Differentiation Of hADSCs Research Articles

Regulatory Effects and Mechanism of Action of Green Tea Polyphenols on Osteogenesis and Adipogenesis in Human Adipose Tissue-Derived Stem Cells.

We previously showed that green tea polyphenols (GTPs) exert antiadipogenic effects on preadipocyte proliferation. Here, we investigated the regulatory effects of GTPs on osteogenesis and adipogenesis during early differentiation of human adipose tissue-derived stem cells (hADSC). Adipogenesis of hADSCs was determined by oil-red-O staining and triglycerides synthesis measurement. Osteoporosis of hADSC was measured using alkaline phosphatase assays and intracellular calcium levels. Immunofluorescence staining and qRT-PCR were used to detect PPARγ-CEBPA regulated adipogenic pathway regulated by PPAR-CEBPA and the osteogenic pathway mediated by RUNX2-BMP2. We found that GTPs treatment significantly decreased lipid accumulation and cellular triglyceride synthesis in mature adipocytes and attenuated pioglitazone-induced adipogenesis in a dose-dependent manner. GTPs downregulated protein and mRNA expression of Pparγ and attenuated pioglitazone-stimulated-Cebpa expression. GTPs treatment significantly enhanced hADSCs differentiation into osteoblasts compared to control and pioglitazone-treated cells. GTPs upregulated RunX2 and Bmp2 proteins and mRNA expression compared to control and significantly attenuated decreased RunX2 and Bmp2 mRNA expression by pioglitazone. In conclusion, our data demonstrates GTPs possesses great ability to facilitate osteogenesis and simultaneously inhibits hADSC differentiation into adipogenic lineage by upregulating the RUNX2-BMP2 mediated osteogenic pathway and suppressing PPARγ-induced signaling of adipogenesis. These findings highlight GTPs' potential to combat osteoporosis associated with obesity.

Down-regulation of hsa-circ-0107593 promotes osteogenic differentiation of hADSCs via miR-20a-5p/SMAD6 signaling.

Increasing evidence indicated circRNAs were involved in stem cells osteogenesis differentiation. Herein, we aimed to clarify the role of hsa-circ-0107593 during the osteogenesis process of human adipose-derived stem cells (hADSCs) and the underlying mechanisms. The ring structure of hsa-circ-0107593 was confirmed using RNase R treatment and Sanger sequencing. Nucleoplasmic separation and fluorescence in situ hybridization detected hsa-circ-0107593 distribution. Lentivirus and siRNA were used to modulate the expression of hsa-circ-0107593, and the binding relationship between hsa-circ-0107593 and miR-20a-5p was verified by luciferase assay and RNA immunoprecipitation. We detected the osteogenic activity of hADSCs through alkaline phosphatase staining, alizarin red S staining, real-time polymerase chain reaction (RT-PCR), western blot, and cellular immunofluorescence experiment. In vivo, micro-computed tomography was performed to analyze bone formation around skull defect. RT-PCR results exhibited that hsa-circ-0107593 was downregulated while miR-20a-5p was upregulated during hADSCs osteogenesis. In vivo and in vitro experiments results indicated that knocking down hsa-circ-0107593 promoted the osteogenic differentiation of hADSCs, while overexpression of hsa-circ-0107593 showed an inhibitory effect on hADSCs osteogenic differentiation. In vitro experiment results showed hsa-circ-0107593 acted as a hADSCs osteogenic differentiation negative factor for it inhibited the suppressing effect of miR-20a-5p on SMAD6. Knocking down hsa-circ-0107593 acts as a positive factor of the osteogenic differentiation of hADSCs via miR-20a-5p/SMAD6 signaling.

