scholarly journals Atractylodes Lancea Volatile Oils Target ADAR2-miR-181a-5p Signaling to Mesenchymal Stem Cells Chondrogenic Differentiation

2021 ◽  
Author(s):  
Shanyu Ye ◽  
Wenwen Si ◽  
Wei Qin ◽  
Ziwei Luo ◽  
Zhen Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chinese Medicine ◽  
Chondrogenic Differentiation ◽  
Volatile Oil ◽  
Luciferase Reporter ◽  
Blue Staining ◽  
Reporter System ◽  
Volatile Oils ◽  
Atractylodes Lancea

Abstract Background：The Rhizoma Atractylodis has long been recommended for the treatment of different diseases in traditional Chinese medicine. The main component of Rhizoma Atractylodis is Atractylodes lancea volatile oils which possesses anti-microorganism, anti-tumor, cognitive protection and immunoregulation. The aim of the study was to elucidate the mechanism of Atractylodes lancea volatile oils promoting mesenchymal stem cells (MSCs) chondrogenic differentiation. Method：Atractylodes lancea volatile oils were extracted from Chinese medicine Cangzhu by volatile oil extractor. MSCs culture were treated with Atractylodes lancea volatile oils medium. Real-time reverse transcription PCR was conducted to verify the candidate microRNAs discovered by microarray analysis. Western-blot analyzed the expressions of mark genes. Sanger sequences identified the changes of the base pairs which would be edited by ADAR2 enzyme. Toluidine blue staining identified the changes of cells chondrogenic differentiation. Result：Treatment of Atractylodes lancea volatile oils increased the cells chondrogenic differentiation of MSCs. Atractylodes lancea volatile oils promoted the expression of ADAR2 enzyme which may edit the precursor of miR-181a-5p. A dual luciferase reporter system assay verified that transcription factors yingyang1(YY1) was targeted by miR-181a-5p which was downregulated in MSCs chondrogenic differentiation. Conclusion：This work demonstrates the mechanism of Atractylodes lancea volatile oils promoting MSCs to chondrogenic differentiation. It may provide an alternative strategy for treatment purposes and diagnosis in clinic.

scholarly journals LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Xiaozhong Zhou ◽  
Wangyang Xu ◽  
Yeyang Wang ◽  
Hui Zhang ◽  
Li Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chondrogenic Differentiation ◽  
Genetic Regulation ◽  
Luciferase Reporter ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Differentiation Markers ◽  
Hypoxic Conditions

Abstract Background Improved chondrogenic differentiation of mesenchymal stem cells (MSCs) by genetic regulation is a potential method for regenerating articular cartilage. MiR-127-5p has been reported to promote cartilage differentiation of rat bone marrow MSCs (rMSCs); however, the regulatory mechanisms underlying hypoxia-stimulated chondrogenic differentiation remain unknown. Methods rMSCs were induced to undergo chondrogenic differentiation under normoxic or hypoxic conditions. Expression of lncRNA DNM3OS, miR-127-5p, and GREM2 was detected by quantitative real-time PCR. Proteoglycans were detected by Alcian blue staining. Western blot assays were performed to examine the relative levels of GREM2 and chondrogenic differentiation related proteins. Luciferase reporter assays were performed to assess the association among DNM3OS, miR-127-5p, and GREM2. Results MiR-127-5p levels were upregulated, while DNM3OS and GREM2 levels were downregulated in rMSCs induced to undergo chondrogenic differentiation, and those changes were attenuated by hypoxic conditions (1% O2). Further in vitro experiments revealed that downregulation of miR-127-5p reduced the production of proteoglycans and expression of chondrogenic differentiation markers (COL1A1, COL2A1, SOX9, and ACAN) and osteo/chondrogenic markers (BMP-2, p-SMAD1/2). MiR-127-5p overexpression produced the opposite results in rMSCs induced to undergo chondrogenic differentiation under hypoxic conditions. GREM2 was found to be a direct target of miR-127-5p, which was suppressed in rMSCs undergoing chondrogenic differentiation. Moreover, DNM3OS could directly bind to miR-127-5p and inhibit chondrogenic differentiation of rMSCs via regulating GREM2. Conclusions Our study revealed a novel molecular pathway (DNM3OS/miR-127-5p/GREM2) that may be involved in hypoxic chondrogenic differentiation.

