Overexpression of β-NGF promotes differentiation of bone marrow mesenchymal stem cells into neurons through regulation of AKT and MAPK pathway

Bone marrow stromal stem cells (BMSCs) are fibroblastic in shape and capable of self-renewal and have the potential for multi-directional differentiation. Nerve growth factor (NGF), a homodimeric polypeptide, plays an important role in the nervous system by supporting the survival and growth of neural cells, regulating cell growth, promoting differentiation into neuron, and neuron migration. Adenoviral vectors are DNA viruses that contain 36 kb of double-stranded DNA allowing for transmission of the genes to the host nucleus but not inserting them into the host chromosome. The present study aimed to investigate the induction efficiency and differentiation of neural cells from BMSCs by β-NGF gene transfection with recombinant adenoviral vector (Ad-β-NGF) in vitro. The results of immunochemical assay confirmed the induced cells as neuron cells. Moreover, flow cytometric analysis, Annexin-V-FITC/PI, and BrdU assay revealed that chemical inducer β-mercaptoethanol (β-met) triggered apoptosis of BMSCs, as evidenced by inhibition of DNA fragmentation, nuclear condensation, translocation of phospholipid phosphatidylserine, and activation of caspase-3. Furthermore, the results of western blotting showed that β-met suppressed AKT signaling pathway and regulated the MAPKs during differentiation of BMSCs. In contrast, Ad-β-NGF effectively induced the differentiation of BMSCs without causing any cytopathic phenomenon and apoptotic cell death. Moreover, Ad-β-NGF recovered the expression level of phosphorylated AKT and MAPKs in cells exposed to chemical reagents. Taken together, these results suggest that β-NGF gene transfection promotes the differentiation of BMSCs into neurons through regulation of AKT and MAPKs signaling pathways.     

Neurogenesis of bone marrow-derived mesenchymal stem cells onto β-mercaptoethanol-loaded PLGA film

Bone marrow-derived mesenchymal stem cells (BMSCs) are of particular interest in the field of tissue engineering because of their potential to differentiate into osteoblasts, chondrocytes, and neuronal cells. In order to promote the differentiation of BMSCs into specific cell types, appropriate scaffold biomaterials and bioactive molecules that can support the differentiation of BMSCs into specific cell types are needed. We hypothesized that β-mercaptoethanol (BME), which has been reported to induce the differentiation of BMSCs into neural-like cells, promotes BMSCs to differentiate into neural-like cells when BME is added to polymeric scaffolds containing the BMSCs. We fabricated biocompatible film shaped scaffolds composed of poly(lacti-co-glycolic) acid (PLGA) and various concentrations of BME to confirm that BME-promoted differentiation of BMSCs is concentration-dependent. Cell proliferation increased as the BME concentration in the films increased at the early stage, and the proliferation rate remained similar on the PLGA films for 3 weeks following the BMSC seeding. The expression of neuronal markers in differentiated BMSCs was assessed by RT-PCR. At 2- and 3-week time-points, mRNA expression of neurofilament and neuron specific enolase was significantly increased in PLGA/BME films containing 400 μM BME compared to PLGA films. Thus, we have identified BMSC-seeded PLGA/BME films with 200 μM and 400 μM BME as potentially useful candidates for neural tissue engineering applications by promoting BMSC proliferation and differentiation towards neural-like cells.     

Semaphorin 3A promotes the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells in inflammatory environments by suppressing the Wnt/β-catenin signaling pathway

Journal of Molecular Histology - After periodontal treatment, the local inflammatory environment surrounding periodontal tissues cannot be entirely eliminated. The means by which alveolar bone...     