Porous cellulose-collagen scaffolds for soft tissue regeneration: influence of cellulose derivatives on mechanical properties and compatibility with adipose-derived stem cells

This study compares two types of bioresorptive cellulose, i.e., calcium-sodium salt of oxidized cellulose (OC) and sodium salt of carboxymethylcellulose (CMC). It investigates which type would be preferable as an implant material in terms of biocompatibility, biomechanical and biological properties, and also in terms of its behavior in combination with collagen fibrils (Col) in composite Col/OC or Col/CMC scaffolds. OC significantly supported the stiffness and elasticity of Col fibrils, whereas CMC significantly reduced these properties. OC also enabled a strong interaction with Col fibrils even in a moist environment, accompanied by a significant drop in elastic modulus. The addition of cellulose did not significantly influence scaffold porosity; however, changes in surface morphology and the lower swelling capacity of OC, with a degree of oxidation of its chains between 16 and 24%, supported the idea of improved cell-material interaction. The elasticity and the stiffness of Col/OC guided human adipose-derived stem cells (hADSCs) to significantly higher adhesion, proliferation, and metabolic activity. On the contrary, the Col/CMC provided only limited mechanical support for the cells and inhibited their attachment and proliferation, although without any signs of cytotoxicity. This phenomenon could be used for future control of the differentiation of hADSCs towards a desired phenotype to generate advanced tissue replacements using modern methods of tissue engineering. The oxidation of cellulose resulted in a firmer scaffolding material, as required in vascular or skin tissue engineering. CMC is suitable for moist wound healing, e.g. as a mucoadhesive gel, where cell adhesion is not desirable.Graphical abstract

Effect and Related Mechanism of Platelet-Rich Plasma on the Osteogenic Differentiation of Human Adipose-Derived Stem Cells.

Objective Human adipose-derived stem cells (hADSCs) are ideal seed cells for the regeneration of alveolar bone defects. Platelet-rich plasma (PRP), which is rich in growth factors, promotes tissue repair. The purpose of the present study was to investigate whether PRP promotes the osteogenic differentiation of hADSCs and to perform high-throughput sequencing to explore the possible mechanism. Methods hADSCs were divided into the three following groups: CON group, OM group, and PRP group. Osteogenesis was detected by Alizarin Red staining on day 14. Total RNA was extracted from the OM and PRP groups for high-throughput sequencing. The target genes of the differentially expressed osteogenic-related miRNAs were predicted, and combined miRNA/mRNA analysis was then performed. The mRNA and protein expression levels of hsa-miR-212-5p, type 1 cannabinoid receptor (CNR1), alkaline phosphatase (ALP), Runx2, osteocalcin (OCN), and collagen 1 A1 (COL1A1) in the OM and PRP groups were detected by qRT–PCR and Western blot analyses. The binding between hsa-miR-212-5p and CNR1 was detected by a dual-luciferase reporter assay. Results Both the OM and PRP groups exhibited enhanced proliferation of hADSCs, and the differences at 48 h and 72 h were statistically significant (P < 0.05). The PRP group had significantly more calcium nodules than the CON group (P < 0.05). Through high-throughput sequencing analysis, differential miRNA and mRNA expression profiles were obtained. During hADSC osteogenesis, the expression of hsa-miR-212-5p was downregulated, and the expression of CNR1 was upregulated. hsa-miR-212-5p was found to bind directly to the 3′ UTR of CNR1. Conclusions The present findings indicated that downregulation of hsa-miR-212-5p and upregulation of CNR1 may be involved in the process by which PRP promotes the osteogenic differentiation of hADSCs.

Characteristic and Chondrogenic Differentiation Analysis of Hybrid Hydrogels Comprised of Hyaluronic Acid Methacryloyl (HAMA), Gelatin Methacryloyl (GelMA), and the Acrylate-Functionalized Nano-Silica Crosslinker

Developing a biomaterial suitable for adipose-derived stem cell (ADSCs)-laden scaffolds that can directly bond to cartilage tissue surfaces in tissue engineering has still been a significant challenge. The bioinspired hybrid hydrogel approaches based on hyaluronic acid methacryloyl (HAMA) and gelatin methacryloyl (GelMA) appear to have more promise. Herein, we report the cartilage tissue engineering application of a novel photocured hybrid hydrogel system comprising HAMA, GelMA, and 0~1.0% (w/v) acrylate-functionalized nano-silica (AFnSi) crosslinker, in addition to describing the preparation of related HAMA, GelMA, and AFnSi materials and confirming their related chemical evidence. The study also examines the physicochemical characteristics of these hybrid hydrogels, including swelling behavior, morphological conformation, mechanical properties, and biodegradation. To further investigate cell viability and chondrogenic differentiation, the hADSCs were loaded with a two-to-one ratio of the HAMA-GelMA (HG) hybrid hydrogel with 0~1.0% (w/v) AFnSi crosslinker to examine the process of optimal chondrogenic development. Results showed that the morphological microstructure, mechanical properties, and longer degradation time of the HG+0.5% (w/v) AFnSi hydrogel demonstrated the acellular novel matrix was optimal to support hADSCs differentiation. In other words, the in vitro experimental results showed that hADSCs laden in the photocured hybrid hydrogel of HG+0.5% (w/v) AFnSi not only significantly increased chondrogenic marker gene expressions such as SOX-9, aggrecan, and type II collagen expression compared to the HA and HG groups, but also enhanced the expression of sulfated glycosaminoglycan (sGAG) and type II collagen formation. We have concluded that the photocured hybrid hydrogel of HG+0.5% (w/v) AFnSi will provide a suitable environment for articular cartilage tissue engineering applications.

