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

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.

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

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.

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.

Differential Expression of MicroRNA-3148 in Patients with Osteoporosis and Its Impacts on the Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells

2021 ◽  
Vol 11 (5) ◽  
pp. 957-962
Author(s):  
Ainiwaerjiang Damaola ◽  
Maerdan Aierken ◽  
Mieralimu Muertizha ◽  
Abudouaini Abudoureheman ◽  
Haishan Lin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Serum Samples ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Qrt Pcr ◽  
Marrow Mesenchymal Stem Cells ◽  
Rat Bone

We aimed to explore the effects of rat bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation via analyzing miR-3148 expression in patients with osteoporosis. Realtime quantitative PCR was conducted for assessing microRNA-3148 expression. BMSCs from SD rats were transfected with microRNA-3148 mimics and microRNA-3148 inhibitor via liposomal trans-fection method utilizing Lipo2000, followed by analysis of microRNA-3148 level. After 10-days of osteogenic differentiation induction, alkaline phosphatase (ALP) staining and alizarin red (ARS) staining were done to investigate the osteogenic differentiation potential. Simultaneously, qRT-PCR measured the expression of osteogenesis marker genes (BMP and Runx2) in each group. qRT-PCR analysis revealed a high expression of miR-3148 in the bone tissue and the serum samples from patients with osteoporosis in comparison with healthy individuals. In addition, miRNA-3148 mimics could retard the osteogenic differentiation of BMSCs, while microRNA-3148 inhibitor could prompt the procedure. MicroRNA-3148 was highly expressed in the skeletal tissues and the serum samples from patients with osteoporosis and it could restrain the differentiation of BMSCs into osteoblasts, suggesting that it might be a novel therapeutic target for treating osteoporosis.

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 Pre-Conditioned Bone Marrow Mesenchymal Stem Cells with Nisin on the Modified Poly-L-Lactic-Acid Nanofibers

2021 ◽  
Author(s):  
Fariba Sadraei ◽  
Marzieh Ghollasi ◽  
Fatemeh Khakpai ◽  
Raheleh Halabian
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Lactic Acid ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Alizarin Red ◽  
Marrow Mesenchymal Stem Cells

Abstract Background: Human bone marrow-derived mesenchymal stem (MSCs) cells are undifferentiated cells with the self-renewing ability and multi-lineage differentiation beneficial for regenerative medicine. Nano scaffolds are novel materials employed in bone repair and regeneration. Nisin is a prebiotic that can increase stem cells’ life span and proliferation. This study attempted to provide a proper strategy for bone marrow mesenchymal stem cells differentiation into the Osteocytes on a Poly‐L‐lactic‐acid scaffold (PLLA) after pretreating with probiotic Nisin. Methods: MSC osteogenic differentiation was evaluated by measuring Calcium, Alkaline phosphatase, and quantitative tests such as Real-Time PCR, Acridine Orange, Alizarin Red, Von Kossa, and others. Results: The result of the MTT test showed that the optimal dose of Nisin probiotic for the MSCs’ preconditioning was 200 IU/mL on the 1st, 3rd, and 5th days of culture. Real-time PCR data indicated that the expression rate of ALP, Osteonectin, Osteocalcin, and Collagen I have increased in the presence of Nisin, while the RUNX-2 gene expression has decreased. Furthermore, the results of Alizarin Red and Von Kossa tests, as well as Scanning electron microscopy (SEM), revealed that the cell proliferation in the preconditioned samples with Nisin increased significantly. Conclusions: The study concluded that the cell proliferation and differentiation increased in samples pretreated with Nisin on the PLLA Nano scaffolds.

scholarly journals Long non-coding RNA taurine upregulated gene 1 is downregulated in osteoporosis and influences the osteogenic differentiation of bone marrow mesenchymal stem cells

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11251
Author(s):  
Zhaowei Teng ◽  
Yun Zhu ◽  
Qinggang Hao ◽  
Xiaochao Yu ◽  
Yirong Teng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Non Coding Rna ◽  
Marrow Mesenchymal Stem Cells ◽  
Long Non Coding Rna ◽  
Taurine Upregulated Gene 1

