Induction Of Osteogenic Differentiation Research Articles

Carbon monoxide releasing molecule‑3 promotes the osteogenic differentiation of rat bone marrow mesenchymal stem cells by releasing carbon monoxide.

Stem cell‑based therapies are promising strategies to stimulate bone regeneration.Carbon monoxide releasing molecule‑3 (CORM‑3) exhibits multiple regulatory effects in a number of cells by releasing carbon monoxide (CO). The present study aimed to investigate the influence of CORM‑3 on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). BMSCs were divided into five groups: A CORM‑3‑osteogenic group, in which cells were pretreated with CORM‑3 and subjected to osteogenic differentiation induction using osteogenic medium; an osteogenic group, in which cells were cultured in osteogenic medium; a degassed CORM‑3‑osteogenic group, in which cells were pretreated with degassed CORM‑3 and subjected to osteogenic differentiation induction; a CORM‑3 group, in which cells were cultured in control medium containing CORM‑3; and a control group, in which cells were cultured in control medium alone. The osteo‑specific mRNA and protein expression of runt‑related transcription factor2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) were assessed using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Alkaline phosphatase (ALP) activity was also examined and mineralization was detected using alizarin red staining. Levels of Runx2, OCN and OPN mRNA and protein in the CORM‑3‑osteogenic group were significantly increased compared with the osteogenic group (P<0.05), with the exception of OCN protein levels on day3. The mRNA and protein expression of Runx2, OCN and OPN in the degassed CORM‑3‑osteogenic and osteogenic groups were similar. In addition, the mRNA and protein expression of Runx2, OCN and OPN in the CORM‑3 and control group were similar. ALP activity in the CORM‑3‑osteogenic group was increased from day3 and remained significantly higher compared with all other groups on days3,5and7 (P<0.05). Additionally, the results indicated that the optical density value of alizarin red staining in the CORM‑3‑osteogenic group was significantly increased compared with the other groups (P<0.05). Therefore, the present study demonstrated that CORM‑3 may promote the osteogenic differentiation of BMSCs by releasing CO.

MicroRNA-223 Suppresses Osteoblast Differentiation by Inhibiting DHRS3

Background/Aims: In this study, we aimed to use bioinformatics tools to identify the specific miRNAs and mRNAs involved in osteogenic differentiation and to further explore the way in which miRNA regulates osteogenic differentiation. Methods: The microarray GSE80614, which includes data from 3 human mesenchymal stromal cells (hMSCs) and 3 hMSCs after 72 hours (hr) of osteogenic differentiation, was used to screen for differentially expressed mRNAs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of these mRNAs were conducted using Gene Set Enrichment Analysis (GSEA). Then, the miRanda website was employed to detect the binding sites of DHRS3. In vitro experiments, including RT-PCR and western blotting, were used to detect miR-233 and DHRS3 expression levels 7 and 14 days (d) after the induction of osteogenic differentiation using human bone marrow-derived mesenchymal stem cells (hBMSCs). The target relationship between miR-223 and DHRS3 was confirmed by a dual luciferase assay. ALP (alkaline phosphatase) staining, ARS (Alizarin Red S) staining and western blotting (Runx2, OPN, OCN) were used to detect the level of osteogenic differentiation after transfection with miR-223 mimics and DHRS3 cDNA. Results: In this study, 127 mRNAs differentially expressed during osteogenic differentiation were identified in GSE80614. GO term and KEGG pathway enrichment analyses found that the retinol metabolism pathway was activated during osteogenic differentiation and that DHRS3, which is involved in the pathway, was upregulated. During osteogenic differentiation in hBMSCs, miR-223 was gradually downregulated, while DHRS3 was upregulated. After 14 days of osteogenic differentiation, ALP and ARS staining assay results showed strong ALP activity and extracellular matrix calcification with the inhibition of miR-223 or the overexpression of DHRS3. Furthermore, the expression levels of Runx2, OPN, and OCN were upregulated with the knockdown of miR-223 or the overexpression of DHRS3, while the simultaneous transfection of a miR-223 agomir and DHRS3 cDNA resulted in no significant difference from the negative control (NC) group. Conclusion: The inhibition of miR-223 promotes the osteogenic differentiation of hBMSCs via the upregulation of DHRS3.