Silica nano particles embedded in random and aligned PLGA/gelatin electrospun nano fibers improve growth and differentiation of human adipose-derived stem cells into anterior neuroectodermal cells

Stem cell therapy is a promising approach involving the application of pluripotent cells for the treatment of a vast variety of diseases. There are several reports on designing and development of methods for suitable differentiation of stem cells to neural lineage. In this study, we investigated the potency of align and random poly(lactic-co-glycolic acid) (PLGA) nano-fibers containing mesoporous silica nanoparticles (MSNPs) in differentiation of human adipose derived stem cells (hADSCs) into anterior neuroectodermal cells. The expression level of four anterior neuroectodermal markers including OTX2, SIX3, PAX6 and RAX was quantified for better understanding of the effect of scaffolds on cell differentiation. According to the obtained results, it seems that align PLGA/MSNPs scaffolds are suitable for anterior neuroectodermal induction of hADSCs. Considering the absence of chemical agents for cell differentiation and with respect to the surprisingly high level of all four markers in the aligned fibers, using these scaffolds is introduced as a safe, cheap and powerful method for easy differentiation of hADSCs into anterior neuroectodermal cells.

Next-Generation Sequencing of Circular RNAs Reveals the Molecular Mechanisms of Chondrogenic Differentiation in Human Adipose-derived Stem Cells.

Adipose-derived stem cells are one of the potential sources of cells for the treatment of cartilage defects. This study aimed to investigate the molecular mechanisms that account for the chondrogenic differentiation of human adipose-derived stem cells (hADSCs). We employed integrin β1 (ITGB1) overexpression to induce chondrogenic differentiation of hADSCs. Next-generation sequencing was used to determine the mRNAs and circular RNAs (circRNAs) expression profiles in ITGB1-overexpresing and negative control cells. The potential functions of differentially expressed mRNAs were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses. Moreover, differentially expressed circRNAs with the greatest fold change were validated by polymerase chain reaction (PCR), Sanger sequencing, and quantitative real-time PCR (qRT-PCR). These three circRNAs and their downstream microRNAs and mRNAs were used to construct a circRNA-microRNA-mRNA interaction network. The results showed that we identified 713 differentially expressed circRNAs (150 upregulated and 563 downregulated in ITGB1-overexpressing hADSCs versus negative control cells, respectively). Meanwhile, 2383 mRNAs were differentially expressed between two groups (1672 upregulated and 711 downregulated in ITGB1-overexpressing cells compared with the negative control cells). The GO and KEGG analysis results showed that the differentially expressed mRNAs were enriched in biological processes, cellular components, and molecular functions, especially in the phosphatidylinositol 3-kinase (PI3K)-AKT and mitogen-activated protein kinase signaling pathways. Three differentially expressed circRNAs, including hsa_circ_0071127, hsa_circ_0008637, and hsa_circ_0020028, were validated by qRT-PCR. Moreover, the circRNA-microRNA-mRNA network predicted that fibroblast growth factor 2 (FGF2) was a common node regulated by these three circRNAs through several microRNAs, including miR-195-3p, miR-205-3p, and miR-152-3p. We further found that the knockdown of hsa_circ_0020028, but not the two other circRNAs, significantly reduced FGF2 mRNA expression in hADSCs. Furthermore, the knockdown of hsa_circ_0020028 significantly inhibited the protein expression of FGF2, chondrogenic differentiation markers (COL II, aggrecan, and SOX9), and PI3K/AKT signaling in ITGB1-overexpressing hADSCs. This study uncovered the differentially expressed mRNA and circRNA profiles in the chondrogenic differentiation of hADSCs induced by ITGB1 overexpression. Our findings demonstrate that hsa_circ_0020028 regulates the ITGB1 overexpression-mediated chondrogenic differentiation of hADSCs through regulation of FGF2-related signaling pathways.