scholarly journals Sox9-Increased miR-322-5p Facilitates BMP2-Induced Chondrogenic Differentiation by Targeting Smad7 in Mesenchymal Stem Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Yongsheng Zeng ◽  
Chengcheng Du ◽  
Pengcheng Xiao ◽  
Yiting Lei ◽  
Piao Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chondrogenic Differentiation ◽  
In Vitro Study ◽  
Underlying Mechanism ◽  
Bone Morphogenetic Protein 2 ◽  
Luciferase Reporter ◽  
Smad7 Expression

Bone morphogenetic protein 2 (BMP2) induces effective chondrogenesis of mesenchymal stem cells (MSCs) by promoting Sox9 expression. However, BMP2 also induces chondrocyte hypertrophy and endochondral ossification by upregulating Smad7 expression, which leads to the disruption of chondrogenesis. In addition, Smad7 can be inhibited by Sox9. Therefore, the underlying mechanism is not clear. Currently, an increasing number of studies have shown that microRNAs play a pivotal role in chondrogenic and pathophysiological processes of cartilage. The purpose of this study was to determine which microRNA is increased by Sox9 and targets Smad7, thus assisting BMP2 in maintaining stable chondrogenesis. We found that miR-322-5p meets the requirement through next-generation sequencing (NGS) and bioinformatic analysis. The targeting relationship between miR-322-5p and Smad7 was confirmed by dual-luciferase reporter assays, qPCR, and western blotting (WB). The in vitro study indicated that overexpression of miR-322-5p significantly inhibited Smad7 expression, thus causing increased chondrogenic differentiation and decreased hypertrophic differentiation, while silencing of miR-322-5p led to the opposite results. Flow cytometry (FCM) analysis indicated that overexpression of miR-322-5p significantly decreased the rate of early apoptosis in BMP2-stimulated MSCs, while silencing of miR-322-5p increased the rate. A mouse limb explant assay revealed that the expression of miR-322-5p was negatively correlated with the length of the BMP2-stimulated hypertrophic zone of the growth plate. An in vivo study also confirmed that miR-322-5p assisted BMP2 in chondrogenic differentiation. Taken together, our results suggested that Sox9-increased miR-322-5p expression can promote BMP2-induced chondrogenesis by targeting Smad7, which can be exploited for effective tissue engineering of cartilage.

scholarly journals Chondrogenic Differentiation of Mesenchymal Stem Cells in Three-Dimensional Chitosan Film Culture

2017 ◽  
Vol 26 (3) ◽  
pp. 417-427 ◽  
Author(s):  
Tsai-Jung Lu ◽  
Fang-Yao Chiu ◽  
Hsiao-Ying Chiu ◽  
Ming-Chau Chang ◽  
Shih-Chieh Hung
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chondrogenic Differentiation ◽  
Chitosan Film ◽  
Cartilage Tissue ◽  
Blue Staining ◽  
Cartilage Defects ◽  
Induction Medium ◽  
Alcian Blue Staining ◽  
Film Culture

Articular cartilage has a very limited capacity for self-repair, and mesenchymal stem cells (MSCs) have the potential to treat cartilage defects and osteoarthritis. However, in-depth mechanistic studies regarding their applications are required. Here we demonstrated the use of chitosan film culture for promoting chondrogenic differentiation of MSCs. We found that MSCs formed spheres 2 days after seeding on dishes coated with chitosan. When MSCs were induced in a chondrogenic induction medium on chitosan films, the size of the spheres continuously increased for up to 21 days. Alcian blue staining and immunohistochemistry demonstrated the expression of chondrogenic proteins, including aggrecan, type II collagen, and type X collagen at 14 and 21 days of differentiation. Importantly, chitosan, with a medium molecular weight (size: 190–310 kDa), was more suitable than other sizes for inducing chondrogenic differentiation of MSCs in terms of sphere size and expression of chondrogenic proteins and endochondral markers. We identified that the mechanistic target of rapamycin (mTOR) signaling and its downstream S6 kinase (S6K)/S6 were activated in chitosan film culture compared to that of monolayer culture. The activation of mTOR/S6K was continuously upregulated from days 2 to 7 of differentiation. Furthermore, we found that mTOR/S6K signaling was required for chondrogenic differentiation of MSCs in chitosan film culture through rapamycin treatment and mTOR knockdown. In conclusion, we showed the suitability of chitosan film culture for promoting chondrogenic differentiation of MSCs and its potential in the development of new strategies in cartilage tissue engineering.

