotrophic lateral sclerosis (ALS) and ALS-frontotemporal dementia (ALS-FTD). They found several missense mutations, including K40I, which present the weakest functional activity in ANG variants. In this study, we investigate whether human wild type ANG (wANG) and its variant K40I (mANG) maintain their divergent functional capacities in neuronal cells. To evaluate this, SH-SY5Y neuroblastoma cells were transfected with wANG and mANG DNA and identified both wild and mutant ANG are localized to nuclei and have no effects on proliferation. We have shown that human wANG prevented cell death under H(2)O(2)-induced oxidative stress in both SH-SY5Y and NSC-34 cells, tested by MTT assay. These effects were more enhanced in motor neuron cell NSC-34. wANG also played a role in cell migration, while mANG decreased these functional activities. Immunoblot analysis revealed that the intracellular signaling of ERK1/2 (at Thr183/Tyr185) was increased following transfection of the wANG gene, and significantly decreased by mANG in neuronal cells. These findings suggest that human ANG plays a critical role in cell protection and migration following alterations in ERK1/2 signaling in SH-SY5Y cells. This may provide the possible relationship between mutations in hANG and other neurodegenerative diseases as well as ALS.

Synthesis and evaluation of tricyclic derivatives containing a non-aromatic amide as inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1).

Highly potent poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors, including 9-hydroxy-1,2-dihydro-4H-thiopyrano[3,4-c]quinolin-5(6H)-one derivatives with a non-aromatic A-ring, were synthesized. Among the derivatives, 12a showed low nanomolar enzyme and cellular activity (IC(50) = 42 nM, ED(50) = 220 nM) with good water solubility. Further, 12a exhibited microsomal stability in vitro and brain permeability in vivo.

Transduction of human EPO into human bone marrow mesenchymal stromal cells synergistically enhances cell-protective and migratory effects.

Human bone marrow mesenchymal stromal cells (hBM-MSCs) are a promising tools for cell therapy. However, the poor viability of the transplanted cells is a major limiting factor. Human erythropoietin (hEPO) has been extensively studied in non-hematopoietic tissues for its neurotrophic, anti-oxidant, anti-apoptotic, and antiinflammatory effects. In this study, we evaluate whether transduction of the hEPO gene into MSCs provides protection and affects their migration. hBM-MSCs transduced with the hEPO gene (EPO-MSCs) stably secreted high levels of hEPO (10 IU/ml) with no alteration of their mesenchymal phenotype. MSCs were also treated with 10 IU rhEPO, an amount similar to what was secreted by EPO-MSCs, to generate 10U-MSCs. Protection against H2O2-induced oxidative stress and staurosporine-induced apoptosis was registered for both EPO-MSCs and 10U-MSCs, but the protective effects were higher for the EPO-MSCs than for the 10U-MSCs. EPO-MSCs had significantly higher migration rates compared to MSCs and 10U-MSCs. We confirmed that the intracellular signaling of ERK1/2 was higher in the EPO-MSCs than 10U-MSCs. This data demonstrates that the endogenous expression of EPO may efficiently initiate the ERK1/2 signaling pathway, resulting in synergistic effects on the production of neurotrophic factors. Thus, EPO-MSCs are a good candidate for cell therapy in ischemic and neurodegenerative diseases.

Caloric restriction maintains stem cells through niche and regulates stem cell aging.

The functional loss of adult stem cells is a major cause of aging and age-related diseases. Changes in the stem cell niche, increased energy metabolic rate, and accumulation of cell damage severely affect the function and regenerative capacity of stem cells. Reducing the cellular damage and maintaining a pristine stem cell niche by regulating the energy metabolic pathways could be ideal for the proper functioning of stem cells and tissue homeostasis. Numerous studies point out that caloric restriction (CR) has beneficiary effects on stem cell maintenance and tissue regeneration. Recent researches indicate the preventive nature of calorie restriction in stem cells by modulating the stem cell niche through the reduction of energy metabolism and eventually decrease stem cell damage. In this review, we have focused on the general stimuli of stem cell aging, particularly the energy metabolism as an intrinsic influence and stem cell niche as an extrinsic influence in different adult stem cells. Further, we discussed the mechanism behind CR in different adult stem cells and their niche. Finally, we conclude on how CR can enhance the stem cell function and tissue homeostasis through the stem cells niche.

Neuroprotective effects of donepezil through inhibition of GSK-3 activity in amyloid-beta-induced neuronal cell death.

