Senescent hMSCs Research Articles

Leveraging Cell Migration Dynamics to Discriminate Between Senescent and Presenescent Human Mesenchymal Stem Cells

PurposeThe suboptimal clinical performance of human mesenchymal stem cells (hMSCs) has raised concerns about their therapeutic potential. One major contributing factor to this issue is the heterogeneous nature of hMSCs. Senescent cell accumulation during stem cell expansion is a key driver of MSC heterogeneity. Current methodologies to eradicate senescent hMSCs have either shown limited success or lack clinical relevance. This study leverages the inherent capacity of hMSCs to migrate toward damaged tissues as a means to discern senescent from presenescent stem cells. Given the established deficiency of senescent cells to migrate through physiologically relevant environments, we hypothesized that a microfluidic device, designed to emulate key facets of in vivo cell motility, could serve as a platform for identifying presenescent cells.MethodsWe employed a Y-shaped microchannel assay, which allows fine-tuning of fluid flow rates and the degree of confinement.ResultsHighly migratory hMSCs detected by the device not only demonstrate increased speed, smaller size, and higher proliferative capacity but also manifest reduced DNA damage and senescence compared to non-migratory cells. Additionally, this assay detects presenescent cells in experiments with mixed early and late passage cells. The introduction of fluid flow through the device can further increase the fraction of highly motile stem cells, improving the assay's effectiveness to remove senescent hMSCs.ConclusionsCollectively, this assay facilitates the detection and isolation of a highly potent stem cell subpopulation. Given the positive correlation between the migratory potential of administered MSCs and the long-term clinical outcome, delivering homogeneous, highly motile presenescent hMSCs may benefit patient outcomes.

Chromatin Condensation Delays Senescence in Human Mesenchymal Stem Cells by Safeguarding Nuclear Damages during In Vitro Expansion

Human mesenchymal stem cells (hMSCs) are multipotent cells that differentiate into adipocytes, chondrocytes, and osteoblasts. Owing to their differentiation potential, hMSCs are among the cells most frequently used for therapeutic applications in tissue engineering and regenerative medicine. However, the number of cells obtained through isolation alone is insufficient for hMSC-based therapies and basic research, which necessitates in vitro expansion. Conventionally, this is often performed on rigid surfaces such as tissue culture plates (TCPs). However, during in vitro expansion, hMSCs lose their proliferative ability and multilineage differentiation potential, rendering them unsuitable for clinical use. Although multiple approaches have been attempted to maintain hMSC stemness during prolonged expansion, finding a suitable culture system remains an unmet need. Recently, a few research groups have shown that hMSCs maintain their stemness over long passages when cultured on soft substrates. In addition, it has been shown that hMSCs cultured on soft substrates have more condensed chromatin and lower levels of histone acetylation compared to those cultured on stiff substrates. Furthermore, it has also been shown that condensing/decondensing chromatin by deacetylation/acetylation can delay replicative senescence in hMSCs during long-term expansion on TCPs. However, the mechanism by which chromatin condensation/decondensation influences nuclear morphology and DNA damage, which are strongly related to the onset of senescence, remains unknown. To answer this question, we cultured hMSCs for long duration in the presence of epigenetic modifiers, histone acetyltransferase inhibitor (HATi), which promotes chromatin condensation by preventing histone acetylation, and histone deacetylase inhibitor (HDACi), which promotes chromatin decondensation, and investigated their effects on various nuclear markers related to senescence. We found that consistent acetylation causes severe nuclear abnormalities, whereas chromatin condensation by deacetylation helps to safeguard the nucleus from damage caused by in vitro expansion.

CRL2APPBP2-mediated TSPYL2 degradation counteracts human mesenchymal stem cell senescence.

