scholarly journals Transcriptome-wide m6A methylome during osteogenic differentiation of human adipose-derived stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wentian Sun ◽  
Yidan Song ◽  
Kai Xia ◽  
Liyuan Yu ◽  
Xinqi Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Protein Interaction ◽  
Kegg Pathway ◽  
Mapk Signaling ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
Protein Protein Interaction ◽  
Qrt Pcr

Abstract Objectives Adipose-derived stem cells are frequently used for bone regeneration both in vitro and in vivo. N6-methyladenosine (m6A) is the most abundant post-transcriptional modification on eukaryotic RNAs and plays multifaceted roles in development and diseases. However, the regulatory mechanisms of m6A in osteogenic differentiation of human adipose-derived stem cells (hASCs) remain elusive. The present study aimed to build the transcriptome-wide m6A methylome during the osteogenic differentiation of hASCs. Materials and methods hASCs were harvested after being cultured in a basic or osteogenic medium for 7 days, and the osteogenic differentiation was validated by alkaline phosphatase (ALP) and Alizarin Red S staining, ALP activity assay, and qRT-PCR analysis of ALP, RUNX2, BGLAP, SPP1, SP7, and COL1A1 genes. The m6A level was colorimetrically measured, and the expression of m6A regulators was confirmed by qRT-PCR and western blot. Moreover, m6A MeRIP-seq and RNA-seq were performed to build the transcriptome and m6A methylome. Furthermore, bioinformatic analyses including volcano plots, Venn plots, clustering analysis, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, gene sets enrichment analysis, and protein-protein interaction analysis were conducted. Results In total, 1145 differentially methylated peaks, 2261 differentially expressed genes, and 671 differentially methylated and expressed genes (DMEGs) were identified. GO and KEGG pathway analyses conducted for these DMEGs revealed extensive and osteogenic biological functions. The “PI3K-Akt signaling pathway”; “MAPK signaling pathway”; “parathyroid hormone synthesis, secretion, and action”; and “p53 signaling pathway” were significantly enriched, and the DMEGs in these pathways were identified as m6A-specific key genes. A protein-protein interaction network based on DMEGs was built, and VEGFA, CD44, MMP2, HGF, and SPARC were speculated as the hub DMEGs. Conclusions The total m6A level was reduced with osteogenic differentiation of hASCs. The transcriptome-wide m6A methylome built in the present study indicated quite a few signaling pathways, and hub genes were influenced by m6A modification. Future studies based on these epigenetic clues could promote understanding of the mechanisms of osteogenic differentiation of hASCs.

scholarly journals Endothelial Progenitor Cells Induce Osteogenic Differentiation in Co-cultured Mesenchymal Stem Cells via the MAPK Signaling Pathway

2020 ◽  
Author(s):  
chu xu ◽  
haijie liu ◽  
yuanjia he ◽  
yuanqing li ◽  
xiaoning he
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Progenitor Cells ◽  
Signaling Pathway ◽  
Endothelial Progenitor Cells ◽  
Mapk Signaling ◽  
Nodule Formation ◽  
Qrt Pcr

Abstract Background: The role of bone tissue engineering is to regenerate tissue using biomaterials and stem cell based approaches. Combination of two or more cell types is one of the strategies to promote bone formation. Endothelial progenitor cells (EPCs) may enhance the osteogenic properties of mesenchymal stem cells (MSCs) and promote bone healing, this study aimed to investigate the possible mechanisms of EPCs on promoting osteogenic differentiation of MSCs.Methods: MSCs and EPCs were isolated and co-cultured in Transwell chambers, the effects of EPCs on the regulation of MSC biological properties was investigated. Real-time PCR array, qRT-PCR and western blotting were performed to explore possible signaling pathways involved in osteogenesis. The expression of osteogenesis markers and calcium nodule formation was quantified by qRT-PCR, western blotting and Alizarin Red staining. Results: Results showed that when co-cultured with EPCs, MSCs exhibited greater alkaline phosphatase (ALP) activity and increased calcium mineral deposition significantly. The mitogen-activated protein kinase (MAPK) signaling pathway was involved in this process. p38 gene expression and p38 protein phosphorylation levels showed significant up-regulation in co-cultured MSCs. Silence expression of p38 in co-cultured MSCs reduced osteogenic gene expression, protein synthesis, ALP activity and calcium nodule formation.Conclusions: These data suggest paracrine signaling from EPCs influence the biological function and promote MSCs osteogenic differentiation. Activation of the p38MAPK pathway may be the key to enhancing MSCs osteogenic differentiation via indirect interactions with EPCs.

scholarly journals Strontium Promotes Osteogenic Differentiation of Mesenchymal Stem Cells Through the Ras/MAPK Signaling Pathway

2009 ◽  
Vol 23 (1-3) ◽  
pp. 165-174 ◽  
Author(s):  
Songlin Peng ◽  
Guangqian Zhou ◽  
Keith D.K. Luk ◽  
Kenneth M.C. Cheung ◽  
Zhaoyang Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Mapk Signaling ◽  
Mapk Signaling Pathway

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 Yunnan Baiyao Conditioned Medium Promotes the Odonto/Osteogenic Capacity of Stem Cells from Apical Papilla via Nuclear Factor Kappa B Signaling Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Xiyao Pang ◽  
Yanqiu Wang ◽  
Jintao Wu ◽  
Zhou Zhou ◽  
Tao Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Conditioned Medium ◽  
Signaling Pathway ◽  
Cell Counting ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Apical Papilla ◽  
Yunnan Baiyao