TGFβ1-Induced Differentiation of Human Bone Marrow-Derived MSCs Is Mediated by Changes to the Actin Cytoskeleton.

TGFβ is a potent regulator of several biological functions in many cell types, but its role in the differentiation of human bone marrow-derived skeletal stem cells (hMSCs) is currently poorly understood. In the present study, we demonstrate that a single dose of TGFβ1 prior to induction of osteogenic or adipogenic differentiation results in increased mineralized matrix or increased numbers of lipid-filled mature adipocytes, respectively. To identify the mechanisms underlying this TGFβ-mediated enhancement of lineage commitment, we compared the gene expression profiles of TGFβ1-treated hMSC cultures using DNA microarrays. In total, 1932 genes were upregulated, and 1298 genes were downregulated. Bioinformatics analysis revealed that TGFβl treatment was associated with an enrichment of genes in the skeletal and extracellular matrix categories and the regulation of the actin cytoskeleton. To investigate further, we examined the actin cytoskeleton following treatment with TGFβ1 and/or cytochalasin D. Interestingly, cytochalasin D treatment of hMSCs enhanced adipogenic differentiation but inhibited osteogenic differentiation. Global gene expression profiling revealed a significant enrichment of pathways related to osteogenesis and adipogenesis and of genes regulated by both TGFβ1 and cytochalasin D. Our study demonstrates that TGFβ1 enhances hMSC commitment to either the osteogenic or adipogenic lineages by reorganizing the actin cytoskeleton.

Bone-forming peptide-3 induces osteogenic differentiation of bone marrow stromal cells via regulation of the ERK1/2 and Smad1/5/8 pathways

A bone-remodeling imbalance induced by increased bone resorption and osteoclast formation causes skeletal diseases such as osteoporosis. Induction of osteogenic differentiation of bone marrow stromal cells (BMSCs) leads to bone regeneration. Many researchers have tried to develop new adjuvants as specific stimulators of bone regeneration for therapeutic use in patients with bone resorption. We tried to develop a new adjuvant that has stronger osteogenic differentiation-promoting activity than bone morphogenetic proteins (BMPs). In this study, we identified a new peptide, which we called bone-forming peptide (BFP)-3, derived from the immature precursor of BMP-7. Upon osteogenic differentiation, BMSCs treated with BFP-3 exhibited higher alkaline phosphatase (ALP) activity and mineralization ability and significantly up-regulated expression of osteogenic genes such as ALP, osteocalcin (OC), Osterix, and Runx2 compared with control BMSCs. Furthermore, fluorescence-activated cell sorting (FACS) and immunofluorescence analyses demonstrated that BFP-3 treatment up-regulated CD44 expression. Interestingly, extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad1/5/8 phosphorylation was increased by BFP-3 treatment during osteogenic differentiation. Furthermore, BFP-3-induced osteogenic differentiation was significantly decreased by treatment with ERK1/2- and Smad-specific inhibitors. These results suggest that BFP-3 plays an important role in regulating osteogenic differentiation of BMSCs through increasing levels of osteogenic-inducing factors and regulating the ERK1/2 and Smad1/5/8 signaling pathways. Our finding indicates that BFP-3 may be a potential new therapeutic target for promoting bone formation.

Silk fibroin/chitosan scaffold with tunable properties and low inflammatory response assists the differentiation of bone marrow mesenchymal stem cells

