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 β‐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.     

Spontaneous differentiation of periodontal ligament stem cells into myofibroblast during ex vivo expansion

Periodontitis is characterized by the chronic inflammation and destruction of tooth-supporting tissues. Periodontal ligament stem cell (PDLSC) is the mesenchymal stem cell (MSC) population isolated from periodontal ligament, which is the key tissue for regeneration of periodontal tissues. Although transplantation of PDLSCs is proposed as novel regenerative therapy, limited information is available, regarding the characteristic change of PDLSCs during ex vivo expansion. In this study, we encountered morphological change of PDLSCs during standard cell culture and aimed to investigate the change of PDLSCs in stem cell characteristics and to search for the culture condition to maintain stem cell properties. Characteristics of PDLSCs were examined using in vitro osteoblast and adipocyte differentiation. Myofibroblast differentiation was confirmed using immunohistochemistry and collagen gel contraction assay. Replicative senescence was examined by β-gal staining. PDLSCs changed their morphology from spindle to flat and wide during ex vivo expansion. After the morphological change, PDLSCs showed several features of myofibroblast including extensive stress fiber formation, contraction activity, and myofibroblast marker expression. Upon the morphological change, osteoblastic and adipocyte differentiation capacity were reduced and expression of stem cell-related genes were decreased. β-Gal staining was not always correlated with the morphological change of PDLSCs. Moreover, exogenous addition of bFGF and PDGF-BB served to maintain spindle shape and osteoblastic differentiation potential of PDLSCs. This study demonstrates that spontaneous differentiation of PDLSCs during ex vivo expansion and may provide the important information of cell culture condition of PDLSCs for clinical use.     

Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity

Cell sheet engineering has been developed as an alternative approach to improve mesenchymal stem cell-mediated tissue regeneration. In this study, we found that vitamin C (Vc) was capable of inducing telomerase activity in periodontal ligament stem cells (PDLSCs), leading to the up-regulated expression of extracellular matrix type I collagen, fibronectin, and integrin β1, stem cell markers Oct4, Sox2, and Nanog as well as osteogenic markers RUNX2, ALP, OCN. Under Vc treatment, PDLSCs can form cell sheet structures because of increased cell matrix production. Interestingly, PDLSC sheets demonstrated a significant improvement in tissue regeneration compared with untreated control dissociated PDLSCs and offered an effective treatment for periodontal defects in a swine model. In addition, bone marrow mesenchymal stem cell sheets and umbilical cord mesenchymal stem cell sheets were also well constructed using this method. The development of Vc-mediated mesenchymal stem cell sheets may provide an easy and practical approach for cell-based tissue regeneration.     

Development of a serum‐free system to expand dental‐derived stem cells: PDLSCs and SHEDs

Recently, extracted teeth have been identified as a viable source of stem cells for tissue regenerative approaches. Current expansion of these cells requires incorporation of animal sera; yet, a fundamental issue underlying cell cultivation methods for cell therapy regards concerns in using animal sera. In this study, we investigated the development of a chemically defined, serum‐free media (K‐M) for the expansion of human periodontal ligament stem cells (PDLSCs) and human stem cells from exfoliated deciduous teeth (SHEDs). Proliferation assays were performed comparing cells in serum‐containing media (FBS‐M) with cells cultured in four different serum‐free medium and these demonstrated that in these medium, the cell proliferation of both cell types was significantly less than the proliferation of cells in FBS‐M. Additional proliferation assays were performed using pre‐coated fibronectin (FN) tissue culture plates and of the four serum‐free medium, only K‐M enabled PDLSCs and SHEDs to proliferate at higher rates than cells cultured in FBS‐M. Next, alkaline phosphatase activity showed that PDLSCs and SHEDs exhibited similar osteogenic potential whether cultured in K‐M or FBS‐M, and, additionally, cells retained their multipotency in K‐M as seen by expression of chondrogenic and adipogenic genes, and positive Von Kossa, Alcian blue, and Oil Red O staining. Finally, differential expression of 84 stem cell associated genes revealed that for most genes, PDLSCs and SHEDs did not differ in their expression regardless of whether cultured in K‐M or FBS‐M. Taken together, the data suggest that K‐M can support the expansion of PDLSCs and SHEDs and maintenance of their multipotency. J. Cell. Physiol. 226: 66–73, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibition of phosphatidylcholine‐specific phospholipase C prevents bone marrow stromal cell senescence in vitro