Yunnan Baiyao is a traditional Chinese herbal remedy that has long been used for its characteristics of wound healing, bone regeneration, and anti-inflammation. However, the effects of Yunnan Baiyao on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) and the potential mechanisms remain unclear. The aim of this study was to investigate the odonto/osteogenic differentiation effects of Yunnan Baiyao on SCAPs and the underlying mechanisms involved. SCAPs were isolated and cocultured with Yunnan Baiyao conditioned media. The proliferation ability was determined by cell counting kit 8 and flow cytometry. The differentiation capacity and the involvement of NF-κB pathway were investigated by alkaline phosphatase assay, alizarin red staining, immunofluorescence assay, real-time RT-PCR, and western blot analyses. Yunnan Baiyao conditioned medium at the concentration of 50 μg/mL upregulated alkaline phosphatase activity, induced more mineralized nodules, and increased the expression of odonto/osteogenic genes/proteins (e.g., OCN/OCN, OPN/OPN, OSX/OSX, RUNX2/RUNX2, ALP/ALP, COL-I/COL-I, DMP1, DSP/DSPP) of SCAPs. In addition, the expression of cytoplasmic phos-IκBα, phos-P65, and nuclear P65 was significantly increased in Yunnan Baiyao conditioned medium treated SCAPs in a time-dependent manner. Conversely, the differentiation of Yunnan Baiyao conditioned medium treated SCAPs was obviously inhibited when these stem cells were cocultured with the specific NF-κB inhibitor BMS345541. Yunnan Baiyao can promote the odonto/osteogenic differentiation of SCAPs via the NF-κB signaling pathway.

scholarly journals Changing Expression Profiles and Inclination to Competing Endogenous RNA Networks on MAPK Signaling Pathways of Human Adipose-Derived Stem Cells in a Direct Current Electric Field

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Mingzhu Jin ◽  
Yujing Zhang ◽  
Yuanyuan Bian ◽  
Ruiqun Qi ◽  
Xinghua Gao
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Signaling Pathway ◽  
Mapk Signaling ◽  
Mapk Signaling Pathway ◽  
Keratinocyte Differentiation ◽  
Adipose Derived Stem Cells ◽  
Competing Endogenous Rna ◽  
Direct Current Electric Field ◽  
Current Electric Field

Adipose-derived stem cells (ADSCs) are an abundant cell source and provide an easy way to harvest mesenchymal stem cells, which are the focus of considerable attention in regenerative medicine. Electric fields (EF) play roles in many biological events and have been reported to promote cell proliferation, migration, and differentiation. In this study, ADSCs were treated with a direct current electric field (DCEF) of either 0 (control group) or 300 mV/mm (EF group) for six hours. RNA screening and analysis revealed that 66, 164, 26, and 1310 circRNAs, lncRNAs, miRNAs, and mRNAs, respectively, were differentially expressed in the DCEF-treated ADSCs compared with untreated ADSCs. Differentially expressed mRNAs were enriched in the MAPK signaling pathway, TNF signaling pathway, and some other pathways. ANXA1, ERRFI1, JAG1, EPHA2, PRR9, and H2AFY2 were related to the keratinocyte differentiation process. Competing endogenous RNA (ceRNA) networks were constructed on the basis of genes in the MAPK signaling pathway. Twenty-one RNAs in the above networks were randomly chosen, and their expression was validated using qRT-PCR, which showed the same expression trends as the RNA sequencing analysis. The MAPK signaling pathway is of great importance in the ADSC processes that occur in a DCEF, including keratinocyte differentiation. Several ceRNAs may participate in the regulation of MAPK signaling. This study may give new insight into the proliferation, migration, and differentiation of ADSCs, which will be valuable for tissue engineering and regenerative medicine.

scholarly journals Bioinformatics analysis and identification of circular RNAs promoting the osteogenic differentiation of human bone marrow mesenchymal stem cells on titanium treated by surface mechanical attrition

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9292
Author(s):  
Shanshan Zhu ◽  
Yuhe Zhu ◽  
Zhenbo Wang ◽  
Chen Liang ◽  
Nanjue Cao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Human Bone ◽  
Cell Counting ◽  
Circular Rnas ◽  
Control Group ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Mechanical Attrition

Background To analyze and identify the circular RNAs (circRNAs) involved in promoting the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) on titanium by surface mechanical attrition treatment (SMAT). Methods The experimental material was SMAT titanium and the control material was annealed titanium. Cell Counting Kits-8 (CCK-8) was used to detect the proliferation of hBMSCs, and alkaline phosphatase (ALP) activity and alizarin red staining were used to detect the osteogenic differentiation of hBMSCs on the sample surfaces. The bioinformatics prediction software miwalk3.0 was used to construct competing endogenous RNA (ceRNA) networks by predicting circRNAs with osteogenesis-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The circRNAs located at the key positions in the networks were selected and analyzed by quantitative real-time PCR (QRT-PCR). Results Compared with annealed titanium, SMAT titanium could promote the proliferation and osteogenic differentiation of hBMSCs. The total number of predicted circRNAs was 51. Among these, 30 circRNAs and 8 miRNAs constituted 6 ceRNA networks. Circ-LTBP2 was selected for verification. QRT-PCR results showed that the expression levels of hsa_circ_0032599, hsa_circ_0032600 and hsa_circ_0032601 were upregulated in the experimental group compared with those in the control group; the differential expression of hsa_circ_0032600 was the most obvious and statistically significant, with a fold change (FC) = 4.25 ± 1.60, p-values (p) < 0.05.

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