The physical and chemical properties of the scaffold are known to play important roles in three-dimensional (3D) cell culture, which always determine the cellular fate or the results of implantation. To control these properties becomes necessary for meeting the requirements of a variety of tissue engineering applications. In this study, a series of silk fibroin/chitosan (SF/CS) scaffolds with tunable properties were prepared using freeze-drying method, and the rat bone marrow-derived mesenchymal stem cells (BM-MSCs) were seeded in these scaffolds to evaluate their availability of use in tissue engineering. The 3D structure, mechanical properties and degradation ability of SF/CS scaffold can be tuned by changing the total concentration of the precursor solution and the blending ratio between SF and CS. BM-MSCs cultured in the SF/CS scaffold exhibited excellent proliferation and multiple morphologies. The induction of osteogenic and adipogenic differentiation of BM-MSCs were successful in this scaffold when cultured in vitro. Subcutaneous implantation of the SF/CS scaffolds did not cause any inflammatory response within four weeks, which revealed good compatibility. Moreover, the implanted scaffold allowed host cells to invade, adhere, grow and form new blood vessels. With these excellent performance, SF/CS scaffold has great potential in preparing implants for tissue engineering applications.

Sprouty 2, an Early Response Gene Regulator of FosB and Mesenchymal Stem Cell Proliferation During Mechanical Loading and Osteogenic Differentiation.

Sprouty 2 (Spry2), an inhibitor of MAP kinase signaling was previously shown by our group to be induced during mechanical loading of mesenchymal stem cells (MSCs). Here, we studied the implication of Spry2 activation during mechanical loading and chemically induced MSC differentiation. Spry 2 expression showed an immediate early response during mechanical loading and chemical induction of osteogenic differentiation and followed the same pattern as osteogenic associated gene FosB and was necessary for the induction of FosB, as Spry 2 knock down also abrogated the upregulation of FosB expression. Spry 2 knock down was, also associated with an early response of the osteogenic genes Runx-2 and ALP. Neither the knock-down of Spry 2 nor the subsequent reduction in FosB had any effect on mid-late osteogenesis or mineralization but was associated with a significant increase in proliferation of MSC. These effects were possibly governed by negative regulation of MEK/Erk signaling as Spry 2 knock down resulted in an increase in phosphorylation of Erk1/2. In summary, our results shows the involvement of Spry2 in regulation of FosB and Runx2 genes, MAPK signaling and proliferation of MSC. Taken together these results suggest a possible role for Spry2 in regulation of MSC functions in response to mechanical loading and osteogenic differentiation. J. Cell. Biochem. 118: 2606-2614, 2017. © 2017 Wiley Periodicals, Inc.

Microcarrier culture enhances osteogenic potential of human periodontal ligament stromal cells

Regeneration of periodontal tissue represents a major challenge to modern tissue engineering, since cell-based therapies require large amounts of periodontal ligament stromal cells (PLSC), which can be obtained only by in vitro expansion. Ideally, the period of the in vitro expansion should be optimized for the generation of large enough numbers of pre-specified progenitor cells ready to contribute to the restoration of periodontal tissues.In the present study, we used a commercially available, three-dimensional culturing platform and alginate microcarrier cell culture system for the propagation of human PLSCs, which were derived using the explant outgrowth method. Induction of osteogenic differentiation resulted in rapid and robust mineralization of the extracellular matrix in PLSCs grown on microcarriers, but not in PLSCs grown under standard culture conditions. Gene expression studies revealed upregulation of osteogenesis-related genes, BMP2, ALP, RUNX2, MSX2, cementum protein 23, bone sialoprotein, osteopontin and periostin, in undifferentiated and differentiating microcarrier cultures of PLSCs. In addition, the microcarrier culture enhanced the expression of β-catenin, intermediate filament protein vimentin and focal adhesion proteins vinculin and paxillin.Our study shows that microcarrier culture allows rapid generation of large numbers of PLSCs pre-specified towards an osteogenic-like phenotype. This method may be useful for the development of new tissue engineering protocols for the reconstruction of periodontal tissues.

Epigenetic Signatures at the RUNX2-P1 and Sp7 Gene Promoters Control Osteogenic Lineage Commitment of Umbilical Cord-Derived Mesenchymal Stem Cells.

