Valproic acid, A Potential Inducer of Osteogenesis in Mouse Mesenchymal Stem Cells

Background: Recent reports have unveiled the potential of flavonoids to enhance bone formation and assuage bone resorption due to their involvement in cell signaling pathways. They also act as an effective alternative to circumvent the disadvantages associated with existing treatment methods, which has increased their scope in orthopedic research. Valproic acid (VA, 2-propylpentanoic acid) is one such flavonoid, obtained from an herbaceous plant, used in the treatment of epilepsy and various types of seizures. Objective: In this study, the role of VA in osteogenesis and the molecular mechanisms underpinning its action in mouse mesenchymal stem cells (mMSCs) were determined. Methods & Results: Cytotoxic studies validated VA’s amiable nature in mMSCs. Alizarin red and von Kossa staining results showed an increased deposition of calcium phosphate in VA-treated mMSCs, which confirmed the occurrence of osteoblast differentiation and mineralization at a cellular level. At the molecular level, there were increased levels of expression of Runx2, a vital bone transcription factor, and other major osteoblast differentiation marker genes in the VAtreated mMSCs. Further, VA-treatment in mMSCs upregulated mir-21 and activated the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway, which might be essential for the expression/activity of Runx2. Conclusion: Thus, the current study confirmed the osteoinductive nature of VA at the cellular and molecular levels, opening the possibility for its application in bone therapeutics with mir-21.

Download Full-text

Melatonin Reverses the Loss of Stemness Induced by TNF-α in Human Bone Marrow Mesenchymal Stem Cells through Upregulation of YAP Expression

Stem Cells International ◽

10.1155/2019/6568394 ◽

2019 ◽

Vol 2019 ◽

pp. 1-16 ◽

Cited By ~ 4

Author(s):

Xudong Wang ◽

Tongzhou Liang ◽

Jincheng Qiu ◽

Xianjian Qiu ◽

Bo Gao ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Molecular Mechanisms ◽

Inflammatory Factor ◽

Cell Transplant ◽

Alizarin Red ◽

Marrow Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration and disease treatment. However, long-term in vitro culture results in loss of MSC stemness. The inflammation that occurs at stem cell transplant sites (such as that resulting from TNF-α) is a contributing factor for stem cell treatment failure. Currently, there is little evidence regarding the protective role of melatonin with regard to the negative effects of TNF-α on the stemness of MSCs. In this study, we report a melatonin-based method to reduce the inflammatory effects on the stemness of bone marrow mesenchymal stem cells (BMMSCs). The results of colony formation assays, Alizarin red staining, western blotting, and reverse transcription-polymerase chain reactions suggest that melatonin can reverse the inflammatory damage caused by TNF-α treatment in the third, seventh, and tenth generations of primary BMMSCs (vs. control and the TNF-α-treated group). Meanwhile, a detailed analysis of the molecular mechanisms showed that the melatonin receptor and YAP signaling pathway are closely related to the role that melatonin plays in negative inflammatory effects against BMMSCs. In addition, in vivo experiments showed that melatonin could reverse the damage caused by TNF-α on bone regeneration by BMMSCs in nude mice. Overall, our results suggest that melatonin can reverse the loss of stemness caused by inflammatory factor TNF-α in BMMSCs. Our results also provide a practical strategy for the application of BMMSCs in tissue engineering and cell therapy.

Download Full-text

MicroRNA-346-5p Regulates Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Inhibiting Transmembrane Protein 9

BioMed Research International ◽

10.1155/2020/8822232 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7

Author(s):

