• Biomedical Science Letters
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• v.22 no.1
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• pp.1-8
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• 2016

Several studies have investigated the various effects of dexamethasone (Dex) on the proliferation and differentiation of mesenchymal stem cells (MSCs). Previously, we reported that co-treatment with L-ascorbic acid 2-phosphate and fibroblast growth factor (FGF)-2 maintained differentiation potential in MSCs through expression of hepatocyte growth factor (HGF). In this study, we investigated the effects of co-treatment with FGF-2 and Dex on the proliferation and differentiation potential of MSCs during a 2-month culture period. Co-treatment with FGF-2 and Dex increased approximately a 4.7-fold higher accumulation rate of MSC numbers than that by FGF-2 single treatment during a 2-month culture period. Interestingly, co-treatment with FGF-2 and Dex increased expression of HGF and maintained adipogenic differentiation potential during this culture period. These results suggest that co-treatment with FGF-2 and Dex preserves the proliferation and differentiation potential during long-term culture.

• International Journal of Oral Biology
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• v.49 no.3
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• pp.69-78
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• 2024

Sestrin 2 (SESN2) is a member of the sestrin family of stress-induced proteins that negatively regulate aging-associated biological processes. This study aims to investigate the role of SESN2 in regulating the differentiation potential and senescence of mesenchymal stem cells (MSCs) derived from young and elderly donors. Bulk RNA sequencing revealed a common decline in the SESN2 mRNA levels in MSCs from elderly individuals, which was confirmed via reverse transcription-polymerase chain reaction and western blot analyses. SESN2 knockdown in MSCs from young donors resulted in phenotypic changes similar to those in MSCs from elderly donors, including an enhanced expression of senescence and adipogenic markers and diminished expression of osteogenic markers. To confirm the effect of decreased SESN2 expression on osteogenic and adipogenic differentiation, we induced Sesn2 knockdown in mouse bone marrow-derived MSCs. Sesn2 knockdown suppressed the mRNA expression of osteogenic marker genes, alkaline phosphatase activity, and matrix mineralization. Furthermore, Sesn2 knockdown enhanced mRNA expression of the adipogenic marker genes and intracellular lipid accumulation. These results suggest that a decline in SESN2 expression during aging contributes to the shift of MSC differentiation from osteogenic to adipogenic lineage.

• Journal of Periodontal and Implant Science
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• v.52 no.3
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• pp.242-257
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• 2022

Purpose: This study investigated periodontal ligament (PDL) restoration in osseointegrated implants using stem cells. Methods: Commercial pure titanium and zirconium oxide (zirconia) were coated with beta-tricalcium phosphate (β-TCP) using a long-pulse Nd:YAG laser (1,064 nm). Isolated bone marrow mesenchymal cells (BMMSCs) from rabbit tibia and femur, isolated PDL stem cells (PDLSCs) from the lower right incisor, and co-cultured BMMSCs and PDLSCs were tested for periostin markers using an immunofluorescent assay. Implants with 3D-engineered tissue were implanted into the lower right central incisors after extraction from rabbits. Forty implants (Ti or zirconia) were subdivided according to the duration of implantation (healing period: 45 or 90 days). Each subgroup (20 implants) was subdivided into 4 groups (without cells, PDLSC sheets, BMMSC sheets, and co-culture cell sheets). All groups underwent histological testing involving haematoxylin and eosin staining and immunohistochemistry, stereoscopic analysis to measure the PDL width, and field emission scanning electron microscopy (FESEM). The natural lower central incisors were used as controls. Results: The BMMSCs co-cultured with PDLSCs generated a well-formed PDL tissue that exhibited positive periostin expression. Histological analysis showed that the implantation of coated (Ti and zirconia) dental implants without a cell sheet resulted in a well-osseointegrated implant at both healing intervals, which was confirmed with FESEM analysis and negative periostin expression. The mesenchymal tissue structured from PDLSCs only or co-cultured (BMMSCs and PDLSCs) could form a natural periodontal tissue with no significant difference between Ti and zirconia implants, consequently forming a biohybrid dental implant. Green fluorescence for periostin was clearly detected around the biohybrid implants after 45 and 90 days. FESEM showed the invasion of PDL-like fibres perpendicular to the cementum of the bio-hybrid implants. Conclusions: β-TCP-coated (Ti and zirconia) implants generated periodontal tissue and formed biohybrid implants when mesenchymal-tissue-layered cell sheets were isolated from PDLSCs alone or co-cultured BMMSCs and PDLSCs.