Bone marrow stromal cells (BMSCs) can proliferate in vitro and can be transplanted for treating many kinds of diseases. However, BMSCs become senescent with long‐term culture, which inhibits their application. To understand the mechanism underlying the senescence, we investigated the activity of phosphatidylcholine‐specific phospholipase C (PC‐PLC) and levels of integrin β4, caveolin‐1 and ROS with BMSC senescence. The activity of PC‐PLC and levels of integrin β4, caveolin‐1 and ROS increased greatly during cell senescence. Selective inhibition of increased PC‐PLC activity with D609 significantly decreased the number of senescence‐associated beta galactosidase positive cells in BMSCs. Furthermore, D609 restored proliferation of BMSCs and their differentiation into adipocytes. Moreover, D609 suppressed the elevated levels of integrin β4, caveolin‐1 and ROS. The data suggest that PC‐PLC is involved in senescence of BMSCs, and its function is associated with integrin β4, caveolin‐1 and ROS. J. Cell. Biochem. 108: 519–528, 2009. © 2009 Wiley‐Liss, Inc.     

Senescence determines the fate of activated rat pancreatic stellate cells

In chronic pancreatitis (CP), persistent activation of pancreatic stellate cells (PSC) converts wound healing into a pathological process resulting in organ fibrosis. Here, we have analysed senescence as a novel mechanism involved in the termination of PSC activation and tissue repair. PSC senescence was first studied in vitro by establishing long‐term cultures and by applying chemical triggers, using senescence‐associated β‐Galactosidase (SA β‐Gal) as a surrogate marker. Subsequently, susceptibility of PSC to immune cell‐mediated cytolysis was investigated employing cocultures. Using the model of dibutyltin dichloride‐induced CP in rats, appearance of senescent cells was monitored by immunohistochemistry and immunofluorescence, and correlated with the progression of tissue damage and repair, immune cell infiltration and fibrosis. The results indicated that long‐term culture and exposure of PSC to stressors (doxorubicin, H₂O₂ and staurosporine) induced senescence. Senescent PSC highly expressed CDKN1A/p21, mdm2 and interleukin (IL)‐6, but displayed low levels of α‐smooth muscle actin. Senescence increased the susceptibility of PSC to cytolysis. In CP, the number of senescent cells correlated with the severity of inflammation and the extension of fibrosis. Areas staining positive for SA β‐Gal overlapped with regions of fibrosis and dense infiltrates of immune cells. Furthermore, a close physical proximity of immune cells and activated PSC was observed. We conclude that inflammation, PSC activation and cellular senescence are timely coupled processes which take place in the same microenvironment of the inflamed pancreas. Lymphocytes may play a dual‐specific role in pancreatic fibrogenesis, triggering both the initiation of wound healing by activating PSC, and its completion by killing senescent stellate cells.     

Effect of long-term culture of mouse embryonic stem cells under low oxygen concentration as well as on glycosaminoglycan hyaluronan on cell proliferation and differentiation

Objectives: Maintaining undifferentiated stem cells in defined conditions is of critical importance to improve their in vitro culture. We have evaluated the effects of culturing mouse stem (mES) cells under physiological oxygen concentration as well as by replacing fibroblast feeder layer (mEF) with gelatin or glycosaminoglycan hyaluronan (HA), on cell proliferation and differentiation. Materials and methods: After 3 days culture or after long‐term cell culture under different conditions, levels of apoptotic cell death were determined by cell cycle and TUNEL (TdT‐mediated dUTP nick end labelling) assays and levels of cell proliferation by CFSE (5‐(and‐6)‐carboxyfluorescein diacetate succinimidyl ester) labelling. We assessed spontaneous differentiation into cardiomyocytes and mRNA expression of pluripotency and differentiation biomarkers. Results: After 3 days culture under hypoxic conditions, levels of proliferation and apoptosis of mES cells were higher, in correlation with increase in intracellular reactive oxygen species. However, when cells were continuously grown for 1 month under those conditions, the level of apoptosis was, in all cases, under 4%. Hypoxia reduced spontaneous differentiation of mES into cardiomyocytes. Long‐term culture on HA was more effective in maintaining the pluripotent state of the mES cells when compared to that on gelatin. Level of terminal differentiation was highest on mEF, intermediate on HA and lowest on gelatin. Conclusions: Our data suggest that hypoxia is not necessary for maintaining pluripotency of mES cells and appeared to be detrimental during ES differentiation. Moreover, HA may offer a valuable alternative for long‐term culture of mES cells in vitro.     