Cullin-RING E3 ubiquitin ligases (CRLs), the largest family of multi-subunit E3 ubiquitin ligases in eukaryotic cells, represent core cellular machinery for executing protein degradation and maintaining proteostasis. Here, we asked what roles Cullin proteins play in human mesenchymal stem cell (hMSC) homeostasis and senescence. To this end, we conducted a comparative aging phenotype analysis by individually knocking down Cullin members in three senescence models: replicative senescent hMSCs, Hutchinson-Gilford Progeria Syndrome hMSCs, and Werner syndrome hMSCs. Among all family members, we found that CUL2 deficiency rendered hMSCs the most susceptible to senescence. To investigate CUL2-specific underlying mechanisms, we then applied CRISPR/Cas9-mediated gene editing technology to generate CUL2-deficient human embryonic stem cells (hESCs). When we differentiated these into hMSCs, we found that CUL2 deletion markedly accelerates hMSC senescence. Importantly, we identified that CUL2 targets and promotes ubiquitin proteasome-mediated degradation of TSPYL2 (a known negative regulator of proliferation) through the substrate receptor protein APPBP2, which in turn down-regulates one of the canonical aging marker-P21waf1/cip1, and thereby delays senescence. Our work provides important insights into how CRL2APPBP2-mediated TSPYL2 degradation counteracts hMSC senescence, providing a molecular basis for directing intervention strategies against aging and aging-related diseases.

The effect of growth hormone-induced cellular behavior and signaling properties on induced cellular senescence in human mesenchymal stem cells

Senescence is associated with a decline in physiological function, which is accompanied by onset of diseases. Growth hormone (GH) is a class of growth-promoting cytokines with reduced secretion in aging populations. However, the effect of senescence on GH bioactivity is not fully understood in human mesenchymal stem cells (hMSCs). In this work, GH-induced cellular behavior and intracellular signaling transduction were explored in senescent hMSCs. Therefore, hMSCs were used to establish a senescence model by H2O2 treatment for this study. First, we investigated the effects of cellular senescence on the cell behavior of GH. The experimental results suggested that GH could not be internalized into the nucleus, and a significant reduction in GH internalization into the cytoplasm was observed in senescent hMSCs compared to the control group. Second, the effect of cellular senescence on GH-mediated intracellular signaling pathways was investigated by Western blotting. For this, the signaling molecule activation of Janus kinase 2 (JAK2)/signal transducer and activator transcription (STAT) stimulated by GH was detected. Our data indicated that the signaling intensity of p-JAK2, p-STAT5, p-STAT3 and p-STAT1 was considerably weakened. Taken together, these findings provide important insights into the impaired effects of cellular senescence on the biological activity of GH.

Apoptosis resistance of senescent cells is an intrinsic barrier for senolysis induced by cardiac glycosides

Targeted elimination of senescent cells, senolysis, is one of the core trends in the anti-aging therapy. Cardiac glycosides were recently proved to be a broad-spectrum senolytics. Here we tested senolytic properties of cardiac glycosides towards human mesenchymal stem cells (hMSCs). Cardiac glycosides had no senolytic ability towards senescent hMSCs of various origins. Using biological and bioinformatic approaches we compared senescence development in ‘cardiac glycosides-sensitive’ A549 and ‘-insensitive’ hMSCs. The absence of senolysis was found to be mediated by the effective potassium import and increased apoptosis resistance in senescent hMSCs. Weakening “antiapoptotic defense” predisposes hMSCs to senolysis. We revealed that apoptosis resistance, previously recognized as a common characteristic of senescence, in fact, is not a general feature of senescent cells. Moreover, only apoptosis-prone senescent cells are sensitive to cardiac glycosides-induced senolysis. Thus, we can speculate that the effectiveness of senolysis might depend on whether senescent cells indeed become apoptosis-resistant as compared to their proliferating counterparts.Graphic abstract

MicroRNA-487a-3p Regulates the Senescence of Mesenchymal Stem Cells by Targeting Silencing Information Regulator-Associated Enzyme 1 (SIRT1)