scholarly journals MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Shuang Lv ◽  
Jinying Xu ◽  
Lin Chen ◽  
Haitao Wu ◽  
Wei Feng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Chondrogenic Differentiation ◽  
Hyaline Cartilage ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Luciferase Reporter ◽  
Therapeutic Effects ◽  
Chondrocyte Differentiation

Abstract Background Human bone marrow-derived mesenchymal stem cells (hBMSCs) have chondrocyte differentiation potential and are considered to be a cell source for cell-transplantation-mediated repair of cartilage defects, including those associated with osteoarthritis (OA). However, chondrocyte hypertrophic differentiation is a major obstacle for the application of hBMSCs in articular cartilage defect treatment. We have previously shown that microRNA-27b (miR-27b) inhibits hypertrophy of chondrocytes from rat knee cartilage. In this study, we investigated the role of miR-27b in chondrocyte hypertrophic differentiation of hBMSCs. Methods Chondrogenic marker and microRNA expression in hBMSC chondrogenic pellets were evaluated using RT-qPCR and immunohistochemistry. The hBMSCs were transfected with miR-27b before inducing differentiation. Gene and protein expression levels were analyzed using RT-qPCR and western blot. Coimmunoprecipitation was used to confirm interaction between CBFB and RUNX2. Luciferase reporter assays were used to demonstrate that CBFB is a miR-27b target. Chondrogenic differentiation was evaluated in hBMSCs treated with shRNA targeting CBFB. Chondrogenic hBMSC pellets overexpressing miR-27b were implanted into cartilage lesions in model rats; therapeutic effects were assessed based on histology and immunohistochemistry. Results The hBMSCs showed typical MSC differentiation potentials. During chondrogenic differentiation, collagen 2 and 10 (COL2 and COL10), SOX9, and RUNX2 expression was upregulated. Expression of miR-140, miR-143, and miR-181a increased over time, whereas miR-27b and miR-221 were downregulated. Cartilage derived from hBMSC and overexpressing miR-27b exhibited higher expression of COL2 and SOX9, but lower expression of COL10, RUNX2, and CBFB than did the control cartilage. CBFB and RUNX2 formed a complex, and CBFB was identified as a novel miR-27b target. CBFB knockdown by shRNA during hBMSC chondrogenic differentiation led to significantly increased COL2 and SOX9 expression and decreased COL10 expression. Finally, miR-27b-overexpressing hBMSC chondrogenic pellets had better hyaline cartilage morphology and reduced expression of hypertrophic markers and tend to increase repair efficacy in vivo. Conclusion MiR-27b plays an important role in preventing hypertrophic chondrogenesis of hBMSCs by targeting CBFB and is essential for maintaining a hyaline cartilage state. This study provides new insights into the mechanism of hBMSC chondrocyte differentiation and will aid in the development of strategies for treating cartilage injury based on hBMSC transplantation.

scholarly journals miR-223 promotes cartilage differentiation of bone marrow-derived mesenchymal stem cells and protects against osteoarthritis by suppressing NLRP-3 expression

2020 ◽  
Author(s):  
Nan Min ◽  
Jie Ma ◽  
Lei Shi ◽  
Lin Wang ◽  
Chi Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Group Treatment ◽  
Chondrogenic Differentiation ◽  
Cruciate Ligament ◽  
Treatment Protocol ◽  
Cartilage Tissue ◽  
Blue Staining ◽  
Alcian Blue Staining