Yicai Zhang ◽

Yi Sun ◽

Jinlong Liu ◽

Yu Han ◽

Jinglong Yan

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Protein Level ◽

Molecular Mechanisms ◽

Transmembrane Protein ◽

Luciferase Reporter ◽

Alizarin Red ◽

Protein Levels

The molecular mechanisms how bone marrow-derived mesenchymal stem cells (BMSCs) differentiate into osteoblast need to be investigated. MicroRNAs (miRNAs) contribute to the osteogenic differentiation of BMSCs. However, the effect of miR-346-5p on osteogenic differentiation of BMSCs is not clear. This study is aimed at elucidating the underlying mechanism by which miR-346-5p regulates osteogenic differentiation of human BMSCs. Results of alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining indicated that upregulation of miR-346-5p suppressed osteogenic differentiation of BMSCs, whereas downregulation of miR-346-5p enhanced this process. The protein levels of the osteoblastic markers Osterix and Runt-related transcription factor 2 (Runx2) were decreased in cells treated with miR-346-5p mimic at day 7 and day 14 after being differentiated. By contrast, downregulation of miR-346-5p elevated the protein levels of Osterix and Runx2. Moreover, a dual-luciferase reporter assay revealed that Transmembrane Protein 9 (TMEM9) was a target of miR-346-5p. In addition, the Western Blot results demonstrated that the TMEM9 protein level was significantly reduced by the miR-346-5p mimic whereas downregulation of miR-346-5p improved the protein level of TMEM9. These results together demonstrated that miR-346-5p served a key role in BMSC osteogenic differentiation of through targeting TMEM9, which may provide a novel target for clinical treatments of bone injury.

Download Full-text

Effects of Severe Hypoxia on Bone Marrow Mesenchymal Stem Cells Differentiation Potential

Stem Cells International ◽

10.1155/2013/232896 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 50

Author(s):

Claudia Cicione ◽

Emma Muiños-López ◽

Tamara Hermida-Gómez ◽

Isaac Fuentes-Boquete ◽

Silvia Díaz-Prado ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Molecular Mechanisms ◽

Control Point ◽

Severe Hypoxia ◽

Marker Genes ◽

Differentiation Potential ◽

Differentiation Capacity ◽

Hypoxic Conditions

Background. The interests in mesenchymal stem cells (MSCs) and their application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently hypoxia has been indicated as crucial for complete chondrogenesis. We aimed at analyzing bone marrow MSCs (BM-MSCs) differentiation capacity under normoxic and severe hypoxic culture conditions.Methods. MSCs were characterized by flow cytometry and differentiated towards adipocytes, osteoblasts, and chondrocytes under normoxic or severe hypoxic conditions. The differentiations were confirmed comparing each treated point with a control point made of cells grown in DMEM and fetal bovine serum (FBS).Results. BM-MSCs from the donors displayed only few phenotypical differences in surface antigens expressions. Analyzing marker genes expression levels of the treated cells compared to their control point for each lineage showed a good differentiation in normoxic conditions and the absence of this differentiation capacity in severe hypoxic cultures.Conclusions. In our experimental conditions, severe hypoxia affects thein vitrodifferentiation potential of BM-MSCs. Adipogenic, osteogenic, and chondrogenic differentiations are absent in severe hypoxic conditions. Our work underlines that severe hypoxia slows cell differentiation by means of molecular mechanisms since a decrease in the expression of adipocyte-, osteoblast-, and chondrocyte-specific genes was observed.

Download Full-text

ID:2050 Impacts of polymer-based and graphene-based biomaterials on the characteristics of Wharton jelly’s-derived mesenchymal stem cells

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.284 ◽

2017 ◽

Vol 4 (S) ◽

pp. 80

Author(s):