SIRT1 is known to be closely associated with cellular senescence, while the relationship between miR-487a-3p and SIRT1 and their role in mesenchymal stem cells (MSCs) remains unclear. MiRDB analysis showed SIRT1 is a target of miR-487a-3p. Here we investigated whether miR-487a-3p modulates senescence of mesenchymal stem cells by targeting SIRT1. The human MSCs (hMSCs) were divided into control group (NC group), miR-487a-3p Mimics group, pCMV-SIRT+miR-487a-3p Mimics group followed by analysis of miR-487a-3p expression by qPCR and protein level of SIRT1, P21 and P53 by western blot. Dual luciferin report assay verified the binding of miR-487a-3p to SIRT1 mRNA and β-galactosidase activity staining detected hMSCs senescence. miR-487a-3p level was significantly elevated after miR-487a-3p Mimics treatment (P <0.01) without difference between miR-487a-3p Mimics group and pCMV-SIRT1 group+miR-487a-3pMimics (P >0.05). miR-487a-3p mimics significantly decreased SIRT1 level (P < 0.01), which was reversed by pCMVSIRT1 plasmid transfection (P <0.05). Moreover, miR-487a-3p could bind SIRT1 mRNA 3′-UTR region. Further more, miR-487a-3p Mimics induced cellular senescence as displayed by increased β-galactosidase activity (P <0.01) and increased level of senescence-related proteins P21 and P53 (P < 0.01), which were all reversed by overexpression of SIRT1 (P < 0.05). In conclusion, miR-487a-3p reduced SIRT1 expression, thus promoting hMSCs senescence, while overexpression of SIRT1 could counteract the senescence of hMSCs induced by miR-487a-3p.

An animal model of MGUS/SMM to investigate the role of cellular senescence in progression to MM.

8050 Background: Multiple Myeloma (MM) is an incurable cancer of plasma cells that arises from precursor stages, monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM). Mechanisms for the transformation of MGUS/SMM to MM are not fully known. There is no approved therapy for MGUS/SMM and research is limited by a lack of animal models. Here, we describe the use of a novel animal model of disease progression derived from patients with MGUS/SMM to study the impact of cellular senescence (CS) on the development of MM. Methods: CD 138+ plasma cells and CD138- stromal cells were isolated via magnetic beads from bone marrow aspirates of patients with MGUS/SMM and co-cultured CD138+/CD138- with different ratios. For the animal model CD138+/CD138- cells from MGUS/SMM patients were injected into the tibias of NSG mice (1:10). After 4 weeks, CD138+ cells were harvested and again co-transplanted into the tibia of a 2nd NSG mouse with the CD138- cells (1:10). Mice underwent serial imaging and tibias were histologically examined after 4 weeks in the 2nd mouse. CS gene expression was compared by RNAseq between patients with SMM and healthy older adults. The primary CD138- cells were stained for SA-β-gal. Human mesenchymal stem cells (hMSCs) induced by H2O2 for CS vs control were co-cultured with CD138+ cells. Patient derived CD138- cells were treated with anti-senolytic drugs, dasatinib (D) and quercetin (Q) and cell growth in co-culture with CD138+ plasma cells was measured. D+Q treated patient-derived CD138+/CD138- cells were co-transplanted into our NSG mouse model and followed with serial imaging. Results: CD138- stromal cells from patients with MGUS/SMM support the growth of CD138+ plasma cells (10:1). CD138- cells were found to gain CD138+ expression, suggesting another source for the plasma cell growth. Imaging of our mouse model showed the development of lytic lesions in the tibias of 5/5 mice versus no lytic lesions in mice transplanted with CD138+ cells alone. Staining of the lytic lesions revealed CD138+ plasma cells. Our RNA seq showed significantly increased expression of CS genes, CDKN2A, p16 and p19 (3 fold) in CD138- cells of SMM patients and confirmed by qPCR (8 fold) compared to healthy older adults. The primary stromal cell culture from MGUS/SMM patients showed the presence of SCs by SA-β-gal staining. H2O2 induced senescent hMSCs stimulate cell survival and growth of CD138+ cells. CD138- cells cultured with conditioned media from H2O2 induced hMSCs demonstrated more CD138+ cells than vehicle treated CD138- cells. Conversely, D + Q pre-treated CD138- cells lost the ability to promote CD138+ cells in co-culture. Our animal model treated with D+Q showed less bone erosion in tibias compared to untreated mice. Conclusions: Our work demonstrates a novel animal model for studying disease progression in patients with MGUS or SMM and demonstrates a role for CS in MM disease progression.

SIRT3 consolidates heterochromatin and counteracts senescence.