MiR-216a-3p inhibits osteogenic differentiation of human adipose-derived stem cells via Wnt3a in the Wnt/β-catenin signaling pathway.

The current study aimed to investigate the potential function and mechanism of microRNA (miR)-216a-3p in the osteogenic differentiation of human adipose-derived stem cells (hADSCs). Dynamic expression changes of miR-216a-3p in the osteogenic differentiation of hADSCs were examined by reverse transcription-quantitative PCR (RT-qPCR). Regulatory effects of miR-216a-3p on the relative levels of osteogenesis-associated genes were also detected by RT-qPCR and western blotting. The relationship between miR-216a-3p and Wnt3a was verified through a dual-luciferase reporter assay. Furthermore, the influence of miR-216a-3p on the Wnt/β-catenin signaling pathway during the osteogenic differentiation of hADSCs was investigated by western blotting. The results revealed that during the osteogenic differentiation process of hADSCs, miR-216a-3p was downregulated and Wnt3a was upregulated. It was further verified that Wnt3a was the target of miR-216a-3p. Through inactivation of the Wnt/β-catenin signaling pathway, miR-216a-3p was able to mediate osteogenic differentiation of hADSCs. In conclusion, by targeting Wnt3a, miR-216a-3p mediated the osteogenic differentiation of hADSCs, which negatively regulated the Wnt/β-catenin signaling pathway.

Transcriptomic analysis and biological evaluation reveals that LMO3 regulates the osteogenic differentiation of human adipose derived stem cells via PI3K/Akt signaling pathway.

Autologous bone transplantation which is a common treatment method for bone defects needs a large quantity of bone cells. In order to develop new treatments to regenerating bone tissues, this research aimed at identifying the key genes and finding their mechanism in human adipose-derived stem cells (hADSCs) osteogenesis. GSE63754, GSE89330 and GSE72429 were downloaded to perform GO functional and KEGG pathway analyses, construct a competing endogenous RNA (ceRNA) network, construct a PPI network and identify hub genes. The expression level of LMO3 during the osteogenesis of hADSCs was examined by quantitative reverse transcription polymerase chain reaction and western blot. Lentivirus transfection was used to knock down or overexpress LMO3, which enabled us to investigate the effect of LMO3 on osteogenic differentiation of hADSCs. Wortmannin were used to identify the mechanism of the LMO3/PI3K/Akt axis in regulating osteogenic differentiation of hADSCs. Moreover, ectopic bone formation in nude mice was used to investigate the effect of LMO3 on osteogenesis in vivo. In this study, we found the expression of LMO3 was significantly upregulated during the osteogenic differentiation of hADSCs. LMO3 knockdown remarkably suppressed osteogenic differentiation of hADSCs, while LMO3 overexpression promoted osteogenic differentiation of hADSCs both in vitro and in vivo. Moreover, we discovered that the enhancing effect of LMO3 overexpression on osteogenic differentiation was related to the activation of PI3K/Akt signaling pathway. Inhibition of PI3K/Akt signaling pathway with wortmannin effectively blocked the stimulation of osteogenic differentiation induced by LMO3 overexpression. In conclusion, based on transcriptomic analysis, we identified key genes involved in regulating the osteogenic differentiation of hADSCs. In addition, we found that LMO3 might act as a positive modulator of hADSC osteogenic differentiation by mediating PI3K/Akt signaling pathway. Manipulating the expression of LMO3 and its associated pathways might contribute to advances in bone regeneration and tissue engineering.

RAB37 Promotes Adipogenic Differentiation of hADSCs via the TIMP1/CD63/Integrin Signaling Pathway.

