scholarly journals SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Activating Notch Signaling Via Antagonizing DNMT1

2020 ◽  
Author(s):  
BO JIA ◽  
Jun Chen ◽  
Qin Wang ◽  
Xiang Sun ◽  
Jiusong Han ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Dna Methyltransferases ◽  
Luciferase Reporter ◽  
Physical Interaction ◽  
Alizarin Red ◽  
Alp Activity

Abstract Background: Adipose-derived stem cells (ADSCs) are increasingly accepted as one of ideal seed cells for regenerative medicine for its potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin proteins 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase and plays important roles in a variety of biological processes, including cell differentiation.Methods: Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin Red Staining was performed to explore the roles of SIRT6 in the osteogenic differentiation of ADSCs. Western blot , RT-qPCR,Luciferase reporter assay and Co-Immunoprecipitation assay were applied to confirm the relationship between of Sirt6, DNA methyltransferases (DNMTs) and NOTCHs.Results: SIRT6 leads to increased alkaline phosphatase (ALP) activity, enhanced mineralization and upregulated expression of osteogenic-related genes of human adipose-derived mesenchymal stem cells (hADSCs) in vitro and in vivo. Further mechanistic studies showed that SIRT6 regulated osteogenic differentiation of hADSCs depending on its deacetylase activity. SIRT6 selectively prevents abnormal DNA methylation of NOTCH1, NOTCH2 in hADSCs by antagonizing DNMT1. DNMT1 expression was suppressed in SIRT6 overexpression hADSCs, and knockdown partially rescued abnormal DNA methylation of NOTCH1 and NOTCH2, leading to the increased capable of osteogenic differentiation. Conculsions: SIRT6 promotes the osteogenic differentiation of hADSCs.The SIRT6 protein suppresses DNMT level via physical interaction with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein,leading the activation of NOTCH1 and NOTCH2.

scholarly journals SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Activating Notch Signaling via Antagonizing DNMT1

2020 ◽  
Author(s):  
Bo Jia ◽  
Jun Chen ◽  
Qin Wang ◽  
Xiang Sun ◽  
JiuSong Han ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Dna Methyltransferases ◽  
Luciferase Reporter ◽  
Physical Interaction ◽  
Alizarin Red ◽  
Alp Activity

Abstract Background: Adipose-derived stem cells (ADSCs) are increasingly accepted as one of ideal seed cells for regenerative medicine for its potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin proteins 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase and plays important roles in a variety of biological processes, including cell differentiation.Methods: Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin Red Staining was performed to explore the roles of SIRT6 in the osteogenic differentiation of ADSCs. Western blot , RT-qPCR,Luciferase reporter assay and Co-Immunoprecipitation assay were applied to confirm the relationship between of Sirt6, DNA methyltransferases (DNMTs) and NOTCHs.Results: SIRT6 leads to increased alkaline phosphatase (ALP) activity, enhanced mineralization and upregulated expression of osteogenic-related genes of human adipose-derived mesenchymal stem cells (hADSCs) in vitro and in vivo. Further mechanistic studies showed that SIRT6 regulated osteogenic differentiation of hADSCs depending on its deacetylase activity. SIRT6 selectively prevents abnormal DNA methylation of NOTCH1, NOTCH2 in hADSCs by antagonizing DNMT1. DNMT1 expression was suppressed in SIRT6 overexpression hADSCs, and knockdown partially rescued abnormal DNA methylation of NOTCH1 and NOTCH2, leading to the increased capable of osteogenic differentiation. Conculsions: SIRT6 promotes the osteogenic differentiation of hADSCs.The SIRT6 protein suppresses DNMT level via physical interaction with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein,leading the activation of NOTCH1 and NOTCH2.

scholarly journals SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Antagonizing DNMT1

2021 ◽  
Vol 9 ◽  
Author(s):  
Bo Jia ◽  
Jun Chen ◽  
Qin Wang ◽  
Xiang Sun ◽  
Jiusong Han ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Notch Signaling ◽  
Osteogenic Differentiation ◽  
Dna Methyltransferases ◽  
Luciferase Reporter ◽  
Calcium Deposition ◽  
Alizarin Red