Hiew Vun Vun

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Molecular Mechanisms ◽

Surface Markers ◽

Rt Pcr ◽

Alizarin Red ◽

Runx2 Expression ◽

Gene Profiles ◽

Different Types ◽

Different Characteristics

The combination of mesenchymal stem cells (MSCs) and biomaterials scaffolds hold significant promise in tissue engineering and regenerative medicine field. In recent years, expansion and differentiation of MSCs culturing with polymer-based and graphene-based biomaterials have been intensely studied. However, the underlying molecular mechanisms are still largely unknown. This study was to examine the different characteristics of Wharton’s jelly (WJ)-derived MSCs using two different types of biomaterials including polymer-based (PU157) and graphene oxide-based (GO). In term of proliferation, WJ-MSCs culturing with PU157 and GO showed no significant differences compared to the controls. PU157 and GO had no effects on morphological features of WJ-MSCs. RT-PCR results showed that all WJ-MSCs co-culturing with or without PU157 and GO expressed positive surface markers including CD29, CD44, CD73, CD90, CD105, CD106 and CD166 but the expression of negative markers (CD34, CD45 and CD133) was undetectable. The SOX2 and RUNX2 expression were both upregulated in WJ-MSCs combining with PU157 when compared to the controls at passage 6, where no obvious difference was observed in passage 3. Interestingly, COL2A expression became more noticeable in the presence of PU157 at passage 6 than passage 3. In contrast, GO did not alter the expression of stemness, osteogenic, adipogenic and chondrogenic genes. Both alizarin red and oil red staining showed calcium and lipid deposition in WJ-MSCs co-cultured with PU157 and GO respectively, suggesting the presence of osteoblast- and adipocyte-like cells. In conclusion, PU157 exerted certain effects on the expression gene profiles at later passage and able to enhance differentiation abilities in WJ-MSCs.

Download Full-text

Enhancing the Therapeutic Potential of Mesenchymal Stem Cells with Light-Emitting Diode: Implications and Molecular Mechanisms

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/6663539 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Barbara Sampaio Dias Martins Mansano ◽

Vitor Pocani da Rocha ◽

Ednei Luiz Antonio ◽

Daniele Fernanda Peron ◽

Rafael do Nascimento de Lima ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Protein Kinase ◽

Molecular Mechanisms ◽

Therapeutic Potential ◽

Light Emitting Diode ◽

Janus Kinase ◽

Mitogen Activated Protein Kinase ◽

Light Emitting ◽

Report Quality

This study evaluated the effects of light-emitting diode (LED) on mesenchymal stem cells (MSCs). An electronic search was conducted in PubMed/MEDLINE, Scopus, and Web of Science database for articles published from 1980 to February 2020. Ten articles met the search criteria and were included in this review. The risk of bias was evaluated to report quality, safety, and environmental standards. MSCs were derived from adipose tissue, bone marrow, dental pulp, gingiva, and umbilical cord. Protocols for cellular irradiation used red and blue light spectrum with variations of the parameters. The LED has been shown to induce greater cellular viability, proliferation, differentiation, and secretion of growth factors. The set of information available leads to proposing a complex signaling cascade for the action of photobiomodulation, including angiogenic factors, singlet oxygen, mitogen-activated protein kinase/extracellular signal-regulated protein kinase, Janus kinase/signal transducer, and reactive oxygen species. In conclusion, although our results suggest that LED can boost MSCs, a nonuniformity in the experimental protocol, bias, and the limited number of studies reduces the power of systematic review. Further research is essential to find the optimal LED irradiation parameters to boost MSCs function and evaluate its impact in the clinical setting.

Download Full-text

Exosomes from bone marrow mesenchymal stem cells promoted osteogenic differentiation by delivering miR-196a that targeted Dkk1 to activate Wnt/β-catenin pathway

10.21203/rs.3.rs-25044/v1 ◽

2020 ◽

Author(s):

Zhi Peng ◽

Zhenkai Lou ◽

Zhongjie Li ◽

Shaobo Li ◽

Kaishun Yang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Western Blot ◽

Osteogenic Differentiation ◽

Osteoblast Differentiation ◽

Negative Regulator ◽

Luciferase Assay ◽

Alizarin Red ◽

Marrow Mesenchymal Stem Cells

Abstract Background: Osteoporosis is the most common bone metabolic disease. Emerging evidence suggests that exosomes are secreted by diverse cells such as bone marrow mesenchymal stem cells (BMSCs), and play important role in cell-to-cell communication and tissue homeostasis. Recently, the discovery of exosomes has attracted attention in the field of bone remodeling. Methods: The exosomes were extracted from BMSCs and labeled by PKH-67, and then incubated with hFOB1.19 cells to investigate the miR-196a function on the osteoblast differentiation of hFOB1.19. The osteoblast differentiation was detected via alizarin red staining and the expression of osteoblast genes were detected by western blot. The cell apoptosis was detected by flow cytometer. The target relationship of miR-196a and Dickkopf-1 (Dkk1) were verified by luciferase assay and western blot. Results: Here we demonstrated that exosomes extracted from BMSCs (BMSC-exo) significantly promoted hFOB1.19 differentiation to osteoblasts. We found that BMSC-exo were enriched with miR-196a and delivered miR-196a to hFOB1.19 cells to inhibit its target Dkk1, which is a negative regulator of Wnt/β-catenin pathway. Conclusion: BMSC-exo activated Wnt/β-catenin pathway to promote osteogenic differentiation, while BMSC-exo failed to exert the effects when miR-196a was deprived. In conclusion, miR-196a delivered by exosomes from BMSCs plays an essential role in enhancing osteoblastic differentiation by targeting Dkk1 to activate Wnt/β-catenin pathway.