The adipogenic differentiation ability of human adipose-derived mesenchymal stem cells (hADSCs) is critical for the construction of tissue engineering adipose, which shows promising applications in plastic surgery and regenerative medicine. RAB37 is a member of the small RabGTPase family and plays a critical role in vesicle trafficking. However, the role of RAB37 in adipogenic differentiation of hADSCs remains unclear. Here, we report that both the mRNA and protein levels of RAB37 fluctuated during adipogenic differentiation. Upregulation of RAB37 was observed at the early stage of adipogenic differentiation, which was accompanied by increased expression of transcription factors PPARγ2 and C/EBPα, and lipoprotein lipase (LPL). Overexpression of RAB37 promoted adipogenesis of hADSCs, as revealed by Oil Red O staining and increased expression of PPARγ2, C/EBPα, and LPL. Several upregulated cytokines related to RAB37-mediated adipogenic differentiation were identified using a cytokine array, including tissue inhibitor of matrix metalloproteinase 1 (TIMP1). ELISA confirmed that upregulation of RAB37 increased the secretion of TIMP1 by hADSCs. Proximity ligation assay showed that RAB37 interacts with TIMP1 directly. Knockdown of TIMP1 compromised RAB37-mediated adipogenic differentiation. In addition, TIMP1 binds membrane receptor CD63 and integrin β1. RAB37 promotes Tyr397 phosphorylation of FAK, an important protein kinase of the integrin β1 signaling. Moreover, both knockdown of CD63 and inhibitor of FAK impeded RAB37-mediated adipogenic differentiation. In conclusion, RAB37 positively regulates adipogenic differentiation of hADSCs via the TIMP1/CD63/integrin β1 signaling pathway.

Directed Regeneration of Osteochondral Tissue by Hierarchical Assembly of Spatially Organized Composite Spheroids.

The use of engineered scaffolds or stem cells is investigated widely in the repair of injured musculoskeletal tissue. However, the combined regeneration of hierarchical osteochondral tissue remains a challenge due to delamination between cartilage and subchondral bone or difficulty in spatial control over differentiation of transplanted stem cells. Here, two types of composite spheroids are prepared using adipose‐derived stem cells (hADSCs) and nanofibers coated with either transforming growth factor‐β3 or bone morphogenetic growth factor‐2 for chondrogenesis or osteogenesis, respectively. Each type of spheroid is then cultured within a 3D‐printed microchamber in a spatially arranged manner to recapitulate the bilayer structure of osteochondral tissue. The presence of inductive factors regionally modulates in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct without dedifferentiation. Furthermore, hADSCs from each spheroid proliferate and sprout and successfully connect the two layers mimicking the osteochondral interface without apertures. In vivo transplantation of the biphasic construct onto a femoral trochlear groove defect in rabbit knee joint results in 21.2 ± 2.8% subchondral bone volume/total volume and a cartilage score of 25.0 ± 3.7. The present approach can be an effective therapeutic platform to engineer complex tissue.

Osteogenic Property Regulation of Stem Cells by a Hydroxyapatite 3D-Hybrid Scaffold With Cancellous Bone Structure

Cancellous bone plays an indispensable role in the skeletal system due to its various functions and high porosity. In this work, chitosan and hydroxyapatite nanowires (CS@HAP NWs) hybrid nanostructured scaffolds with suitable mechanical properties, high porosity and a fine porous structure were prepared to simulate the 3-dimensional structure of cancellous bone. The 3D-hybrid scaffolds promote cell adhesion and the migration of human adipose-derived stem cells (hADSCs) inside the scaffolds. The cavities in the scaffolds provide space for the hADSCs proliferation and differentiation. Moreover, the various contents of HAP and the induced mechanical property changes regulate the differentiation of hADSCs toward osteoblasts. Overall, cellular fate regulation of hADSCs via rationally engineered HAP-based hybrid scaffolds is a facile and effective approach for bone tissue engineering.