Background With aging, an imbalance in bone remodeling leading to increased bone resorption and decreased bone formation is thought to contribute to osteoporosis. Osteoblastic differentiation of bone marrow mesenchymal stem cells (BMMSCs) plays a vital role in the pathogenesis of osteoporosis. However, the detailed molecular mechanisms of osteoporosis remain incompletely understood. Given that long non-coding RNA taurine upregulated gene 1 (lnc TUG1) plays a critical role in the osteogenic differentiation, and microRNA-23b (miR-23b) as a putative sponge for lnc TUG1 has upregulated expression in osteoporosis. Therefore, this study investigated the roles of TUG1/miR-23b in osteoporotic pathology. Material and Methods TUG1 and miR-23b expression in the plasma of osteoporotic patients were evaluated by quantitative real-time PCR (qRT-PCR). The osteogenic differentiation in human BMMSCs was evaluated by qRT-PCR, western blot, Alizarin red staining after knockdown of TUG1 by small interfering RNA (siRNA) treatment. Results Decreased expression of TUG1 and increased expression of miR-23b evident in the plasma of patients with osteoporosis than in that of age- and sex-matched healthy controls. Additionally, increased miR-23b expression inhibited runt-related transcription factor 2 (RUNX2), osteocalcin, and osteopontin expression and reduced calcified nodule formation based on the results of qRT-PCR, western blot, and Alizarin Red S staining. Conclusion The study for the first time reported that silence of lncRNA TUG1 significantly suppressed the osteogenic differentiation of BMMSCs possibly by targeting the miR-23b/RUNX2 signaling pathway. This mechanism of TUG1/miR-23b/RUNX2 signaling within the osteogenic differentiation of BMMSCs might provide new insight for the development of lncRNA-directed diagnostic and therapeutic strategies for osteoporosis.

scholarly journals Morusin induces osteogenic differentiation of bone marrow mesenchymal stem cells by canonical Wnt/β-catenin pathway and prevents bone loss in an ovariectomized rat model

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ming Chen ◽  
Hui Han ◽  
Siqi Zhou ◽  
Yinxian Wen ◽  
Liaobin Chen
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Western Blotting ◽  
Nuclear Translocation ◽  
Osteoclast Differentiation ◽  
Alizarin Red ◽  
Marrow Mesenchymal Stem Cells

Abstract Background Osteoporosis (OP) is a metabolic bone disease due to the imbalance of osteogenesis and bone resorption, in which, bone marrow mesenchymal stem cells (BMSCs) have a significant effect as the seed cells. Recent research has shown the function of Morusin on inhibiting osteoclast differentiation in vitro. However, whether Morusin can regulate the osteogenic differentiation in addition to the proliferation of BMSCs remains unclear. Methods BMSCs were isolated from 4-week-old Wistar rats and then treated with different concentrations of Morusin for 3, 5, 7, and 14 days. The proliferation of BMSCs was detected by MTT assay. The effect of Morusin on osteogenic differentiation of BMSCs was detected by RT-qPCR, Western blotting, ALP, and Alizarin Red staining. The effect of Morusin on Wnt/β-catenin signaling pathway was analyzed by RT-qPCR, Western blotting, and immunofluorescence. Finally, in the ovariectomy-induced osteoporosis model, the anti-osteoporosis activity of Morusin was determined by micro-CT, HE, and immunohistochemistry. Results The results showed the function of 2.5–10 μM Morusin in the promotion of the proliferation in addition to osteogenic differentiation of BMSCs. Moreover, it also has an impact in activating the Wnt/β-catenin signaling pathway via inhibition of β-catenin phosphorylation as well as promotion of its nuclear translocation. Upon Dickkopf-related protein-1 (DKK-1, an inhibitor of the Wnt/β-catenin signaling pathway) was added to the Morusin, Morusin had a decreased stimulatory osteogenic effect on BMSCs. Finally, in the rat OP model, we found that Morusin could also exert anti-osteoporosis activity in vivo. Conclusions This study indicates the ability of Morusin in the promotion of osteogenic differentiation of BMSCs via the activation of Wnt/β-catenin signaling pathway and also shows the potential of Morusin to be an agent for osteoporosis treatment.

High glucose inhibits osteogenic differentiation of bone marrow mesenchymal stem cells via regulating miR-493-5p/ZEB2 signalling

2020 ◽  
Vol 167 (6) ◽  
pp. 613-621
Author(s):  
Zhongshu Zhai ◽  
Wanhong Chen ◽  
Qiaosheng Hu ◽  
Xin Wang ◽  
Qing Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
High Glucose ◽  
Underlying Mechanism ◽  
Luciferase Assay ◽  
Downstream Target ◽  
Marrow Mesenchymal Stem Cells ◽  
Diabetic Osteoporosis

Abstract Diabetic osteoporosis (DOP) is attributed to the aberrant physiological function of bone marrow mesenchymal stem cells (BMSCs) under high glucose (HG) environment. MicroRNAs (miRNAs) are involved in the pathological processes of DOP. We aimed to explore the underlying mechanism of miRNA in DOP. BMSCs were cultured in osteogenic medium with HG to induce osteogenic differentiation, and the interaction between miR-493-5p and ZEB2 was assessed by luciferase assay. Herein, we found miR-493-5p is gradually reduced during osteogenic differentiation in BMSCs. HG treatment inhibits osteogenic differentiation and induces an up-regulation of miR-493-5p leading to reduced level of its downstream target ZEB2. Inhibition of miR-493-5p attenuates HG-induced osteogenic differentiation defects by upregulation of ZEB2. Mechanistically, miR-493-5p/ZEB2 signalling mediates HG-inhibited osteogenic differentiation by inactivation of Wnt/β-catenin signalling. More importantly, knockdown of miR-493-5p therapeutically alleviated the DOP condition in mice. HG prevents BMSCs osteogenic differentiation via up-regulation of miR-493-5p, which results in reduced level of ZEB2 by directly targeting its 3′-untranslated region of mRNA. Thus, miR-493-5p/ZEB2 is a potential therapeutic target and provides novel strategy for the treatment and management of DOP.