Acetylcholinesterase inhibitors (AChE-inhibitors) are used for the treatment of Alzheimer's disease. Recently, the AChE-inhibitor donepezil was found to have neuroprotective effects. However, the protective mechanisms of donepezil have not yet been clearly identified. We investigated the neuroprotective effects of donepezil and other AChE-inhibitors against amyloid-beta1-42 (Abeta42)-induced neurotoxicity in rat cortical neurons. To evaluate the neuroprotective effects of AChE-inhibitors, primary cultured cortical neurons were pre-treated with several concentrations of AChE-inhibitors for 24 h and then treated with 20 microM Abeta42 for 6 h. In addition to donepezil, other AChE-inhibitors (galantamine and huperizine A) also showed increased neuronal cell viability against Abeta42 toxicity in a concentration-dependent manner. However, we demonstrated that donepezil has a more potent effect in inhibiting glycogen synthase kinase-3 (GSK-3) activity compared with other AChE-inhibitors. The neuroprotective effects of donepezil were blocked by LY294002 (10 microM), a phosphoinositide 3 kinase inhibitor, but only partially by mecamylamine (10 microM), a blocker of nicotinic acetylcholine receptors. Additionally, donepezil's neuroprotective mechanism was related to the enhanced phosphorylation of Akt and GSK-3beta and reduced phosphorylation of tau and glycogen synthase. These results suggest that donepezil prevents Abeta42-induced neurotoxicity through the activation of phosphoinositide 3 kinase/Akt and inhibition of GSK-3, as well as through the activation of nicotinic acetylcholine receptors.

TCF/beta-catenin plays an important role in HCCR-1 oncogene expression.

BACKGROUND: Oncogene HCCR-1 functions as a negative regulator of the p53 and contributes to tumorigenesis of various human tissues. However, it is unknown how HCCR-1 contributes to the cellular and biochemical mechanisms of human tumorigenesis. RESULTS: In this study, we showed how the expression of HCCR-1 is modulated. The luciferase activity assay indicated that the HCCR-1 5'-flanking region at positions -166 to +30 plays an important role in HCCR-1 promoter activity. Computational analysis of this region identified two consensus sequences for the T-cell factor (TCF) located at -26 to -4 (Tcf1) and -136 to -114 (Tcf2). Mutation at the Tcf1 site led to a dramatic decrease in promoter activity. Mobility shift assays (EMSA) revealed that nuclear proteins bind to the Tcf1 site, but not to the Tcf2 site. LiCl, Wnt signal activator by GSK-3beta inhibition, significantly increased reporter activities in wild-type Tcf1-containing constructs, but were without effect in mutant Tcf1-containing constructs in HEK/293 cells. In addition, endogenous HCCR-1 expression was also increased by treatment with GSK-3beta inhibitor, LiCl or AR-A014418 in HEK/293 and K562 cells. Finally, we also observed that the transcription factor, TCF, and its cofactor, beta-catenin, bound to the Tcf1 site. CONCLUSION: These findings suggest that the Tcf1 site on the HCCR-1 promoter is a major element regulating HCCR-1 expression and abnormal stimulation of this site may induce various human cancers.

Reduced sirtuin 1/adenosine monophosphate-activated protein kinase in amyotrophic lateral sclerosis patient-derived mesenchymal stem cells can be restored by resveratrol.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neuron system. Our previous study has shown that bone marrow-mesenchymal stem cells (BM-MSCs) from ALS patients have functional limitations in releasing neurotrophic factors and exhibit the senescence phenotype. In this study, we examined sirtuin 1/adenosine monophosphate-activated protein kinase (SIRT1/AMPK) activities and identified significant decreases in the ALS-MSCs compared with normal healthy control originated BM-MSCs. This decline was restored by pretreatment with resveratrol (RSV), measured using quantitative polymerase chain reaction, NAD/NADH assay, and immunoblot analysis. Neuroprogenitor markers were increased in RSV-treated ALS-MSCs (RSV/ALS-MSCs). The differentiated ALS-MSCs (ALS-dMSCs) exhibited a cell body and dendritic shape similar to neurons. RSV/ALS-MSCs showed significantly increased differentiation rate as compared with the untreated ALS-dMSCs. The neurite numbers and lengths were also significantly increased. This was confirmed with immunoblot analysis using neuron specific markers such as nestin, NF-M, Tuj-1, and Map-2 in RSV/ALS-dMSCs. Thus, this study shows that ALS-MSCs showed down-regulation of AMPK/SIRT1 signalling, which was recovered by treatment with RSV. This data suggest that RSV can be one of the candidate agents for improving therapeutic efficacy of ALS patients' originated MSCs.

Memantine Attenuates Salicylate-induced Tinnitus Possibly by Reducing NR2B Expression in Auditory Cortex of Rat.

