June 2023
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12 Reads
Journal of Cell Science
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June 2023
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12 Reads
Journal of Cell Science
January 2017
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53 Reads
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46 Citations
Cellular Physiology and Biochemistry
Background: BMP9 induces both osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). Nell1 is a secretory glycoprotein with osteoinductive and anti-adipogenic activities. We investigated the role of Nell1 in BMP9-induced osteogenesis and adipogenesis in MSCs. Methods: Previously characterized MSCs iMEFs were used. Overexpression of BMP9 and NELL1 or silencing of mouse Nell1 was mediated by adenoviral vectors. Early and late osteogenic and adipogenic markers were assessed by staining techniques and qPCR analysis. In vivo activity was assessed in an ectopic bone formation model of athymic mice. Results: We demonstrate that Nell1 expression was up-regulated by BMP9. Exogenous Nell1 potentiated BMP9-induced late stage osteogenic differentiation while inhibiting the early osteogenic marker. Forced Nell1 expression enhanced BMP9-induced osteogenic regulators/markers and inhibited BMP9-upregulated expression of adipogenic regulators/markers in MSCs. In vivo ectopic bone formation assay showed that exogenous Nell1 expression enhanced mineralization and maturity of BMP9-induced bone formation, while inhibiting BMP9-induced adipogenesis. Conversely, silencing Nell1 expression in BMP9-stimulated MSCs led to forming immature chondroid-like matrix. Conclusion: Our findings indicate that Nell1 can be up-regulated by BMP9, which in turn accelerates and augments BMP9-induced osteogenesis. Exogenous Nell1 may be exploited to enhance BMP9-induced bone formation while overcoming BMP9-induced adipogenesis in regenerative medicine.
December 2013
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25 Reads
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23 Citations
Journal of Biomedical Materials Research Part A
Articular cartilage lesions in the knee are common injuries. Chondrocyte transplant represents a promising therapeutic modality for articular cartilage injuries. Here, we characterize the viability and transgene expression of articular chondrocytes cultured in three-dimensional scaffolds provided by four types of carriers. Articular chondrocytes are isolated from rabbit knees and cultured in four types of scaffolds: type I collagen sponge, fibrin glue, hyaluronan, and open-cell polylactic acid (OPLA). The cultured cells are transduced with adenovirus expressing green fluorescence protein (AdGFP) and luciferase (AdGL3-Luc). The viability and gene expression in the chondrocytes are determined with fluorescence microscopy and luciferase assay. Cartilage matrix production is assessed by Alcian blue staining. Rabbit articular chondrocytes are effectively infected by AdGFP and exhibited sustained GFP expression. All tested scaffolds support the survival and gene expression of the infected chondrocytes. However, the highest transgene expression is observed in the OPLA carrier. At 4 weeks, Alcian blue-positive matrix materials are readily detected in OPLA cultures. Thus, our results indicate that, while all tested carriers can support the survival of chondrocytes, OPLA supports the highest transgene expression and is the most conductive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repairs. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.
September 2013
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168 Reads
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41 Citations
Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.
September 2013
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14 Reads
Primers used for sqPCR, ChIP, siRNA and cloning. (XLS)
September 2013
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15 Reads
Top 20 BMP9 up-regulated genes in MSCs. (XLS)
August 2013
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949 Reads
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254 Citations
Journal of Biomedical Science and Engineering
Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and have diverse functions during development and organogenesis. BMPs play a major role in skeletal development and bone formation, and disruptions in BMP signaling cause a variety of skeletal and extraskeletal anomalies. Several knockout models have provided insight into the mechanisms responsible for these phenotypes. Proper bone formation requires the differentiation of osteoblasts from mesenchymal stem cell (MSC) precursors, a process mediated in part by BMP signaling. Multiple BMPs, including BMP2, BMP6, BMP7 and BMP9, promote osteoblastic differentiation of MSCs both in vitro and in vivo. BMP9 is one of the most osteogenic BMPs yet is a poorly characterized member of the BMP family. Several studies demonstrate that the mechanisms controlling BMP9-mediated osteogenesis differ from other osteogenic BMPs, but little is known about these specific mechanisms. Several pathways critical to BMP9-mediated osteogenesis are also important in the differentiation of other cell lineages, including adipocytes and chondrocytes. BMP9 has also demonstrated translational promise in spinal fusion and bone fracture repair. This review will summarize our current knowledge of BMP-mediated osteogenesis, with a focus on BMP9, by presenting recently completed work which may help us to further elucidate these pathways.
July 2013
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101 Reads
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67 Citations
Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.