Activation of epidermal growth factor receptor signaling mediates cellular senescence induced by certain pro‐inflammatory cytokines

It is well established that inflammation in the body promotes organism aging, and recent studies have attributed a similar effect to senescent cells. Considering that certain pro‐inflammatory cytokines can induce cellular senescence, systematically evaluating the effects of pro‐inflammatory cytokines in cellular senescence is an important and urgent scientific problem, especially given the ongoing surge in aging human populations. Treating IMR90 cells and HUVECs with pro‐inflammatory cytokines identified six factors able to efficiently induce cellular senescence. Of these senescence‐inducing cytokines, the activity of five (namely IL‐1β, IL‐13, MCP‐2, MIP‐3α, and SDF‐1α) was significantly inhibited by treatment with cetuximab (an antibody targeting epidermal growth factor receptor [EGFR]), gefitinib (a small molecule inhibitor of EGFR), and EGFR knockdown. In addition, treatment with one of the senescence‐inducing cytokines, SDF‐1α, significantly increased the phosphorylation levels of EGFR, as well as Erk1/2. These results suggested that pro‐inflammatory cytokines induce cellular senescence by activating EGFR signaling. Next, we found that EGF treatment could also induce cellular senescence of IMR90 cells and HUVECs. Mechanically, EGF induced cellular senescence via excessive activation of Ras and the Ras‐BRaf‐Erk1/2 signaling axis. Moreover, EGFR activation induced IMR90 cells to secrete certain senescence‐associated secretory phenotype factors (IL‐8 and MMP‐3). In summary, we report that certain pro‐inflammatory cytokines induce cellular senescence through activation of the EGFR‐Ras signaling pathway. Our study thus offers new insight into a long‐ignored mechanism by which EGFR could regulate cellular senescence and suggests that growth signals themselves may catalyze aging under certain conditions.     

Effect of substrate stiffness on proliferation and differentiation of periodontal ligament stem cells

Objectives

The aim of this study was to understand the effect of substrate stiffness (a mechanical factor of the extracellular matrix) on periodontal ligament stem cells (PDLSCs) and its underlying mechanism.

Materials and methods

Elastic substrates were fabricated by mixing 2 components, a base and curing agent in proportions of 10:1, 20:1, 30:1 or 40:1. PDLSC morphology was observed using scanning electron microscopy (SEM). Cell proliferation and differentiation were assessed after PDLSCs was cultured on various elastic substrates. Data were analysed using one‐way ANOVA.

Results

SEM revealed variations in the morphology of PDLSCs cultured on elastic substrates. PDLSC proliferation increased with substrate stiffness (P < .05). Osteogenic differentiation of PDLSCs was higher on stiff substrates. Notch pathway markers were up‐regulated in PDLSCs cultured on stiff substrates.

Conclusions

Results suggested that the osteogenic differentiation of PDLSCs might be promoted by culturing them in a stiffness‐dependent manner, which regulates the Notch pathway. This might provide a new method of enhancing osteogenesis in PDLSCs.

     

HES6 promotes prostate cancer aggressiveness independently of Notch signalling

Notch signalling is implicated in the pathogenesis of a variety of cancers, but its role in prostate cancer is poorly understood. However, selected Notch pathway members are overrepresented in high‐grade prostate cancers. We comprehensively profiled Notch pathway components in prostate cells and found prostate cancer‐specific up‐regulation of NOTCH3 and HES6. Their expression was particularly high in androgen responsive lines. Up‐ and down‐regulating Notch in these cells modulated expression of canonical Notch targets, HES1 and HEY1, which could also be induced by androgen. Surprisingly, androgen treatment also suppressed Notch receptor expression, suggesting that androgens can activate Notch target genes in a receptor‐independent manner. Using a Notch‐sensitive Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) reporter assay, we found that basal levels of Notch signalling were significantly lower in prostate cancer cells compared to benign cells. Accordingly pharmacological Notch pathway blockade did not inhibit cancer cell growth or viability. In contrast to canonical Notch targets, HES6, a HES family member known to antagonize Notch signalling, was not regulated by Notch signalling, but relied instead on androgen levels, both in cultured cells and in human cancer tissues. When engineered into prostate cancer cells, reduced levels of HES6 resulted in reduced cancer cell invasion and clonogenic growth. By molecular profiling, we identified potential roles for HES6 in regulating hedgehog signalling, apoptosis and cell migration. Our results did not reveal any cell‐autonomous roles for canonical Notch signalling in prostate cancer. However, the results do implicate HES6 as a promoter of prostate cancer progression.     