IntroductionThe present investigation evaluates the role of miR-223 mimic in the treatment of osteoarthritis (OA) and postulates the possible molecular mechanism of its action.Material and methodsBone marrow-derived mesenchymal stem cells (BMSCs) were isolated from rats and cultured in chondrogenic medium to stimulate the differentiation of chondrocytes. Alcian blue staining was performed to determine the chondrogenic differentiation and expression of miR-223 in the BMSCs. Moreover, expression of NLR family pyrin domain containing 3 (NLRP-3), matrix metallopeptidase-13 (MMP-13) and collagen (Col II) was determined in miR-223 mimic and inhibitor treated BMSCs. OA was induced by injecting anterior cruciate ligament transection in rats followed by further treatment with the miR-223 mimic for the period of the treatment protocol. Level and expression of inflammatory cytokines were estimated in the cartilage tissue of OA rats. Moreover, immunohistochemical analysis and histopathology study were also performed.ResultsData of the study reveal that expression of miR-223 was higher in chondrogenic differentiated BMSCs than normal. Expression of MMP-13 and NLRP-3 was lower, and expression of Col II was higher in miR-223 mimic treated BMSCs than normal. Moreover, data of the in-vivo study indicate that the expression level of cytokines was lower in the cartilage tissue of the miR-223 mimic treated group than the OA group. Treatment with the miR-223 mimic ameliorates the altered histopathology and expression of NLRP-3 in the cartilage tissue of OA rats.ConclusionsData of the study reveal that the miR-223 mimic enhances the chondrogenic differentiation of BMSCs by regulating the NLRP-3/IL-18/TGF-b pathway.

scholarly journals Protective Effects of a Hyaluronan-Binding Peptide (P15-1) on Mesenchymal Stem Cells in an Inflammatory Environment

2021 ◽  
Vol 22 (13) ◽  
pp. 7058
Author(s):  
Thorsten Kirsch ◽  
Fenglin Zhang ◽  
Olivia Braender-Carr ◽  
Mary K. Cowman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Chondrogenic Differentiation ◽  
Therapeutic Strategy ◽  
Cartilage Defects ◽  
Mrna Levels ◽  
Protective Effects ◽  
Human Bmscs ◽  
Hyaluronan Binding

Mesenchymal stem cells (MSCs) obtained from various sources, including bone marrow, have been proposed as a therapeutic strategy for the improvement of tissue repair/regeneration, including the repair of cartilage defects or lesions. Often the highly inflammatory environment after injury or during diseases, however, greatly diminishes the therapeutic and reparative effectiveness of MSCs. Therefore, the identification of novel factors that can protect MSCs against an inflammatory environment may enhance the effectiveness of these cells in repairing tissues, such as articular cartilage. In this study, we investigated whether a peptide (P15-1) that binds to hyaluronan (HA), a major component of the extracellular matrix of cartilage, protects bone-marrow-derived MSCs (BMSCs) in an inflammatory environment. The results showed that P15-1 reduced the mRNA levels of catabolic and inflammatory markers in interleukin-1beta (IL-1β)-treated human BMSCs. In addition, P15-1 enhanced the attachment of BMSCs to HA-coated tissue culture dishes and stimulated the chondrogenic differentiation of the multipotential murine C3H/10T1/2 MSC line in a micromass culture. In conclusion, our findings suggest that P15-1 may increase the capacity of BMSCs to repair cartilage via the protection of these cells in an inflammatory environment and the stimulation of their attachment to an HA-containing matrix and chondrogenic differentiation.

Effect of electrical stimulation on chondrogenic differentiation of mesenchymal stem cells cultured in hyaluronic acid – Gelatin injectable hydrogels

2020 ◽  
Vol 134 ◽  
pp. 107536 ◽  
Author(s):  
Juan Jairo Vaca-González ◽  
Sandra Clara-Trujillo ◽  
María Guillot-Ferriols ◽  
Joaquín Ródenas-Rochina ◽  
María J. Sanchis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Electrical Stimulation ◽  
Hyaluronic Acid ◽  
Chondrogenic Differentiation ◽  
Injectable Hydrogels ◽  
Cells Cultured
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