July 2013
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52 Reads
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90 Citations
Journal of Orthopaedic Research
Mesenchymal stem cells (MSCs) are multipotent progenitors and can differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bone morphogenetic proteins (BMPs) play important roles in stem cell proliferation and differentiation. We recently demonstrated that BMP9 is a potent but less understood osteogenic factor. We previously found that BMP9-induced ectopic bone formation is not inhibited by BMP3. Here, we investigate the effect of BMP antagonist noggin on BMP9-induced osteogenic differentiation. BMP antagonists noggin, chording, gremlin, follistatin, and BMP3 are highly expressed in MSCs, while noggin and follistatin are lowly expressed in more differentiated pre-osteoblast C2C12 cells. BMP9-induced osteogenic markers and matrix mineralization are not inhibited by noggin, while noggin blunts BMP2, BMP4, BMP6, and BMP7-induced osteogenic markers and mineralization. Likewise, ectopic bone formation by MSCs transduced with BMP9, but not the other four BMPs, is resistant to noggin inhibition. BMP9-induced nuclear translocation of Smad1/5/8 is not affected by noggin, while noggin blocks BMP2-induced activation of Smad1/5/8 in MSCs. Noggin fails to inhibit BMP9-induced expression of downstream targets in MSCs. Thus, our results strongly suggest that BMP9 may effectively overcome noggin inhibition, which should at least in part contribute to BMP9's potent osteogenic capability in MSCs. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
June 2013
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11 Reads
... Athymic nude mice were obtained from and housed in the Experimental Animal Research Center of Chongqing Medical University. Stem cell-based ectopic bone formation was performed as previously described [69][70][71][72][73][74][75]. Briefly, subconfluent cells were infected with Ad-B9 or Ad-GFP for 36 h, collected and resuspended in sterile PBS for subcutaneous injection into the flanks of athymic nude mice (5-6 week old, male, 5 × 10 6 cells/injection, 6 injections per mouse, and 3 mice per group). ...
January 2017
Cellular Physiology and Biochemistry
... The late inflammatory phase (following the formation of a hematoma and an innate immune response) is marked by the polarization of macrophages to the M2 phenotype, followed by their secretion of osteogenic factors such as transforming growth factor β (TGFβ), which recruits osteoprogenitors to the site of injury [58]. The differentiation of osteoprogenitors into osteoblasts is stimulated by the presence of growth factors such as BMP-2 [59]. During the repair phase, the hematoma is replaced by a soft callus (unmineralized) formed by the chondrocyte secretion of cartilaginous matrix. ...
August 2013
Journal of Biomedical Science and Engineering
... Bone morphogenetic proteins (BMPs) are implicated in osteogenic differentiation and ectopic bone formation [77]. BMP2 and BMP9 induce ER stress to promote the differentiation of BMSCs into osteoblasts [78][79][80][81]. UPR signaling is an essential regulator of bone development [82,83]. ...
September 2013
... As CTM possuem grande potencial em sua aplicabilidade na ciência (REDMAN et al., 2016;ZHANG et al., 2019;VHORA et al., 2019), podendo se diferenciar em determinadas linhagens de origens condrogênicas, adipogênicas e osteogênicas, que em associação as BMPs apresentam funções importantes no processo de proliferação (WANG et al., 2013;LIU et al., 2021;REN et al., 2016). Estudos recentes destacam que as BMPs, em especial a de numeração nove, apresenta grande formação de tecido ósseo (ZHOU et al., 2020;VHORA et al., 2019;KHORSAND et al., 2017). ...
July 2013
Journal of Orthopaedic Research
... Athymic nude mice were obtained from and housed in the Experimental Animal Research Center of Chongqing Medical University. Stem cell-based ectopic bone formation was performed as previously described [69][70][71][72][73][74][75]. Briefly, subconfluent cells were infected with Ad-B9 or Ad-GFP for 36 h, collected and resuspended in sterile PBS for subcutaneous injection into the flanks of athymic nude mice (5-6 week old, male, 5 × 10 6 cells/injection, 6 injections per mouse, and 3 mice per group). ...
July 2013
... Cells labeled with firefly luciferase were harvested with trypsin, suspended in 60-80 uL PBS, and kept on ice until injection into the host mice (Westerman and Leboulch, 1996;Li et al., 2013). Prior to injection, the mice were anesthetized using isoflurane gas. ...
June 2013
International Journal of Medical Sciences
... This regeneration encompasses the renewal of the periodontal ligament and alveolar bone, entailing complex processes such as cell chemotaxis, adhesion, proliferation, and differentiation, each requiring distinct regulatory factors at various stages and tissues [11]. Extensive research [12,13] has validated that FGF2 and BMP9 significantly enhance the proliferation and differentiation of periodontal ligament stem cells, thereby facilitating periodontal tissue regeneration. However, challenges such as the initial "burst release effect" and vulnerability to protease degradation in vivo persist, and the solitary application of FGF2 or BMP9 is insufficient for modulating the entire periodontal regeneration process. ...
May 2013
American Journal of Stem Cells
... Unlike the protein-based treatments which have a short half-life, gene therapies aim to establish persistent, endogenous synthesis of the trans-gene products at target sites through IA injection (Mobasheri, 2019). Recently, this has been carried out in vivo and ex vivo studies, using different delivery vectors (non-viral or viral), target genes (growth factors, transcription factors, antiinflammatory cytokines, cell signaling protein iHH, ECM protein, integrin-β1), and cells (stem cells, chondrocytes) with or without biomaterials (Heiligenstein et al., 2011;Shui et al., 2013). ...
December 2013
Journal of Biomedical Materials Research Part A
... TGF-β, a pleiotropic cytokine abundantly seen in bone remodeling ecosystems has also been extensively studied for its contribution to proliferation and differentiation of human PDLSCs [40]. The Wnt signaling pathway a signal transduction pathway is also known to be crucial for osteogenic differentiation and bone formation in MSCs, playing a role in periodontal homeostasis, pathological process, and tissue regeneration [41]. In hPDLSCs, Wnt signaling pathway served as a key regulator of osteogenic differentiation [42]. ...
February 2013
... After being stimulated mechanically or by BMP binding, these receptors operate on the Smad protein in the cytoplasm, which is their downstream target. Smad protein is a recently identified intracellular signal transduction protein (Hu et al., 2013). The phosphorylation of Smad proteins is further encouraged by activating these two receptor types. ...
November 2012
Journal of Cell Science