Proteostasis failure and cellular senescence in long‐term cultured postmitotic rat neurons

Cellular senescence, a stress‐induced irreversible cell cycle arrest, has been defined for mitotic cells and is implicated in aging of replicative tissues. Age‐related functional decline in the brain is often attributed to a failure of protein homeostasis (proteostasis), largely in postmitotic neurons, which accordingly is a process distinct by definition from senescence. It is nevertheless possible that proteostasis failure and cellular senescence have overlapping molecular mechanisms. Here, we identify postmitotic cellular senescence as an adaptive stress response to proteostasis failure. Primary rat hippocampal neurons in long‐term cultures show molecular changes indicative of both senescence (senescence‐associated β‐galactosidase, p16, and loss of lamin B1) and proteostasis failure relevant to Alzheimer's disease. In addition, we demonstrate that the senescent neurons exhibit resistance to stress. Importantly, treatment of the cultures with an mTOR antagonist, protein synthesis inhibitor, or chemical compound that reduces the amount of protein aggregates relieved the proteotoxic stresses as well as the appearance of senescence markers. Our data propose mechanistic insights into the pathophysiological brain aging by establishing senescence as a primary cell‐autonomous neuroprotective response.     

Rho‐kinase inhibitor Y‐27632 facilitates the proliferation,migration and pluripotency of human periodontal ligament stem cells

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell‐based regenerative therapies, while technical challenges have limited cell yield and thus affected the success of these potential treatments. The Rho GTPases and downstream Rho kinases are central regulators of cytoskeletal dynamics during cell cycle and determine the balance between stem cells self‐renewal, lineage commitment and apoptosis. Trans‐4‐[(1R)‐aminoethyl]‐N‐(4‐pyridinyl)cylohexanecarboxamidedihydrochloride (Y‐27632), Rho‐associated kinase (ROCK) inhibitor, involves various cellular functions that include actin cytoskeleton organization, cell adhesion, cell motility and anti‐apoptosis. Here, human periodontal ligament stem cells (PDLSCs) were isolated by limiting dilution method. Cell counting kit‐8 (CCK8), 5‐ethynyl‐2′‐deoxyuridine (EdU) labelling assay, cell apoptosis assay, cell migration assay, wound‐healing assay, alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, Oil Red O staining, quantitative real‐time polymerase chain reaction (qRT‐PCR) were used to determine the effects of Y‐27632 on the proliferation, apoptosis, migration, stemness, osteogenic and adipogenic differentiation of PDLSCs. Afterwards, Western blot analysis was performed to elucidate the mechanism of cell proliferation. The results indicated that Y‐27632 significantly promoted cell proliferation, chemotaxis, wound healing, fat droplets formation and pluripotency, while inhibited ALP activity and mineral deposition. Furthermore, Y‐27632 induced PDLSCs proliferation through extracellular‐signal‐regulated kinase (ERK) signalling cascade. Therefore, control of Rho‐kinase activity may enhance the efficiency of stem cell‐based treatments for periodontal diseases and the strategy may have the potential to promote periodontal tissue regeneration by facilitating the chemotaxis of PDLSCs to the injured site, and then enhancing the proliferation of these cells and maintaining their pluripotency.     

Oestrogen retains human periodontal ligament stem cells stemness in long‐term culture

Objectives

During long‐term culture, loss of stemness is observed which greatly restricts the application of human periodontal ligament stem cells (hPDLSCs) in tissue regeneration. Oestrogen (E2) was found to significantly enhance the proliferation and osteogenic differentiation capacity in mesenchymal stem cells. Therefore, in this study, we investigated effects of E2 on hPDLSCs stemness in long‐term culture.

Materials and methods

Effects of E2 on hPDLSCs stemness were systematically evaluated. To characterize underlying the mechanisms, its effects on PI3K/AKT signalling pathway were determined.

Results

Our results showed that E2 was able to enhance the proliferation, modify cell cycle, up‐regulate stemness‐related genes expression, promote osteogenic differentiation and elevate the positive rate of CD146 and STRO‐1 over 10 passages in hPDLSCs. Importantly, PI3K/AKT signing pathway might play a role in these effects.