Sirtuin 3 (SIRT3) is an NAD+-dependent deacetylase linked to a broad range of physiological and pathological processes, including aging and aging-related diseases. However, the role of SIRT3 in regulating human stem cell homeostasis remains unclear. Here we found that SIRT3 expression was downregulated in senescent human mesenchymal stem cells (hMSCs). CRISPR/Cas9-mediated depletion of SIRT3 led to compromised nuclear integrity, loss of heterochromatin and accelerated senescence in hMSCs. Further analysis indicated that SIRT3 interacted with nuclear envelope proteins and heterochromatin-associated proteins. SIRT3 deficiency resulted in the detachment of genomic lamina-associated domains (LADs) from the nuclear lamina, increased chromatin accessibility and aberrant repetitive sequence transcription. The re-introduction of SIRT3 rescued the disorganized heterochromatin and the senescence phenotypes. Taken together, our study reveals a novel role for SIRT3 in stabilizing heterochromatin and counteracting hMSC senescence, providing new potential therapeutic targets to ameliorate aging-related diseases.

Functional Regulatory Mechanisms Underlying Bone Marrow Mesenchymal Stem Cell Senescence During Cell Passages.

Mesenchymal stem cell (MSC) transplantation is an effective periodontal regenerative therapy. MSCs are multipotent, have self-renewal ability, and can differentiate into periodontal cells. However, senescence is inevitable for MSCs. In vitro, cell senescence can be induced by long-term culture with/without cell passage. However, the regulatory mechanism of MSC senescence remains unclear. Undifferentiated MSC-specific transcription factors can regulate MSC function. Herein, we identified the regulatory transcription factors involved in MSC senescence and elucidated their mechanisms of action. We cultured human MSCs (hMSCs) with repetitive cell passages to induce cell senescence and evaluated the mRNA and protein expression of cell senescence-related genes. Additionally, we silenced the cell senescence-induced transcription factors, GATA binding protein 6 (GATA6) and SRY-box 11 (SOX11), and investigated senescence-related signaling pathways. With repeated passages, the number of senescent cells increased, while the cell proliferation capacity decreased; GATA6 mRNA expression was upregulated and that of SOX11 was downregulated. Repetitive cell passages decreased Wnt and bone morphogenetic protein (BMP) signaling pathway-related gene expression. Silencing of GATA6 and SOX11 regulated Wnt and BMP signaling pathway-related genes and affected cell senescence-related genes; moreover, SOX11 silencing regulated GATA6 expression. Hence, we identified them as pair of regulatory transcription factors for cell senescence in hMSCs via the Wnt and BMP signaling pathways.

FZD5 regulates cellular senescence in human mesenchymal stem/stromal cells.

Human mesenchymal stem/stromal cells (hMSCs) have garnered enormous interest as a potential resource for cell‐based therapies. However, the molecular mechanisms regulating senescence in hMSCs remain unclear. To elucidate these mechanisms, we performed gene expression profiling to compare clonal immature MSCs exhibiting multipotency with less potent MSCs. We found that the transcription factor Frizzled 5 (FZD5) is expressed specifically in immature hMSCs. The FZD5 cell surface antigen was also highly expressed in the primary MSC fraction (LNGFR+THY‐1+) and cultured MSCs. Treatment of cells with the FZD5 ligand WNT5A promoted their proliferation. Upon FZD5 knockdown, hMSCs exhibited markedly attenuated proliferation and differentiation ability. The observed increase in the levels of senescence markers suggested that FZD5 knockdown promotes cellular senescence by regulating the noncanonical Wnt pathway. Conversely, FZD5 overexpression delayed cell cycle arrest during the continued culture of hMSCs. These results indicated that the intrinsic activation of FZD5 plays an essential role in negatively regulating senescence in hMSCs and suggested that controlling FZD5 signaling offers the potential to regulate hMSC quality and improve the efficacy of cell‐replacement therapies using hMSCs.