• Biomolecules & Therapeutics
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• v.21 no.6
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• pp.447-453
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• 2013

Chondroitin sulfate proteoglycan (CSPG) inhibits neurite outgrowth of various neuronal cell types, and CSPG-associated inhibition of neurite outgrowth is mediated by the Rho/ROCK pathway. Mesenchymal stromal/stem cells (MSCs) have the potential to differentiate into neuron-like cells under specific conditions and have been shown to differentiate into neuron-like cells by co-treatment with the ROCK inhibitor Y27632 and the hypoxia condition mimicking agent $CoCl_2$. In this study, we addressed the hypothesis that a ROCK inhibitor might be beneficial to regenerate neurons during stem cell therapy by preventing transplanted MSCs from inhibition by CSPG in damaged tissues. Indeed, dose-dependent inhibition by CSPG pretreatment was observed during morphological changes of Wharton's jelly-derived MSCs (WJ-MSCs) induced by Y27632 alone. The formation of neurite-like structures was significantly inhibited when WJ-MSCs were pre-treated with CSPG before induction under Y27632 plus $CoCl_2$ conditions, and pretreatment with a protein kinase C inhibitor reversed such inhibition. However, CSPG treatment resulted in no significant inhibition of the WJ-MSC morphological changes into neuron-like cells after initiating induction by Y27632 plus $CoCl_2$. No marked changes were detected in expression levels of neuronal markers induced by Y27632 plus $CoCl_2$ upon CSPG treatment. CSPG also blocked the morphological changes of human bone marrow-derived MSCs into neuron-like cells under other neuronal induction condition without the ROCK inhibitor, and Y27632 pre-treatment blocked the inhibitory effect of CSPG. These results suggest that a ROCK inhibitor can be efficiently used in stem cell therapy for neuronal induction by avoiding hindrance from CSPG.

• Toxicological Research
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• v.35 no.1
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• pp.83-91
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• 2019

Nanoparticles (NPs) have been recognized as both useful tools and potentially toxic materials in various industrial and medicinal fields. Previously, we found that zinc oxide (ZnO) NPs that are neurotoxic to human dopaminergic neuroblastoma SH-SY5Y cells are mediated by lipoxygenase (LOX), not cyclooxygenase-2 (COX-2). Here, we examined whether human bone marrow-derived mesenchymal stem cells (MSCs), which are different from neuroblastoma cells, might exhibit COX-2- and/or LOX-dependent cytotoxicity of ZnO NPs. Additionally, changes in annexin V expression, caspase-3/7 activity, and mitochondrial membrane potential (MMP) induced by ZnO NPs and ZnO were compared at 12 hr and 24 hr after exposure using flow cytometry. Cytotoxicity was measured based on lactate dehydrogenase activity and confirmed by trypan blue staining. Rescue studies were executed using zinc or iron chelators. ZnO NPs and ZnO showed similar dose-dependent and significant cytotoxic effects at concentrations ${\geq}15{\mu}g/mL$, in accordance with annexin V expression, caspase-3/7 activity, and MMP results. Human MSCs exhibited both COX-2 and LOX-mediated cytotoxicity after exposure to ZnO NPs, which was different from human neuroblastoma cells. Zinc and iron chelators significantly attenuated ZnO NPs-induced toxicity. Conclusively, these results suggest that ZnO NPs exhibit both COX-2- and LOX-mediated apoptosis by the participation of mitochondrial dysfunction in human MSC cultures.

• Molecules and Cells
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• v.43 no.10
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• pp.848-855
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• 2020

Cells assemble stress granules (SGs) to protect their RNAs from exposure to harmful chemical reactions induced by environmental stress. These SGs release RNAs, which resume translation once the stress is relieved. During stem cell differentiation, gene expression is altered to allow cells to adopt various functional and morphological features necessary to differentiate. This process induces stress within a cell, and cells that cannot overcome this stress die. Here, we investigated the role of SGs in the progression of stem cell differentiation. SGs aggregated during the neuronal differentiation of human bone marrow-mesenchymal stem cells, and not in cell lines that could not undergo differentiation. SGs were observed between one and three hours post-induction; RNA translation was restrained at the same time. Immediately after disassembly of SGs, the expression of the neuronal marker neurofilament-M (NF-M) gradually increased. Assembled SGs that persisted in cells were exposed to salubrinal, which inhibited the dephosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), and in eIF2α/S51D mutant cells. When eIF2α/S51A mutant cells differentiated, SGs were not assembled. In all experiments, the disruption of SGs was accompanied by delayed NF-M expression and the number of neuronally differentiated cells was decreased. Decreased differentiation was accompanied by decreased cell viability, indicating the necessity of SGs for preventing cell death during neuronal differentiation. Collectively, these results demonstrate the essential role of SGs during the neuronal differentiation of stem cells.