Platelet-rich plasma promotes the regeneration of cartilage engineered by mesenchymal stem cells and collagen hydrogel via the TGF-β/SMAD signaling pathway

The tissue engineering technique using mesenchymal stem cells (MSCs) and scaffolds is promising. Transforming growth factor-β1 (TGF-β1) is generally accepted as an chondrogenic agent, but immunorejection and unexpected side effects, such as tumorigenesis and heterogeneity, limit its clinical application. Autogenous platelet-rich plasma (PRP), marked by low immunogenicity, easy accessibility, and low-cost, may be favorable for cartilage regeneration. In our study, the effect of PRP on engineered cartilage constructed by MSCs and collagen hydrogel in vitro and in vivo was investigated and compared with TGF-β1. The results showed that PRP promoted cell proliferation and gene and protein expressions of chondrogenic markers via the TGF-β/SMAD signaling pathway. Meanwhile, it suppressed the expression of collagen type I, a marker of fibrocartilage. Furthermore, PRP accelerated cartilage regeneration on defects with engineered cartilage, advantageous over TGF-β1, as evaluated by histological analysis and immunohistochemical staining. Our work demonstrates that autogenous PRP may substitute TGF-β1 as a potent and reliable chondrogenic inducer for therapy of cartilage defect.     

Interferon-γ enhances the immunosuppressive ability of canine bone marrow-derived mesenchymal stem cells by activating the TLR3-dependent IDO/kynurenine pathway

Molecular Biology Reports - The immunomodulatory function of mesenchymal stem cells (MSCs) has been considered to be vital for MSC-based therapies. Many works have been devoted to excavate...     

MicroRNA-200c promotes osteogenic differentiation of human bone mesenchymal stem cells through activating the AKT/β-Catenin signaling pathway via downregulating Myd88

During the human bone formation, the event of osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) is vital, and recent evidence has emphasized the important role of microRNAs (miRNAs) in osteogenic differentiation of hBMSCs. This study aims to examine the potential effects of miR-200c in osteogenic differentiation of hBMSCs and understand their underlying mechanisms. HBMSCs were obtained via human bone marrow. During osteogenic induction and differentiation, cells were transfected with different plasmids with the intention of investigating the roles of miR-200c on osteogenic differentiation, calcium salt deposition, alkaline-phosphatase (ALP) activity, mineralized nodule formation, osteocalcin (OCN) content, and proliferation of osteoblasts. Following transfection, dual luciferase reporter gene assay was conducted so as to explore the correlation between miR-200c and Myd88. Moreover, the AKT/β-Catenin signaling pathway was blocked with an AKT/β-Catenin inhibitor, AKTi, to investigate its involvement. The hBMSCs were successfully isolated from human bone marrow. Myd88 was determined as a target gene of miR-200c. Gain and loss-of-function assays confirmed that overexpression of miR-200c, or silencing of Myd88 promoted osteogenic differentiation, increased calcium salt deposition, ALP activity, mineralized nodule formation, and enhanced the proliferation of osteoblasts following osteogenic differentiation of hBMSCs. Meanwhile, the downregulation of miR-200c has been shown to have the opposite effect. Furthermore, these findings showed that the miR-200c overexpression activated the AKT/β-Catenin signaling pathway by targeting Myd88. To sum up, the miR-200c upregulation induces osteogenic differentiation of hBMSCs by activating the AKT/β-Catenin signaling pathway via the inhibition of Myd88, providing a target for treatment of bone repair.     

Study of Wnt2 secreted by A-549 cells in paracrine activation of β-catenin in co-cultured mesenchymal stem cells

The canonical Wnt signal pathway is a key regulator of self-renewal and cell fate determination in various types of stem cells. The total pool of β-catenin consists of two different forms: the signaling form of the protein transmits the Wnt signals from the cell membrane to the target genes, whereas the membrane β-catenin is involved in formation of cell-to-cell contact at cadherin junctions. Earlier we developed an in vitro model of epithelial differentiation of mesenchymal stem cells (MSCs) co-cultured with epithelial A-549 cells. The purpose of the present work was to study the role of Wnt2 secreted by the A-549 cells in paracrine induction of β-catenin in co-cultured MSCs. Using the somatic gene knockdown technique, we obtained A-549 cell cultures with down-regulated WNT2. The MSCs co-cultured with the control A-549 cells displayed an increase in the levels of total cellular and signaling β-catenin and transactivation of a reporter construction containing the Lef/Tcf protein family binding sites. In contrast, β-catenin was not induced in the MSCs co-cultured with the A-549 cells with down-regulated WNT2 expression, but the total protein level was increased. We suggest that Wnt2 secreted by A-549 cells induces in co-cultured MSCs the Wnt/β-catenin signaling pathway, whereas the associated increase in total β-catenin level should be due to another mechanism.     