NAD+/NADH redox alterations reconfigure metabolism and rejuvenate senescent human mesenchymal stem cells in vitro

Human mesenchymal stem cells (hMSCs) promote endogenous tissue regeneration and have become a promising candidate for cell therapy. However, in vitro culture expansion of hMSCs induces a rapid decline of stem cell properties through replicative senescence. Here, we characterize metabolic profiles of hMSCs during expansion. We show that alterations of cellular nicotinamide adenine dinucleotide (NAD + /NADH) redox balance and activity of the Sirtuin (Sirt) family enzymes regulate cellular senescence of hMSCs. Treatment with NAD + precursor nicotinamide increases the intracellular NAD + level and re-balances the NAD + /NADH ratio, with enhanced Sirt-1 activity in hMSCs at high passage, partially restores mitochondrial fitness and rejuvenates senescent hMSCs. By contrast, human fibroblasts exhibit limited senescence as their cellular NAD + /NADH balance is comparatively stable during expansion. These results indicate a potential metabolic and redox connection to replicative senescence in adult stem cells and identify NAD + as a metabolic regulator that distinguishes stem cells from mature cells. This study also suggests potential strategies to maintain cellular homeostasis of hMSCs in clinical applications.

METTL3 counteracts premature aging via m6A-dependent stabilization of MIS12 mRNA.

N6-Methyladenosine (m6A) messenger RNA methylation is a well-known epitranscriptional regulatory mechanism affecting central biological processes, but its function in human cellular senescence remains uninvestigated. Here, we found that levels of both m6A RNA methylation and the methyltransferase METTL3 were reduced in prematurely senescent human mesenchymal stem cell (hMSC) models of progeroid syndromes. Transcriptional profiling of m6A modifications further identified MIS12, for which m6A modifications were reduced in both prematurely senescent hMSCs and METTL3-deficient hMSCs. Knockout of METTL3 accelerated hMSC senescence whereas overexpression of METTL3 rescued the senescent phenotypes. Mechanistically, loss of m6A modifications accelerated the turnover and decreased the expression of MIS12 mRNA while knockout of MIS12 accelerated cellular senescence. Furthermore, m6A reader IGF2BP2 was identified as a key player in recognizing and stabilizing m6A-modified MIS12 mRNA. Taken together, we discovered that METTL3 alleviates hMSC senescence through m6A modification-dependent stabilization of the MIS12 transcript, representing a novel epitranscriptional mechanism in premature stem cell senescence.

Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells

The use of human mesenchymal stem cells (hMSCs) in clinical applications requires large-scale cell expansion prior to administration. However, the prolonged culture of hMSCs results in cellular senescence, impairing their proliferation and therapeutic potentials. To understand the role of microRNAs (miRNAs) in regulating cellular senescence in hMSCs, we globally depleted miRNAs by silencing the DiGeorge syndrome critical region 8 (DGCR8) gene, an essential component of miRNA biogenesis. DGCR8 knockdown hMSCs exhibited severe proliferation defects and senescence-associated alterations, including increased levels of reactive oxygen species (ROS). Transcriptomic analysis revealed that the antioxidant gene superoxide dismutase 2 (SOD2) was significantly downregulated in DGCR8 knockdown hMSCs. Moreover, we found that DGCR8 silencing in hMSCs resulted in hypermethylation in CpG islands upstream of SOD2. 5-aza-2′-deoxycytidine treatment restored SOD2 expression and ROS levels. We also found that these effects were dependent on the epigenetic regulator DNA methyltransferase 3 alpha (DNMT3A). Using computational and experimental approaches, we demonstrated that DNMT3A expression was regulated by miR-29a-3p and miR-30c-5p. Overexpression of miR-29a-3p and/or miR-30c-5p reduced ROS levels in DGCR8 knockdown hMSCs and rescued proliferation defects, mitochondrial dysfunction, and premature senescence. Our findings provide novel insights into hMSCs senescence regulation by the miR-29a-3p/miR-30c-5p/DNMT3A/SOD2 axis.

ZKSCAN3 counteracts cellular senescence by stabilizing heterochromatin.