Download Full-text

JAK2 Inhibition by Fedratinib Reduces Osteoblast Differentiation and Mineralisation of Human Mesenchymal Stem Cells

Molecules ◽

10.3390/molecules26030606 ◽

2021 ◽

Vol 26 (3) ◽

pp. 606

Author(s):

Nihal AlMuraikhi ◽

Hanouf Alaskar ◽

Sarah Binhamdan ◽

Amal Alotaibi ◽

Moustapha Kassem ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Molecular Mechanisms ◽

Osteoblastic Differentiation ◽

Molecular Signature ◽

Signalling Pathways ◽

Alizarin Red ◽

Jak2 Inhibitor

Several signalling pathways, including the JAK/STAT signalling pathway, have been identified to regulate the differentiation of human bone marrow skeletal (mesenchymal) stem cells (hBMSCs) into bone-forming osteoblasts. Members of the JAK family mediate the intracellular signalling of various of cytokines and growth factors, leading to the regulation of cell proliferation and differentiation into bone-forming osteoblastic cells. Inhibition of JAK2 leads to decoupling of its downstream mediator, STAT3, and the subsequent inhibition of JAK/STAT signalling. However, the crucial role of JAK2 in hBMSCs biology has not been studied in detail. A JAK2 inhibitor, Fedratinib, was identified during a chemical biology screen of a small molecule library for effects on the osteoblastic differentiation of hMSC-TERT cells. Alkaline phosphatase activity and staining assays were conducted as indicators of osteoblastic differentiation, while Alizarin red staining was used as an indicator of in vitro mineralised matrix formation. Changes in gene expression were assessed using quantitative real-time polymerase chain reaction. Fedratinib exerted significant inhibitory effects on the osteoblastic differentiation of hMSC-TERT cells, as demonstrated by reduced ALP activity, in vitro mineralised matrix formation and downregulation of osteoblast-related gene expression, including ALP, ON, OC, RUNX2, OPN, and COL1A1. To identify the underlying molecular mechanisms, we examined the effects of Fedratinib on a molecular signature of several target genes known to affect hMSC-TERT differentiation into osteoblasts. Fedratinib inhibited the expression of LIF, SOCS3, RRAD, NOTCH3, TNF, COMP, THBS2, and IL6, which are associated with various signalling pathways, including TGFβ signalling, insulin signalling, focal adhesion, Notch Signalling, IL-6 signalling, endochondral ossification, TNF-α, and cytokines and inflammatory response. We identified a JAK2 inhibitor (Fedratinib) as a powerful inhibitor of the osteoblastic differentiation of hMSC-TERT cells, which may be useful as a therapeutic option for treating conditions associated with ectopic bone formation or osteosclerotic metastases.

Download Full-text

Leonurine Promotes the Osteoblast Differentiation of Rat BMSCs by Activation of Autophagy via the PI3K/Akt/mTOR Pathway

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.615191 ◽

2021 ◽

Vol 9 ◽

Author(s):