LncRNA NONHSAT030515 promotes the chondrogenic differentiation of human adipose-derived stem cells via regulating the miR-490-5p/BMPR2 axis

BackgroundChondrogenic differentiation of human adipose-derived stem cells (hADSCs) is important for cartilage generation and degradation. LncRNAs play an essential role in stem cell differentiation. However, the role and mechanism of lncRNA in hADSCs remain unclear. Our previous study showed that miR-490-5p was downregulated during chondrogenic differentiation of hADSCs. In this study, we investigated the effect and mechanism of lncRNA NONHSAT030515 interacting with miR-490-5p on chondrogenic differentiation of hADSCs.MethodsAlcian blue staining was used to assess the deposition of chondromatrix proteins following chondrogenic differentiation of human adipose stem cells. Immunohistochemistry was used to evaluate the expression of collagenII. TargetScan, miRTarBase and miRDB database analyses were applied to find the miRNA and target genes of lncRNA NONHSAT030515. A dual luciferase experiment was conducted to identify the direct target of NONHSAT030515. pcDNA3.1- NONHSAT030515 transfection and sh- NONHSAT030515 treatment were conducted to verify the role of lncRNA NONHSAT030515 in chondrogenic differentiation. The levels of Aggrecan, SOX9 and COL2A1 were analyzed by qRT-PCR and Western blot assay.ResultsAlcian blue staining, immunocytochemical, qRT-PCR, and Western blot have determined that lncRNA NONHSAT030515 can promote the chondrogenic differentiation of hADSCs. MiR-490- 5p was the direct target of NONHSAT030515, while BMPR2 was the target gene. This result was confirmed by luciferase reporter assay. Up-regulation of NONHSAT030515 promoted BMPR2 protein expression and promoted chondrogenic differentiation, whereas down-regulation of NONHSAT030515 caused completely opposite results.ConclusionLncRNA NONHSAT030515 promotes the chondrogenic differentiation of hADSCs through increasing BMPR2 expression by regulating miR-490- 5p.

Porous BMP-2 immobilized PLGA/Glycol chitosan scaffold with enhanced hydrophilicity, mineralization and osteogenesis

In this study, we investigated the effectiveness of poly (lactic-co-glycolic acid) (PLGA) porous scaffold in enhancing bone regeneration through surface modification with glycol chitosan (GCH) and bone morphogenetic protein-2(BMP-2). Immobilization of GCH on PLGA (PLGA-GCH) and simple immobilization of osteogenic BMP-2 on PLGA-GCH (PLGA-GCH-BMP-2) via EDC/NHS as crosslinker was highly efficient. The surface morphologies of the scaffolds were observed using a scanning electron microscopy (SEM). The immobilization of GCH and BMP-2 on PLGA was characterized using Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS), which confirmed the conjugation of GCH and BMP-2 peptides on PLGA. Human adipose derived stem cells (hADSCs) were cultured on the scaffolds to determine the effect of surface modification on mineralization and cell differentiation. PLGA-GCH-BMP-2 scaffold showed enhanced in-vitro calcium mineralization and osteogenic differentiation of hADSCs using osteogenic medium. The developed scaffold might be effective filler materials for enhancing osteogenesis in 3D printed vertebra spacers targeting the aged patients.

Effect of Eucomis autumnalis on the Osteogenic Differentiation of Adipose-Derived Stem Cells.

Eucomis autumnalis subsp. autumnalis (Mill.) Chitt. (EASA) is a commonly used medicinal plant for the treatment of fractures, osteoarthritis, back pain, and wound healing in Southern Africa. In this study, the effects of water and acetone extracts of EASA on the viability, osteogenic differentiation, and mineralization of human adipose-derived stem cells (hADSCs) were investigated in vitro. The results showed that both water and acetone extracts of EASA increased cell viability at concentrations between 10 and 50 μg/mL on day 7 and 14 of treatment. Osteogenic differentiation and mineralization of hADSCs were optimal at 5 μg/mL for the water extract and at 5-10 μg/mL for the acetone extract. A 5 μg/mL acetone extract upregulated the expression of the ALP, Runx2, Col1a1, and osteopontin genes. In addition, EASA upregulated β-catenin, cyclin D1, and osteoprotegerin (OPG). The results suggest that EASA may likely upregulate the expression of β-catenin, which subsequently upregulates the osteogenic marker genes through Runx2. However, EASA also upregulates cyclin D1 supporting the growth of precursor cells. Additionally, EASA upregulated the expression of OPG suggesting that it may inhibit bone resorption. The results of this study support the traditional use of the plant in bone healing. Impact statement Herbal remedies are used to treat various ailments by almost 80% of the developing world. Eucomis autumnalis is one of the most used medicinal plants in Southern Africa for the treatment of backache, osteoarthritis, and healing of fracture. There is a scarcity of scientific evidence to prove the efficacy of E. autumnalis for fracture repair. This study sought to assess the effect of the crude extracts in vitro and found that E. autumnalis promoted viability and osteogenic differentiation of human adipose-derived stem cells as demonstrated by biochemical and genetic markers. This medicinal plant could therefore have potential to regenerate bone tissue.