Zinc finger protein with KRAB and SCAN domains 3 (ZKSCAN3) has long been known as a master transcriptional repressor of autophagy. Here, we identify a novel role for ZKSCAN3 in alleviating senescence that is independent of its autophagy-related activity. Downregulation of ZKSCAN3 is observed in aged human mesenchymal stem cells (hMSCs) and depletion of ZKSCAN3 accelerates senescence of these cells. Mechanistically, ZKSCAN3 maintains heterochromatin stability via interaction with heterochromatin-associated proteins and nuclear lamina proteins. Further study shows that ZKSCAN3 deficiency results in the detachment of genomic lamina-associated domains (LADs) from the nuclear lamina, loss of heterochromatin, a more accessible chromatin status and consequently, aberrant transcription of repetitive sequences. Overexpression of ZKSCAN3 not only rescues premature senescence phenotypes in ZKSCAN3-deficient hMSCs but also rejuvenates physiologically and pathologically senescent hMSCs. Together, these data reveal for the first time that ZKSCAN3 functions as an epigenetic modulator to maintain heterochromatin organization and thereby attenuate cellular senescence. Our findings establish a new functional link among ZKSCAN3, epigenetic regulation, and stem cell aging.

Telomere-dependent and telomere-independent roles of RAP1 in regulating human stem cell homeostasis

RAP1 is a well-known telomere-binding protein, but its functions in human stem cells have remained unclear. Here we generated RAP1-deficient human embryonic stem cells (hESCs) by using CRISPR/Cas9 technique and obtained RAP1-deficient human mesenchymal stem cells (hMSCs) and neural stem cells (hNSCs) via directed differentiation. In both hMSCs and hNSCs, RAP1 not only negatively regulated telomere length but also acted as a transcriptional regulator of RELN by tuning the methylation status of its gene promoter. RAP1 deficiency enhanced self-renewal and delayed senescence in hMSCs, but not in hNSCs, suggesting complicated lineage-specific effects of RAP1 in adult stem cells. Altogether, these results demonstrate for the first time that RAP1 plays both telomeric and nontelomeric roles in regulating human stem cell homeostasis.

Identification of new genes associated to senescent and tumorigenic phenotypes in mesenchymal stem cells

Although human mesenchymal stem cells (hMSCs) are a powerful tool for cell therapy, prolonged culture times result in replicative senescence or acquisition of tumorigenic features. To identify a molecular signature for senescence, we compared the transcriptome of senescent and young hMSCs with normal karyotype (hMSCs/n) and with a constitutional inversion of chromosome 3 (hMSC/inv). Senescent and young cells from both lineages showed differentially expressed genes (DEGs), with higher levels in senescent hMSCs/inv. Among the 30 DEGs in senescent hMSC/inv, 11 are new candidates for biomarkers of cellular senescence. The functional categories most represented in senescent hMSCs were related to cellular development, cell growth/proliferation, cell death, cell signaling/interaction, and cell movement. Mapping of DEGs onto biological networks revealed matrix metalloproteinase-1, thrombospondin 1, and epidermal growth factor acting as topological bottlenecks. In the comparison between senescent hMSCs/n and senescent hMSCs/inv, other functional annotations such as segregation of chromosomes, mitotic spindle formation, and mitosis and proliferation of tumor lines were most represented. We found that many genes categorized into functional annotations related to tumors in both comparisons, with relation to tumors being highest in senescent hMSCs/inv. The data presented here improves our understanding of the molecular mechanisms underlying the onset of cellular senescence as well as tumorigenesis.

Antioxidant effects of selenocysteine on replicative senescence in human adipose-derived mesenchymal stem cells

In most clinical applications, human mesenchymal stem cells (hMSCs) are expanded in large scale before their administration. Prolonged culture in vitro results in cellular senescence-associated phenotypes, including accumulation of reactive oxygen species (ROS) and decreased cell viabilities. Profiling of stem cell-related genes during in vitro expansion revealed that numerous canonical pathways were significantly changed. To determine the effect of selenocysteine (Sec), a rare amino acid found in several antioxidant enzymes, on the replicative senescence in hMSCs, we treated senescent hMSCs with Sec. Supplementation of Sec in the culture medium in late-passage hMSCs reduced ROS levels and improved the survival of hMSCs. In addition, a subset of key antioxidant genes and Sec-containing selenoproteins showed increased mRNA levels after Sec treatment. Furthermore, ROS metabolism and inflammation pathways were predicted to be downregulated. Taken together, our results suggest that Sec has antioxidant effects on the replicative senescence of hMSCs.