Bingkun Zhao ◽

Qian Peng ◽

Enoch Hin Lok Poon ◽

Fubo Chen ◽

Rong Zhou ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Western Blot ◽

Osteogenic Differentiation ◽

Osteoblast Differentiation ◽

Mtor Pathway ◽

Control Group ◽

Alizarin Red ◽

Qrt Pcr

BackgroundLeonurine, a major bioactive component from Herba leonuri, has been shown to exhibit anti-inflammatory and antioxidant effects. The aim of this study was to investigate the effect of leonurine on bone marrow-derived mesenchymal stem cells (BMSCs) as a therapeutic approach for treating osteoporosis.Materials and MethodsRat bone marrow-derived mesenchymal stem cells (rBMSCs) were isolated from 4-weeks-old Sprague–Dawley rats. The cytocompatibility of leonurine on rBMSCs was tested via CCK-8 assays and flow cytometric analyses. The effects of leonurine on rBMSC osteogenic differentiation were analyzed via ALP staining, Alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Additionally, autophagy-related markers were examined via qRT-PCR and Western blot analyses of rBMSCs during osteogenic differentiation with leonurine and with or without 3-methyladenine (3-MA) as an autophagic inhibitor. Finally, the PI3K/Akt/mTOR signaling pathway was evaluated during rBMSC osteogenesis.ResultsLeonurine at 2–100 μM promoted the proliferation of rBMSCs. ALP and Alizarin red staining results showed that 10 μM leonurine promoted rBMSC osteoblastic differentiation, which was consistent with the qRT-PCR and Western blot results. Compared with those of the control group, the mRNA and protein levels of Atg5, Atg7, and LC3 were upregulated in the rBMSCs upon leonurine treatment. Furthermore, leonurine rescued rBMSC autophagy after inhibition by 3-MA. Additionally, the PI3K/AKT/mTOR pathway was activated in rBMSCs upon leonurine treatment.ConclusionLeonurine promotes the osteoblast differentiation of rBMSCs by activating autophagy, which depends on the PI3K/Akt/mTOR pathway. Our results suggest that leonurine may be a potential treatment for osteoporosis.

Download Full-text

T-Box20 inhibits osteogenic differentiation in adipose-derived human mesenchymal stem cells: the role of T-Box20 on osteogenesis

Journal of Biological Research-Thessaloniki ◽

10.1186/s40709-019-0099-5 ◽

2019 ◽

Vol 26 (1) ◽

Author(s):

Samaneh Mollazadeh ◽

Bibi Sedigheh Fazly Bazzaz ◽

Vajiheh Neshati ◽

Antoine A. F. de Vries ◽

Hojjat Naderi-Meshkin ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Transcription Factors ◽

Osteogenic Differentiation ◽

Heart Development ◽

Fluorescent Protein ◽

Lentiviral Vector ◽

Human Mesenchymal Stem Cells ◽

Marker Genes ◽

Alizarin Red

Abstract Background Skeletal development and its cellular function are regulated by various transcription factors. The T-box (Tbx) family of transcription factors have critical roles in cellular differentiation as well as heart and limbs organogenesis. These factors possess activator and/or repressor domains to modify the expression of target genes. Despite the obvious effects of Tbx20 on heart development, its impact on bone development is still unknown. Methods To investigate the consequence by forced Tbx20 expression in the osteogenic differentiation of human mesenchymal stem cells derived from adipose tissue (Ad-MSCs), these cells were transduced with a bicistronic lentiviral vector encoding Tbx20 and an enhanced green fluorescent protein. Results Tbx20 gene delivery system suppressed the osteogenic differentiation of Ad-MSCs, as indicated by reduction in alkaline phosphatase activity and Alizarin Red S staining. Consistently, reverse transcription-polymerase chain reaction analyses showed that Tbx20 gain-of-function reduced the expression levels of osteoblast marker genes in osteo-inductive Ad-MSCs cultures. Accordingly, Tbx20 negatively affected osteogenesis through modulating expression of key factors involved in this process. Conclusion The present study suggests that Tbx20 could inhibit osteogenic differentiation in adipose-derived human mesenchymal stem cells.

Download Full-text

Spheroid Culture of Mesenchymal Stem Cells

Stem Cells International ◽

10.1155/2016/9176357 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 138

Author(s):

Zoe Cesarz ◽

Kenichi Tamama

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Biology ◽

Molecular Mechanisms ◽

Replicative Senescence ◽

Marker Genes ◽

Adherent Cell ◽

Pluripotency Marker ◽

Adherent Cell Culture ◽

Microarray Analyses

Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown.

Download Full-text