Cardiomyocyte-like cells differentiation from non β-catenin expression mesenchymal stem cells

Recent studies have shown that block wnt/β-catenin signaling pathway is integrant for cardiomyocytes differentiation from bone marrow mesenchymal stem cells (MSCs). By transducing the MSCs with lentivirus which contain β-catenin interference RNA, we screened out the non β-catenin expression clone. In the establishment of knockdown β-catenin in MSCs, we investigated the role of 5-azacytidine (5-aza), salvianolic acid B (salB), and cardiomyocytes lysis medium (CLM) in inducing MSCs to differentiate into cardiomyocyte-like cells. A method for culturing MSCs and cardiomyocytes was established. Purified MSCs were investigated by flow cytometry. The MSCs were positive for CD90 and CD29, but negative for CD34 and CD45. Meanwhile, the cardiomyocytes contracted spontaneously after 24 h of seeding into the plates. The fourth-passage non-β-catenin expression MSCs were divided into eight groups: control group, 5-aza, salB, CLM, 5-aza + salB, 5-aza + CLM, salB + CLM, and 5-aza + salB + CLM. The gene and protein expression of cTnT, α-actin, β-myosin, β-catenin, and GSK-3β were detected by quantitative real-time PCR and Western blotting. Our results showed that cTnT expression in 5-aza + salB + CLM group was ninefold higher than in the control group in the non-β-catenin MSCs model, implying that cardiomyocytes differentiation from MSCs is an extremely complicated process and it is necessary to consider the internal and external environmental conditions, such as suitable pharmaceutical inducers, cardiomyocytes microenvironments, inhibition of the negative signaling pathway and so on.     

Epidermal growth factor can signal via β-catenin to control proliferation of mesenchymal stem cells independently of canonical Wnt signalling

Bone marrow mesenchymal stem/stromal cells (MSCs) maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal lineages. Signalling mechanisms that enable precise control of MSC function remain unclear. Here we report that by initially examining differences in signalling pathway expression profiles of individual MSC clones, we identified a previously unrecognised signalling mechanism regulated by epidermal growth factor (EGF) in primary human MSCs. We demonstrate that EGF is able to activate β-catenin, a key component of the canonical Wnt signalling pathway. EGF is able to induce nuclear translocation of β-catenin in human MSCs but does not drive expression of Wnt target genes or T cell factor (TCF) activity in MSC reporter cell lines. Using an efficient Design of Experiments (DoE) statistical analysis, with different combinations and concentrations of EGF and Wnt ligands, we were able to confirm that EGF does not influence the Wnt/β-catenin pathway in MSCs. We show that the effects of EGF on MSCs are temporally regulated to initiate early “classical” EGF signalling mechanisms (e.g via mitogen activated protein kinase) with delayed activation of β-catenin. By RNA-sequencing, we identified gene sets that were exclusively regulated by the EGF/β-catenin pathway, which were distinct from classical EGF-regulated genes. However, subsets of classical EGF gene targets were significantly influenced by EGF/β-catenin activation. These signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by classical EGF signalling.     

Circ_FBLN1 promotes the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by regulating let-7i-5p/FZD4 axis and Wnt/β-catenin pathway

Background

Recently, more and more circular RNAs (circRNAs) have been identified in osteogenesis. In this study, we aimed to explore the effect of circ_FBLN1 on the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs).