MiR-149-3p can improve the osteogenic differentiation of human adipose-derived stem cells via targeting AKT1.

The study aims to investigate the role of microRNA-149-3p (miR-149-3p) in regulating osteogenic differentiation of human adipose-derived stem cells (hADSCs) by targeting v-akt murine thymoma viral oncogene homolog 1 (AKT1). Bioinformatics websites and a dual luciferase reporter assay were used to predict and verify the targeting relationship between miR-149-3p and AKT1. The hADSCs were divided into the blank, negative control (NC), mimic, control siRNA, AKT1 siRNA, and miR-149-3p inhibitors + AKT1 siRNA groups and then subjected to Alizarin Red staining, Alkaline phosphatase (ALP) staining, ALP activity detections, MTT assay, and EdU cell proliferation assay. Gene or protein expression was quantified using quantitative real-time PCR (qRT-PCR) or Western blotting, respectively. The miR-149-3p expression increased gradually and AKT1 expression decreased gradually during osteogenic differentiation of hADSCs. The prediction of bioinformatics websites miRTarBase and TargetScan and the dual luciferase reporter assay indicated that miR-149-3p can directly target AKT1. After hADSCs were transfected with miR-149-3p mimic, AKT1 expression was significantly downregulated. However, transfection with AKT1 siRNA did not have an impact on miR-149-3p in hADSCs. In comparison with the AKT1 siRNA group, the miR-149-3p inhibitors + AKT1 siRNA group showed decreased miR-149-3p expression but increased AKT1 expression. In addition, AKT1 siRNA enhanced the cell viability and proliferation of hADSCs and increased mineral calcium deposition and ALP activity, resulting in higher expression of osteogenic differentiation-related genes, which was reversed by miR-149-3p inhibition. The miR-149-3p can increase the expression of osteogenic differentiation-related genes by targeting AKT1 and thereby enhance the osteogenic differentiation of hADSCs.

MiR-125a-5p Regulates Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells under Oxidative Stress.

Adipose-derived mesenchymal stem cells (ADSCs) are a well-recognized multilineage stem cell with vital clinical feasibility for tissue regeneration. Extensive evidence indicates that oxidative stress and microRNAs (miRNAs/miRs) play an important role in the osteoinduction of adipose-derived mesenchymal stem cells. In this study, we investigated the mechanism of miR-125a-5p in regulating the osteogenesis of human adipose-derived mesenchymal stem cells (hADSCs) under oxidative stress. The expression of miR-125a-5p lessened gradually during the osteogenic differentiation of hADSCs. Relative to the negative group, the expression levels of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and osterix in the miR-125a-5p group were marked lower than those in the miR-125a-5p inhibitor group. The levels of p16, p21, p53, miR-125a-5p, and ROS during osteoinduction of hADSCs were assessed in vitro under oxidative stress and were observed to be upregulated. Further experiments showed that oxidative stress and miR-125a-5p together suppressed the expression of VEGF during osteogenic differentiation of hADSCs and that the inhibition of miR-125a-5p reversed the effect of oxidative stress. In short, our study indicated that miR-125a-5p is induced under oxidative stress and inhibits the expression of VEGF, leading to the reduction of osteogenic differentiation of hADSCs. Our outcomes showed that miR-125a-5p could be a potential clinical target for bone repairing.