scholarly journals miR-206 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells by targetting glutaminase

2019 ◽  
Vol 39 (3) ◽  
Author(s):  
Ying Chen ◽  
Yu-Run Yang ◽  
Xiao-Liang Fan ◽  
Peng Lin ◽  
Huan Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Osteoblast Differentiation ◽  
Bone Diseases ◽  
Glutamine Metabolism ◽  
Mrna Levels ◽  
Marrow Mesenchymal Stem Cells ◽  
Osteogenic Induction

AbstractOsteoblast-mediated bone formation is a complex process involving various pathways and regulatory factors, including cytokines, growth factors, and hormones. Investigating the regulatory mechanisms behind osteoblast differentiation is important for bone regeneration therapy. miRNAs are known as important regulators, not only in a variety of cellular processes, but also in the pathogenesis of bone diseases. In the present study, we investigated the potential roles of miR-206 during osteoblast differentiation. We report that miR-206 expression was significantly down-regulated in human bone marrow mesenchymal stem cells (BMSCs) at days 7 and 14 during osteogenic induction. Furthermore, miR-206 overexpressing BMSCs showed attenuated alkaline phosphatase (ALP) activity, Alizarin Red staining, and osteocalcin secretion. The mRNA levels of osteogenic markers, Runx2 and Osteopontin (OPN), were significantly down-regulated in miR-206 overexpressing BMSCs. We observed that significantly increased glutamine uptake at days 7 and 14 during the osteogenic induction and inhibition of glutamine metabolism by knocking down glutaminase (GLS)-suppressed osteogenic differentiation of BMSCs. Here, we discover that miR-206 could directly bind to the 3′-UTR region of GLS mRNA, resulting in suppressed GLS expression and glutamine metabolism. Finally, restoration of GLS in miR-206 overexpressing BMSCs led to recovery of glutamine metabolism and osteogenic differentiation. In summary, these results reveal a new insight into the mechanisms of the miR-206-mediated osteogenesis through regulating glutamine metabolism. Our study may contribute to the development of therapeutic agents against bone diseases.

scholarly journals Effect of miR-192-5p on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Idiopathic Scoliosis by Regulating Wnt/β-catenin Signaling Pathway via RSPO1

2021 ◽  
Author(s):  
Yifan Yang ◽  
Jing Xu ◽  
Qingxin Su ◽  
Yiran Wu ◽  
Qizheng Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Alizarin Red ◽  
Alkaline Phosphatase Staining ◽  
Marrow Mesenchymal Stem Cells ◽  
Related Proteins

Abstract BackgroundIdiopathic scoliosis (IS) is the most common structural scoliosis, which seriously affects not only patient’s physical and mental health but also quality of patient’s life. Abnormal osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is one of the causes of IS. However, the regulation mechanism of osteogenic differentiation of BMSCs in patients with IS remains to be further studied.MethodsSerum samples of 135 patients with IS were collected, and the expression of miRNA were detected by RT-qPCR. BMSCs from patients with IS were collected and the expression of miR-192-5p in BMSCs from IS patients and normal BMSCs was detected by RT-qPCR. Double luciferase reporter genes assay was used to verify the targeting relationship between miR-192-5p and RSPO1. The levels of RSPO1, osteogenic related proteins (OC, OPN and RUNX2) and Wnt/β-catenin signaling pathway related proteins (WNT3A and β-catenin) were detected by Western blotting. Alkaline phosphatase staining and alizarin red staining were used to evaluate the osteogenesis of BMSCs.ResultsmiR-192-5p was significantly up-regulated in serum and BMSCs of patients with IS. Alkaline phosphatase staining and alizarin red staining showed that miR-192-5p inhibitor promoted the osteogenic differentiation of BMSCs from IS patients. miR-192-5p targeted down-regulated the expression of RSPO1 in BMSCs from IS patients. In addition, overexpression of RSPO1 activated Wnt/β-catenin signaling pathway in BMSCs from IS patients. Furthermore, miR-192-5p/RSPO1 axis regulated levels of osteogenic related proteins (OC, OPN and RUNX2) in BMSCs from IS patients through Wnt/β-catenin signaling pathway, and affected the osteogenic differentiation of BMSCs.ConclusionmiR-192-5p, which was highly expressed in patients with IS, inhibited Wnt/β-catenin signaling pathway by down-regulating RSPO1 protein and then reduced the osteogenic differentiation ability of BMSCs.

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