Methods

The protein levels of osteogenesis-related genes, let-7i-5p, frizzled class receptor 4 (FZD4), Ki67, Wnt6 and β-catenin were measured by western blot assay. The levels of circ_FBLN1, FBLN1 mRNA and FZD4 mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR) assay. The feature of circ_FBLN1 was investigated by RNase R and Actinomycin D assays. Cell proliferation ability was evaluated by colony formation assay and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The targeting relationship between let-7i-5p and circ_FBLN1 or FZD4 was verified by dual-luciferase reporter assay.

Results

Circ_FBLN1 level was enhanced during the osteogenic differentiation of hBMSCs. Silencing of circ_FBLN1 repressed cell proliferation and osteogenic differentiation in hBMSCs. For mechanism analysis, circ_FBLN1 was found to act as a sponge for let-7i-5p and FZD4 served as a direct target gene of let-7i-5p. Let-7i-5p was downregulated during the osteogenic differentiation of hBMSCs and let-7i-5p inhibition restored the effects of circ_FBLN1 knockdown on the proliferation and osteogenesis of hBMSCs. Moreover, let-7i-5p overexpression suppressed cell proliferation and osteogenesis in hBMSCs through targeting FZD4. In addition, circ_FBLN1 knockdown reduced the levels of Wnt6 and β-catenin in hBMSCs, indicating the inactivation of Wnt/β-catenin pathway.

Conclusion

Knockdown of circ_FBLN1 inhibited the proliferation and osteogenesis of hBMSCs by regulating let-7i-5p/FZD4 axis and repressing Wnt/β-catenin pathway.

     

BMAL1 regulates balance of osteogenic–osteoclastic function of bone marrow mesenchymal stem cells in type 2 diabetes mellitus through the NF-κB pathway

In bone marrow mesenchymal stem cell (BMSCs), type 2 diabetes mellitus (T2DM) induces metabolic and functional disorders, leading to imbalanced bone resorption and formation and bone loss. Brain and muscle ARNT-like protein 1 (BMAL1) is involved in regulating T2DM-related suppression of BMSCs osteogenesis and bone formation. However, the relationship between BMAL1 and bone remodelling, especially bone resorption in T2DM, is unclear. We investigated the antergic role played by BMAL1 in T2DM-prompted imbalance in BMSCs osteogenic–osteoclastic function. BMAL1 was inhibited and the receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio was increased in diabetic BMSCs. Inhibitor κB (IκB) expression was decreased, whereas phosphorylated-p65 (p-p65), caspase-3, and p-IκB expression were increased in diabetic BMSCs. BMAL1 overexpression recovered the osteogenesis ability and suppressed osteoclastic induction capability of BMSCs to improve bone metabolism and function, which was partially due to NF-κB pathway activity inhibition. Our results provide evidence about the role of BMAL1 in T2DM-prompted BMSCs differentiation dysfunction, i.e. partially decreasing NF-κB pathway expression. In T2DM, it might be possible to use overexpressed BMAL1 to re-establish the homeostasis of bone metabolism.     

Overexpression of αCGRP promotes osteogenesis of periodontal ligament cells by regulation of YAP signaling

Human periodontal ligament cells (hPDLCs) are considered as an ideal cell type for periodontal tissue engineering as hPDLCs own mesenchymal stem cell-like properties. Additionally, it is suggested that α-calcitonin gene-related peptide (αCGRP) plays a pivotal role in the pathogenesis of periodontitis. However, the specific role of αCGRP on the regulation of alveolar bone regeneration which is essential for treatment of periodontitis remains unclear. In this study, lentiviral αCGRP expression vector was first transfected into hPDLCs. αCGRP expression and the osteogenesis-related gene (ALP, RUNX2, OCN, and BSP) expressions were detected. The results showed that expressions of osteogenic phenotypes were upregulated in αCGRP-transfected hPDLCs combined with an increased expression of Yes-associated protein (YAP), which is the key downstream effectors of Hippo pathway. Our observations suggest that αCGRP-mediated hPDLCs’ osteogenesis might relate with the activity of YAP signaling. These observations may reflect intrinsic functions of αCGRP in hPDLCs’ osteogenesis and its promising role in the treatment of bone deficiency in periodontal regeneration.