Guiding Stem Cell Differentiation and Proliferation Activities Based on Nanometer-Thick Functionalized Poly-p-xylylene Coatings

Modifications of biomaterials based on the combination of physical, chemical, and biological cues for manipulating stem cell growth are needed for modern regenerative medicine. The exploitation of these sophisticated modifications remains a challenge, including substrate limitation, biocompatibility, and versatile and general cues for stem cell activities. In this report, a vapor-phase coating technique based on the functionalization of poly-p-xylylene (PPX) was used to generate a surface modification for use with stem cells in culture. The coating provided the ability for covalent conjugation that immobilized bone morphogenetic protein 2 (BMP-2) and fibroblast growth factor 2 (FGF-2), and the modified coating surfaces enabled direct stem cell differentiation and controlled proliferation because of the specific activities. The ligations were realized between the growth factors and the maleimide-modified surface, and the conjugation reactions proceeded with high specificity and rapid kinetics under mild conditions. The conjugation densities were approximately 140 ng·cm−2 for BMP-2 and 155 ng·cm−2 for FGF-2. Guiding the activities of the human adipose-derived stem cells (hADSCs) was achieved by modifying surfaces to promote the hADSC differentiation capacity and proliferation rate. The reported coating system demonstrated biocompatibility, substrate-independent conformity, and stability, and it could provide an effective and versatile interface platform for further use in biomedical applications.

MiR-105 enhances osteogenic differentiation of hADSCs via the targeted regulation of SOX9

ObjectiveTo investigate whether miR-105 can regulate the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) by targeting SOX9. MethodsThe hADSCs were grouped for subsequent transfection and induction of osteogenic differentiation as follows: control, miR-NC, miR-105 mimics, miR-105 inhibitors, SOX9, SOX9 siRNA, miR-105 mimics + SOX9 and miR-105 inhibitors + SOX9 siRNA groups. Next, hADSCs were stained for alkaline phosphatase (ALP), and Alizarin Red S staining (ARS) was performed. Osteogenic differentiation-related genes and miR-105 expression were assessed by qRT-PCR, while SOX9 protein expression was determined by Western blotting. ResultsMiR-105 expression was increased and SOX9 protein expression was decreased during the osteogenic differentiation of hADSCs. A dual-luciferase reporter assay confirmed SOX9 to be a target gene of miR-105. Compared with the control group, the miR-105 mimics and SOX9 siRNA groups had elevated BMP2, OPN, OCN, BSP, Osx and Runx2 mRNA expression with reduced SOX9 expression, as well as increased ARS intensity and ALP activity. After transfection of miR-105 inhibitors/SOX9 into hADSCs, the results were the opposite. Overexpressing SOX9 reversed the effect of miR-105 in promoting the osteogenic differentiation of hADSCs. ConclusionMiR-105 could target SOX9 to improve the expression of osteogenic differentiation genes and thus enhance the osteogenic differentiation of hADSCs.

Synergic effects of extremely low-frequency electromagnetic field and betaine on in vitro osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.

Human adipose tissue-derived mesenchymal stem cells (hADSCs) due to easy extraction, relative abundance, in vitro expansion and differentiation potential, frozen storage capability, and ability to secrete cytokines, compared to other stem cells, are appropriate candidate in regenerative medicine. Extremely low-frequency electromagnetic fields (ELF-EMF) and betaine are two safe factors in bone lesions repair. This study was designed to assess the osteogenic differentiation potential of these factors on hADSCs. The samples were collected from women undergoing liposuction after obtaining written consent. The hADSCs were extracted and treated with osteogenesis differentiation medium (OD) as the positive control, with OD and betaine (BET group), with OD and EMF (EMF group), and with OD and betaine and EMF (BET+EMF group) for 21 d; the negative control consisted of cells without treatment. Betaine 10 mM and EMF with 50-Hz frequency, 1-mT intensity (8 h daily), and in the form of sinus wave were used. Osteogenic differentiation was evaluated by Alizarin Red staining, alkaline phosphatase activity, calcium deposition, and real-time PCR. A significant increase in calcium deposition in the BET+EMF group was observed compared to the other groups. The activity of alkaline phosphatase in the positive control and BET groups was increased significantly compared to EMF and BET + EMF groups and a significant increase of this enzyme activity in the BET + EMF compared to EMF group was observed. The expression of RUNX2 and OCN genes in the EMF-treated groups were significantly reduced compared to the non-EMF-treated groups, and BET+EMF showed a significant increase of RUNX2 gene expression as compared the EMF group. The ELF-EMF leads to a decrease in the osteogenic differentiation and the expression RUNX2 and OCN genes in hADSCs. But osteogenic differentiation and RUNX2 gene expression were increased post-induction by betaine. The synergic effect of betaine and EMF on the osteogenic differentiation and related genes expression of hADSCs was higher than EMF.