BackgroundAdipose-derived stem cells (ADSCs) are increasingly used in regenerative medicine because of their potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin protein 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that plays important roles in cell differentiation. NOTCH signaling has also been reported to involve in osteogenic differentiation. However, the function of SIRT6 in osteogenic differentiation of ADSCs and its relation to the NOTCH signaling pathways are yet to be explored.MethodsThe in vitro study with human ADSCs (hADSCs) and in vivo experiments with nude mice have been performed. Alkaline phosphatase (ALP) assays and ALP staining were used to detect osteogenic activity. Alizarin Red staining was performed to detect calcium deposition induced by osteogenic differentiation of ADSCs. Western blot, RT-qPCR, luciferase reporter assay, and co-immunoprecipitation assay were applied to explore the relationship between of SIRT6, DNA methyltransferases (DNMTs) and NOTCHs.ResultsSIRT6 promoted ALP activity, enhanced mineralization and upregulated expression of osteogenic-related genes of hADSCs in vitro and in vivo. Further mechanistic studies showed that SIRT6 deacetylated DNMT1, leading to its unstability at protein level. The decreased expression of DNMT1 prevented the abnormal DNA methylation of NOTCH1 and NOTCH2, resulting in the upregulation of their transcription. SIRT6 overexpression partially suppressed the abnormal DNA methylation of NOTCH1 and NOTCH2 by antagonizing DNMT1, leading to an increased capacity of ADSCs for their osteogenic differentiation.ConclusionThis study demonstrates that SIRT6 physical interacts with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein, leading to the activation of NOTCH1 and NOTCH2, Which in turn promotes the osteogenic differentiation of ADSCs.

scholarly journals Thrombin-activated platelet rich plasma (PRP) enhanced osteogenic differentiation of bone marrow mesenchymal stem cells by activing autophagy through miR-140-3p/SPRED2 axis

2020 ◽  
Author(s):  
Shuting Jiang ◽  
Hongyan Liu ◽  
Weiyan Zhu ◽  
Hui Yan ◽  
Beizhan Yan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells

Abstract Background Mesenchymal stem cells transplantation gradually become a potential treatment for bone defect in clinic practice. This study aimed to investigate the molecular mechanism of PRP and autophagy for osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). Methods Thrombin activated PRP was prepared and the BMSCs were treated with activated PRP with different concentration and transfected with miR-140-3p vector (mimics or inhibitor), si-SPRED2 or co-transfected with miR-140-3p inhibitor and si-SPRED2, respectively. qRT-PCR and Western blotting were used to determine the mRNA expression and protein expression. A luciferase reporter assay was conducted to identified the targeting relationship between iR-140-3p and SPRED2 Subsequently, cell proliferation was detected by MTT and ALP activity was also determined. Alizarin red staining was used for the evaluating the formation of calcium nodules. Results MiR-140-3p expression was found to be inhibited by PRP in a dose-dependent manner, besides, cell proliferation, ALP activity, the expression of COL-I, OPN, Runx2 and OCN, and the formation of calcium nodules related to osteogenic differentiation were enhanced by PRP. Subsequently, we found that PRP activated autophagy and up-regulated SPRED2 expression in BMSCs through suppressing miR-140-3p expression. Moreover, we confirmed that miR-140-3p targeted SPRED2 and negatively regulation its expression. Finally, the findings showed that inhibition of miR-140-3p enhanced cell proliferation, osteogenic differentiation and autophagy of BMSCs by negatively regulating SPRED2 expression. Conclusion Thrombin activated PRP accelerated osteogenic differentiation of BMSCs by activing autophagy through miR-140-3p/SPRED2 axis.

scholarly journals Macromolecule Biomaterials Promote the Osteogenic Differentiation Capacity of Equine Adipose -Derived Mesenchymal Stem Cells

2020 ◽  
Author(s):  
Mohamed I. Elashry ◽  
Nadine Baulig ◽  
Alena-Svenja Wagner ◽  
Michele C. Klymiuk ◽  
Benjamin Kruppke ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Fluid Shear Stress ◽  
Basal Medium ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Alp Activity

Abstract Background: Combination of mesenchymal stem cells (MSCs) and biomaterials is a rapidly growing approach in regenerative medicine particularly for chronic degenerative disorders including osteoarthritis and osteoporosis. In the present study, the effect of biomaterial bone substitutes on equine adipose derived MSCs morphology, viability, adherence, migration and osteogenic differentiation were investigated. Methods: MSCs were cultivated in conjunction with collagen CultiSpher-S Microcarrier (MC), nanocomposite xerogels B30 and B30Str biomaterials in osteogenic differentiate medium either under static or mechanical fluid shear stress (FSS) culture conditions. The data were generated by histological means, life cell imaging, cell viability, adherence and migration assays. Osteogenic differentiation was detected by semi-quantification of alkaline phosphatase (ALP) activity, matrix mineralization using Alizarin Red S (ARS) staining and quantification of the osteogenic markers; runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) expression using RT-qPCR. All data were statistically analyzed using ANOVA. Results: The data revealed that combined mechanical stress with MC but not B30 enhanced MSCs viability and promoted their migration. Combined osteogenic medium with MC, B30 and B30Str increased ALP activity compared to cultivation in basal medium. Osteogenic induction with MC, B30 and B30Str resulted in diffused matrix mineralization by means of ARS. FSS increased the viability in the presence of the osteogenic medium with MC but not B30 or B30Str. FSS enhanced osteogenic differentiation in the presence of B30Str. Upregulation of Runx2 and ALP expression was detected with osteogenic differentiation together with B30 and B30Str regardless of static or FSS culture. Conclusions: Taken together, the data revealed that FSS in conjunction with biomaterials promoted osteogenic differentiation of MSCs. This combination may be considered as a marked improvement for clinical applications to cure bone defects.