• Journal of Pharmaceutical Investigation
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• v.40 no.6
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• pp.333-337
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• 2010

Combining cell- and gene-based therapy is a promising therapeutic strategy in regenerative medicine. The aim of this study was to develop genetically modified mesenchymal stem cell (MSC) aggregates using a poly(ethylene glycol) (PEG) hydrogel micro-well array technique. Stable PEG hydrogel micro-well arrays with diameters of 200 to $500\;{\mu}m$ were fabricated and used to generate genetically engineered MSC aggregates. Rat bone marrow-derived MSCs were transfected with a green fluorescent protein (GFP) plasmid as a reporter gene, and aggregated by culturing in the PEG hydrogel micro-well arrays. The resultant cell aggregates had a mean diameter of less than $200\;{\mu}m$, and maintained the mesenchymal phenotype even after genetic modification and cell aggregation. Transplantation of MSC aggregates that are genetically modified to express therapeutic or cell-survival genes may be a potential therapeutic approach for regenerative medicine.

• Journal of Periodontal and Implant Science
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• v.35 no.2
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• pp.263-269
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• 2005

Bone morphogenetic protein(BMP) and platelet-derived growth factor(PDGF) have been demonstrated tostimulate bone formation when applied locally in vivo. To explore whether or not the combined use of BMP and PDGF could have promotive effect and synergic interaction on bone formation in vivo, bone marrow mesenchymal stem cells were treated with BMP-2, PDGF-BB, or BMP-2 plus PDGF-BB, and then these cells were injected into the subcutaneous space on the dorsum of nude mice. The bone formation was evaluated after 12 weeks. Histomorphometric analysis demonstrated that the subcutaneous nodules formed in nude mice contained 25.3% newly formed bone in the BMP-2 treated cells, 14.4% newly formed bone in the PDGF-BB treated cells, and 8.9% newly formed bone in the RMP-2 plus PDGF-BB treated cells. The results showed that the combination of BMP-2 and PDGF-BB had neither a promotive effect nor synergic interact on bone formation in vivo.

• International Journal of Stem Cells
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• v.15 no.4
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• pp.405-414
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• 2022

Background and Objectives: Chronic inflammation of bone tissue often results in bone defects and hazards to tissue repair and regeneration. Cannabidiol (CBD) is a natural cannabinoid with multiple biological activities, including anti-inflammatory and osteogenic potential. This study aimed to investigate the efficacy and mechanisms of CBD in the promotion of bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation in the inflammatory microenvironment. Methods and Results: BMSCs isolated from C57BL/6 mice, expressed stem cell characteristic surface markers and presented multidirectional differentiation potential. The CCK-8 assay was applied to evaluate the effects of CBD on BMSCs' vitality, and demonstrating the safety of CBD on BMSCs. Then, BMSCs were stimulated with lipopolysaccharide (LPS) to induce inflammatory microenvironment. We found that CBD intervention down-regulated mRNA expression levels of inflammatory cytokines and promoted cells proliferation in LPS-treated BMSCs, also reversed the protein and mRNA levels downregulation of osteogenic markers caused by LPS treatment. Moreover, CBD intervention activated the cannabinoid receptor 2 (CB2) and the p38 mitogen-activated protein kinase (MAPK) signaling pathway. While AM630, a selective CB2 inhibitor, reduced phosphorylated (p)-p38 levels. In addition, AM630 and SB530689, a selective p38 MAPK inhibitor, attenuated the enhancement of osteogenic markers expression levels by CBD in inflammatory microenvironment, respectively. Conclusions: CBD promoted osteogenic differentiation of BMSCs via the CB2/p38 MAPK signaling pathway in the inflammatory microenvironment.

• Journal of Life Science
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• v.25 no.6
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• pp.724-731
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• 2015

Mesenchymal stem cells (MSCs) are characterized by their multipotency capacity, which allows them to differentiate into diverse cell types (bone, cartilage, fat, tendon, and neuron-like cells) and secrete a variety of trophic factors (ANG, FGF-2, HGF, IGF-1, PIGF, SDF-1α, TGF-β, and VEGF). MSCs can be easily isolated from human bone-marrow, fat, and umbilical-cord tissues. These features indicate that MSCs might be of use in stem-cell therapy. However, MSCs undergo cellular senescence during long-term expansion, and this is accompanied by functional declines in stem-cell potency. In the human body, because of their senescence and declines in their microenvironmental niches stem cells fail to maintain tissue homeostasis, and as a result, senescent cells accumulate in tissues. This can lead to age-related diseases, including degenerative disorders and cancers. Recent studies suggest that the number of histone modifications to stem cells’ genomes and aberrant alterations to their DNA methylation increase as stem cells progress into senescence. These epigenetic alterations have been partly reversed with treatments in which DNA methyltransferase (DNMT) inhibitors or histone deacetylase (HDAC) inhibitors are introduced into replicative senescent-MSCs. This review focuses on epigenetic alteration in replicative senescent-MSCs and explains how epigenetic modifications are widely associated with stem-cell senescences such as differentiation, proliferation, migration, calcium signaling, and apoptosis.