Conclusions

These findings suggest that E2 retains hPDLSCs stemness in long‐term culture, which might enhance its application in tissue engineering.     

Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast‐like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF‐β, fibronectin (FN), α‐SMA, and NG2. LPS also increased protein and gene expression levels of anti‐inflammatory COX‐2 and pro‐inflammatory IL‐6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS‐treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen‐stimulated proliferation of CD4⁺ and the ratio of CD4⁺CD25^high/CD4⁺CD25^low lymphocytes. LPS‐treated PDLSCs did not change the frequency of CD34⁺ and CD45⁺ cells, but decreased the frequency of CD33⁺ and CD14⁺ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU‐GM number. The results indicated that LPS‐activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features.     

HIF1α‐mediated AIMP3 suppression delays stem cell aging via the induction of autophagy

Senescence in stem cells, which occurs as a consequence of chronic responses to the environment, defines the capacity of stem cells for proliferation and differentiation as well as their potential for tissue regeneration and homeostasis maintenance. Although stem cells reside under low oxygen pressure and the availability of oxygen is known to be a crucial determinant in their fate, the key modulators in stem cell aging and the underlying mechanism have yet to be unraveled. Human placenta‐derived mesenchymal stem cells (hpMSCs) were cultured under hypoxia (3% O₂) or normoxia (21% O₂) to investigate the key factors that regulate stem cell senescence under hypoxic conditions. RNA sequencing results suggested that the expression of aminoacyl‐tRNA synthetase‐interacting multifunctional protein 3 (AIMP3, EEF1E1), an aging inducer, in the hpMSCs was dramatically repressed under hypoxia with concurrent suppression of the aging marker p16^INK4a. The hpMSCs that overexpressed AIMP3 under hypoxic conditions displayed significantly decreased proliferation and fewer stem cell characteristics, whereas the downregulation of AIMP3 ameliorated the age‐related senescence of MSCs. Consistent with the results of the hpMSCs, MSCs isolated from the adipose tissue of AIMP3‐overexpressing mice exhibited decreased stem cell functions. Interestingly, AIMP3‐induced senescence is negatively regulated by hypoxia‐inducible factor 1α (HIF1α) and positively regulated by Notch3. Furthermore, we showed that AIMP3 enhanced mitochondrial respiration and suppressed autophagic activity, indicating that the AIMP3‐associated modulation of metabolism and autophagy is a key mechanism in the senescence of stem cells and further suggesting a novel target for interventions against aging.     

Tumor Necrosis Factor‐α Stimulates Cell Proliferation in Adipose Tissue‐Derived Stromal‐Vascular Cell Culture: Promotion of Adipose Tissue Expansion by Paracrine Growth Factors

Objective: Elevated levels of tumor necrosis factor‐α (TNF‐α) protein and mRNA have been reported in adipose tissue from obese humans and rodents. However, TNF‐α has catabolic and antiadipogenic effects on adipocytes. Addressing this paradox, we tested the hypothesis that paracrine levels of TNF‐α, alone or together with insulin‐like growth factor‐I (IGF‐I), support preadipocyte development. Research Methods and Procedures: Cultured stromal‐vascular cells from rat inguinal fat depots were exposed to serum‐free media containing insulin and 0.2 nM TNF‐α, 2.0 nM TNF‐α, or 0.2 nM TNF‐α + 1.0 nM IGF‐I at different times during 7 days of culture. Results: TNF‐α inhibited adipocyte differentiation as indicated by a reduction in both immunocytochemical reactivity for the preadipocyte‐specific antigen (AD3; early differentiation marker) and glycerol‐3‐phosphate dehydrogenase activity (late differentiation marker). Early exposure (Days 1 through 3 of culture) to 0.2 nM TNF‐α did not have a long term effect on inhibiting differentiation. Continuous exposure to 0.2 nM TNF‐α from Days 1 through 7 of culture resulted in a 75% increase in cell number from control. There was a synergistic effect of 0.2 nM TNF‐α + 1 nM IGF‐I on increasing cell number by Day 7 of culture to levels greater than those observed with either treatment applied alone. Discussion: These data suggest that paracrine levels (0.2 nM) of TNF‐α alone or in combination with IGF‐I may support adipose tissue development by increasing the total number of stromal‐vascular and/or uncommitted cells within the tissue. These cells may then be recruited to become preadipocytes or may alternatively serve as infrastructure to support adipose tissue growth.