miRNA-126 Inhibits Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells (BMSCs)

2022 ◽  
Vol 12 (4) ◽  
pp. 794-799
Author(s):  
Le Chang ◽  
Wei Duan ◽  
Chuang Wang ◽  
Jian Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Mrna Level ◽  
Alizarin Red ◽  
Runx2 Expression ◽  
Alp Activity ◽  
Smad4 Protein ◽  
Marrow Mesenchymal Stem Cells

This study was to determine whether microRNA (miRNA)-126 regulates osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Rat BMSCs were extracted and stimulated for osteogenic differentiation. Functional experiments were conducted to assess miR-126’s impact on BMSCs differentiation. Western blot and RT-qPCR determined miR-126 expression. ALP activity detection and alizarin red staining detection were also performed. After osteogenic differentiation of BMSCs, miR-126 expression was gradually decreased over time. Overexpression of miR-26 decreased ALP activity, Notch signaling activity as well as declined Runx2 expression and calcium Salt nodules after treatment. Importantly, we found that Smad4 serves as a target of miR-126 while upregulation of the miRNA was accompanied with the decreased Smad4 protein expression without affecting the Smad4 mRNA level. In conclusion, miR-126 restrains osteogenic differentiation through inhibition of SMAD4 signaling, providing a novel insight into the mechanism.

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

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.

scholarly journals Two Transcripts of FBXO5 Promote Migration and Osteogenic Differentiation of Human Periodontal Ligament Mesenchymal Stem Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Lin Liu ◽  
Kun Liu ◽  
Yanzhe Yan ◽  
Zhuangzhuang Chu ◽  
Yi Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Tissue Regeneration ◽  
Periodontal Ligament ◽  
Periodontal Tissue ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Alp Activity ◽  
Periodontal Tissue Regeneration

Objectives. Enhanced migration and osteogenic differentiation of mesenchymal stem cells (MSCs) are beneficial for MSC-mediated periodontal tissue regeneration, a promising method for periodontitis treatment. FBXO5, a member of the F-box protein family, is involved in the osteogenic differentiation of MSCs. Here, we investigated the effect of FBXO5 on human periodontal ligament stem cells (hPDLSCs). Materials and Methods. hPDLSCs were isolated from periodontal ligament tissue. Lentivirus FBXO5 shRNA was used to silence FBXO5 expression. Two transcripts of FBXO5 were overexpressed and transduced into hPDLSCs via retroviral infection. Migration and osteogenic differentiation of hPDLSCs were evaluated using the scratch migration assay, alkaline phosphatase (ALP) activity, ALP staining, alizarin red staining, western blotting, and real-time polymerase chain reaction. Results. The expression of FBXO5 was upregulated after osteogenic induction in hPDLSCs. FBXO5 knockdown attenuated migration, inhibited ALP activity and mineralization, and decreased RUNX2, OSX, and OCN expression, while the overexpression of two transcript isoforms significantly accelerated migration, enhanced ALP activity and mineralization, and increased RUNX2, OSX, and OCN expression in hPDLSCs. Conclusions. Both isoforms of FBXO5 promoted the migration and osteogenic differentiation potential of hPDLSCs, which identified a potential target for improving periodontal tissue regeneration.

scholarly journals Polypeptides IGF-1C and P24 synergistically promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro through the p38 and JNK signaling pathways

2021 ◽  
Author(s):  
Gaoying Ran ◽  
Wei Fang ◽  
Lifang Zhang ◽  
Yuting Peng ◽  
Jiatong Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Bone Morphogenetic Protein 2 ◽  
Cell Counting ◽  
Signal Pathways ◽  
Alizarin Red ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells

Objectives: Insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein 2 (BMP-2) both promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs). IGF-1C, the C domain peptide of IGF-1, and P24, a BMP-2-derived peptide, both have similar biological activities as their parent growth factors. This study aimed to investigate the effects and their mechanisms of polypeptides IGF-1C and P24 on the osteogenic differentiation of BMSCs. Methods: The optimum concentrations of IGF-IC and P24 were explored. The effects of the two polypeptides on the proliferation and osteogenic differentiation of BMSCs were examined using the Cell Counting Kit-8 (CCK-8), Alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red S staining, qPCR, and western blotting. In addition, specific pathway inhibitors were utilized to explore whether p38 and JNK pathways were involved in this process. Results: The optimal concentrations of action were both 50 g/ml. IGF-1C and P24 synergistically promoted the proliferation of BMSCs, increased ALP activity and the formation of calcified nodules and upregulated the mRNA and protein levels of osterix (Osx), runt-related transcription factor 2 (Runx2), and osteocalcin (Ocn), phosphorylation level of p38 and JNK proteins also improved. Inhibition of the pathways significantly reduced the activation of p38 and JNK, blocked the expression of Runx2 while inhibiting ALP activity and the formation of calcified nodules. Conclusions: These findings suggest IGF-1C and P24 synergistically promote the osteogenesis of BMSCs through activation of p38 and JNK signal pathways.

scholarly journals Osteogenic differentiation of rat bone mesenchymal stem cells cultured on poly (hydroxybutyrate-co-hydroxyvalerate), poly (ε-caprolactone) scaffolds

2021 ◽  
Vol 32 (11) ◽  
Author(s):  
Ana A. Rodrigues ◽  
Nilza A. Batista ◽  
Sônia M. Malmonge ◽  
Suzan A. Casarin ◽  
José Augusto M. Agnelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Mtt Assay ◽  
Vero Cells ◽  
Sem Analysis ◽  
Bone Mesenchymal Stem Cells ◽  
Alizarin Red ◽  
Cytochemical Study ◽  
Alp Activity

AbstractBioresorbable biomaterials can fill bone defects and act as temporary scaffold to recruit MSCs to stimulate their differentiation. Among the different bioresorbable polymers studied, this work focuses on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL). Were prepared blends of PHBV and PCL to obtain PHBV based biomaterials with good tenacity, important for bone tissue repair, associated with biocompatible properties of PCL. This study assesses the viability of Vero cells on scaffolds of PHBV, PCL, and their blends and the osteogenic differentiation of mesenchymal stem cells (MSCs). Materials were characterized in surface morphology, DSC and Impact Strength (IS). Vero cells and MSCs were assessed by MTT assay, cytochemical and SEM analysis. MSC osteogenic differentiation was evaluated through alizarin red staining and ALP activity. We found some roughness onto surface materials. DSC showed that the blends presented two distinct melting peaks, characteristic of immiscible blends. IS test confirmed that PHBV-PCL blends is an alternative for increase the tenacity of PHBV. MTT assay showed cells with high metabolic activities on extract toxicity test, but with low activity in the direct contact test. SEM analysis showed spreading cells with irregular and flattened morphology on different substrates. Cytochemical study revealed that MSCs maintained their morphology, although in smaller number for MSCs. The development of nodules of mineralized organic matrix in MSC cultures was identified by alizarin red staining and osteogenic differentiation was confirmed by the quantification of ALP activity. Thus, our scaffolds did not interfere on viability of Vero cells or the osteogenic differentiation of MSCs.

scholarly journals Thymoquinone loading into hydroxyapatite/alginate scaffolds accelerated the osteogenic differentiation of the mesenchymal stem cells

2021 ◽  
Author(s):  
Ebrahim Rahmani-Moghadam ◽  
Tahereh Talaei-Khozani ◽  
Vahideh Zarrin ◽  
Zahra Vojdani
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Physical And Mechanical Properties ◽  
Calcium Deposition ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Alp Activity ◽  
Sintering Method

Abstract Background: Hydroxyapatite (HA) can be loaded by some osteogenic inducing agents such as thymoquinone (TQ) and alginate. This study was performed to investigate the effect of TQ loading into HA/alginate scaffolds on osteogenic differentiation capability of mesenchymal stem cells.Methods: HA scaffolds were fabricated by casting and sintering method and impregnated by TQ containing alginate. The stem cells were loaded onto the scaffolds and induced to differentiate into osteoblasts. Alkaline Phosphatase (ALP) activity, Alizarin Red S, Real-Time qRT-PCR, and MTT assessments were done. Finally, the cells were examined with a light microscope, confocal microscope, and SEM.Results: The results showed that the presence of the alginate decelerates the degradation rate and reinforces the mechanical strength. while the presence of TQ had no significant influence on physical and mechanical properties of the HA/alginate scaffolds, it led to a significant increase in ALP activity and expression of collagen, osteopontin, and osteocalcin at early phase of differentiation. Also, TQ administration had no impact on calcium deposition and proliferation as well as bone-marker expression at long term differentiation.Conclusion: TQ accelerates the differentiation of the stem cells into the osteoblasts without changing the properties of the scaffolds, and the HA/alginate/TQ scaffold can be used as a scaffold with osteogenic properties in bone tissue engineering applications.

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