Lactoferrin Protects Human Mesenchymal Stem Cells from Oxidative Stress-Induced Senescence and Apoptosis.

Mesenchymal stem cells (MSCs) have been suggested as a primary candidate for cell therapy applications because they have self-renewal and differentiation capabilities. Although they can be expanded in ex vivo system, clinical application of these cells is still limited because they survive poorly and undergo senescence or apoptosis when transplanted and exposed to environmental factors such as oxidative stress. Thus, reducing oxidative stress is expected to improve the efficacy of MSC therapy. The milk protein lactoferrin is a multifunctional iron-binding glycoprotein that plays various roles, including reduction of oxidative stress. Thus, we explored the effect of lactoferrin on oxidative stress-induced senescence and apoptosis of human MSCs (hMSCs). Measurement of reactive oxygen species (ROS) revealed that lactoferrin inhibited the production of hydrogen peroxide-induced intracellular ROS, suggesting lactoferrin as a good candidate as an antioxidant in hMSCs. Pretreatment of lactoferrin suppressed hydrogen peroxide-induced senescence of hMSCs. In addition, lactoferrin reduced hydrogen peroxide-induced apoptosis via inhibition of caspase-3 and Akt activation. These results demonstrate that lactoferrin can be a promising factor to protect hMSCs from oxidative stress-induced senescence and apoptosis, thus increasing the efficacy of MSC therapy.

Usage of Human Mesenchymal Stem Cells in Cell-based Therapy: Advantages and Disadvantages.

ABSTRACTThe use of human mesenchymal stem cells (hMSCs) in cell-based therapy has attracted extensive interest in the field of regenerative medicine, and it shows applications to numerous incurable diseases. hMSCs show several superior properties for therapeutic use compared to other types of stem cells. Different cell types are discussed in terms of their advantages and disadvantages, with focus on the characteristics of hMSCs. hMSCs can proliferate readily and produce differentiated cells that can substitute for the targeted affected tissue. To maximize the therapeutic effects of hMSCs, a substantial number of these cells are essential, requiring extensive ex vivo cell expansion. However, hMSCs have a limited lifespan in an in vitro culture condition. The senescence of hMSCs is a double-edged sword from the viewpoint of clinical applications. Although their limited cell proliferation potency protects them from malignant transformation after transplantation, senescence can alter various cell functions including proliferation, differentiation, and migration, that are essential for their therapeutic efficacy. Numerous trials to overcome the limited lifespan of mesenchymal stem cells are discussed.

Epigallocatechin-3-gallate prevents oxidative stress-induced cellular senescence in human mesenchymal stem cells via Nrf2.

Human mesenchymal stem cells (hMSCs) have great therapeutic potential due to their high plasticity, immune privileged status and ease of preparation, as well as a lack of ethical barriers to their use. However, their ultimate usefulness is limited by cellular senescence occurring secondary to increased cellular levels of reactive oxygen species (ROS) during their propagation in culture. The underlying molecular mechanisms responsible for this process in hMSCs remain unclear. An antioxidant polyphenol epigallocatechin-3-gallate (EGCG) found in green tea, is known to activate nuclear factor-erythroid 2-related factor 2 (Nrf2), a master transcriptional regulator of antioxidant genes. Herein, we examined the EGCG-mediated antioxidant mechanism in hMSCs exposed to ROS which involves Nrf2 activation. The H2O2-exposed hMSCs showed cellular senescence with significantly increased protein levels of acetyl-p53 and p21 in comparison with the untreated hMSCs, and these effects were prevented by pre-treatment with EGCG. By contrast, in Nrf2-knockdown hMSCs, EGCG lost its antioxidant effect, exhibiting high levels of acetyl-p53 and p21 following EGCG pre-treatment and H2O2 exposure. This indicates that Nrf2 and p53/p21 may be involved in the anti-senescent effect of EGCG in hMSCs. Taken together, these findings indicate the important role of EGCG in preventing oxidative stress-induced cellular senescence in hMSCs through Nrf2 activation, which has applications for the massive production of more suitable hMSCs for cell-based therapy.