Memantine, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, suppresses the release of excessive levels of glutamate that may induce neuronal excitation. Here we investigated the effects of memantine on salicylate-induced tinnitus model. The expressions of the activity-regulated cytoskeleton-associated protein (ARC) and tumor necrosis factor-alpha (TNFα) genes; as well as the NMDA receptor subunit 2B (NR2B) gene and protein, were examined in the SH-SY5Y cells and the animal model. We also used gap-prepulse inhibition of the acoustic startle reflex (GPIAS) and noise burst prepulse inhibition of acoustic startle, and the auditory brainstem level (electrophysiological recordings of auditory brainstem responses, ABR) and NR2B expression level in the auditory cortex to evaluate whether memantine could reduce salicylate-mediated behavioral disturbances. NR2B was significantly upregulated in salicylate-treated cells, but downregulated after memantine treatment. Similarly, expression of the inflammatory cytokine genes TNFα and immediate-early gene ARC was significantly increased in the salicylate-treated cells, and decreased when the cells were treated with memantine. These results were confirmed by NR2B immunocytochemistry. GPIAS was attenuated to a significantly lesser extent in rats treated with a combination of salicylate and memantine than in those treated with salicylate only. The mean ABR threshold in both groups was not significant different before and 1 day after the end of treatment. Additionally, NR2B protein expression in the auditory cortex was markedly increased in the salicylate-treated group, whereas it was reduced in the memantine-treated group. These results indicate that memantine is useful for the treatment of salicylate-induced tinnitus.

HCCR-1, a novel oncogene, encodes a mitochondrial outer membrane protein and suppresses the UVC-induced apoptosis.

BACKGROUND: The Human cervical cancer oncogene (HCCR-1) has been isolated as a human oncoprotein, and has shown strong tumorigenic features. Its potential role in tumorigenesis may result from a negative regulation of the p53 tumor suppressor gene. RESULTS: To investigate the biological function of HCCR-1 in the cell, we predicted biological features using bioinformatic tools, and have identified a LETM1 homologous domain at position 75 to 346 of HCCR-1. This domain contains proteins identified from diverse species predicted to be mitochondrial proteins. Fluorescence microscopy and fractionation experiments showed that HCCR-1 is located in mitochondria in the COS-7, MCF-7 and HEK/293 cell lines, and subcompartamentally at the outer membrane in the HEK/293 cell line. The topological structure was revealed as the NH2-terminus of HCCR-1 oriented toward the cytoplasm. We also observed that the D1-2 region, at position 1 to 110 of HCCR-1, was required and sufficient for posttranslational mitochondrial import. The function of HCCR-1 on mitochondrial membrane is to retard the intrinsic apoptosis induced by UVC and staurosporine, respectively. CONCLUSION: Our experiments show the biological features of HCCR-1 in the cell, and suggest that uncontrolled expression of HCCR-1 may cause mitochondrial dysfunction that can result in resisting the UVC or staurosporine-induced apoptosis and progressing in the tumor formation.

Functional restoration of replicative senescent mesenchymal stem cells by the brown alga Undaria pinnatifida.

The brown alga Undaria pinnatifida, which is called Mi-Yoek in Korea, has been traditionally consumed as a health food in East Asian countries. Recent studies have reported that U. pinnatifida has beneficial effects on arteriosclerosis, inflammation, fat metabolism, and tumors. In this study, we examined the anti-senescence effects of ethanol extracts of U. pinnatifida (UP-Ex) in human bone marrow mesenchymal stem cells (hBM-MSCs). UP-Ex protected hBM-MSCs against oxidative injury, as determined by MTT assays. This effect was confirmed by immunoblot analysis of the oxidation-sensitive protein p53 and the apoptotic protein cleaved caspase-3. Excessive intracellular reactive oxygen species (ROS) accumulation induced by oxidative stress was moderated in UP-Ex-treated hBM-MSCs (UP-Ex-MSCs). Similarly, expression of the ROS-scavenging enzymes superoxide dismutase 1 (SOD1), SOD2, and catalase was recovered in UP-Ex-MSCs. Excessive ROS induced by long-term cell expansion (passage 17) was significantly decreased along with restoration of the senescence proteins p53, p21, and p16 in UP-Ex-MSCs. UP-Ex treatment also improved the ability of these replicative, senescent hBM-MSCs (passage 17) to differentiate into osteocytes or adipocytes, suggesting that UP-Ex ameliorates the functional decline of senescent stem cells and may provide better therapeutic efficacy in stem cell therapy. Abbreviations: hBM-MSCs: human bone marrow mesenchymal stem cells; DCF: 2',7'-dichlorodihydrofluorescein; DCFH-DA: 2',7'-dichlorofluorescein diacetate; MTT: 3-(4,5-dimethylthiazol-2-yl-)2,5-diphenyltetrazolium bromide; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RIPA: radioimmunoprecipitation assay; ROS: reactive oxygen species; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2.