Wharton's Jelly mesenchymal stem cells (WJ-MSCs) are an attractive potential source of multipotent stem cells for bone tissue replacement therapies. However, the molecular mechanisms involved in their osteogenic conversion are poorly understood. Particularly, epigenetic control operating at the promoter regions of the two master regulators of the osteogenic program, RUNX2/P57 and SP7 has not yet been described in WJ-MSCs. Via quantitative PCR profiling and chromatin immunoprecipitation (ChIP) studies, here we analyze the ability of WJ-MSCs to engage osteoblast lineage. In undifferentiated WJ-MSCs, RUNX2/P57 P1, and SP7 promoters are found deprived of significant levels of the histone post-translational marks that are normally associated with transcriptionally active genes (H3ac, H3K27ac, and H3K4me3). Moreover, the RUNX2 P1 promoter lacks two relevant histone repressive marks (H3K9me3 and H3K27me3). Importantly, RUNX2 P1 promoter is found highly enriched in the H3K4me1 mark, which has been shown recently to mediate gene repression of key regulatory genes. Upon induction of WJ-MSCs osteogenic differentiation, we found that RUNX2/P57, but not SP7 gene expression is strongly activated, in a process that is accompanied by enrichment of activating histone marks (H3K4me3, H3ac, and H3K27ac) at the P1 promoter region. Histone mark analysis showed that SP7 gene promoter is robustly enriched in epigenetic repressive marks that may explain its poor transcriptional response to osteoblast differentiating media. Together, these results point to critical regulatory steps during epigenetic control of WJ-MSCs osteogenic lineage commitment that are relevant for future applications in regenerative medicine. J. Cell. Physiol. 232: 2519-2527, 2017. © 2016 Wiley Periodicals, Inc.

Low-serum culture with novel medium promotes maxillary/mandibular bone marrow stromal cell proliferation and osteogenic differentiation ability.

The purpose of this study was to evaluate the effect of low-serum STK2 medium on the isolation and osteogenic differentiation of human maxillary/mandibular bone marrow stromal cells (MBMSCs). Human MBMSCs were obtained from patients undergoing dental implant treatment. These cells were cultured in serum-free medium or STK2 medium containing 1% fetal bovine serum (low-serum) or α-MEM containing 10% fetal bovine serum (control). Proliferation on the culture surface, cell surface antigen expression, and mRNA levels of neural crest and osteogenic markers were examined. Alkaline phosphatase assay and Alizarin red staining were used to assess osteogenic differentiation potential. Immunoblotting analysis was performed to detect ERK phosphorylation. Low-serum and control MBMSCs were positive for CD73, CD90, and CD105 and negative for CD14, CD34, CD45, CD271, and HLA-DR. CD140a was absent in low-serum cells but present in control cells. Low-serum MBMSCs proliferated more than control MBMSCs. After induction of osteogenic differentiation, alkaline phosphatase activity and osteocalcin mRNA levels were higher in low-serum MBMSCs than in control cells, and Alizarin red staining was stronger in low-serum MBMSCs than in control cells. Low-serum culture promoted ERK phosphorylation. MBMSCs precultured in low-serum medium exhibited a greater cumulative cell number and a higher osteogenic differentiation capacity than those cultured in control medium. These findings indicate that low-serum STK2 culture might be useful to promote MBMSC proliferation and osteogenic differentiation. This method requires less autologous blood collection for cell expansion than conventional methods, thus reducing the burden on patients.

The shift of macrophages toward M1 phenotype promotes aortic valvular calcification

ObjectiveThe purpose of the present study was to comprehensively compare the phenotype profile of infiltrated macrophages in human noncalcified and calcific aortic valves, and to determine whether the shift of macrophage polarization modulates valvular calcification in vitro. MethodsCell surface markers of macrophages and inflammatory cytokines expression in 90 cases of human noncalcified and calcific aortic valve leaflets were analyzed. The normal aortic valve interstitial cells were isolated and cultured in vitro. After incubation with nonconditioned medium and conditioned medium from unstimulated or lipopolysaccharide-stimulated U937 monocytes, valve interstitial cells were evaluated by osteogenic differentiation markers. ResultsInfiltration of macrophages was enhanced in the calcific aortic valves, and M1 phenotype was the predominant macrophage subsets. In addition, both proinflammatory and anti-inflammatory cytokines were significantly upregulated in the calcific aortic valves. Furthermore, lipopolysaccharide-stimulated monocytes presented with increased expression of inducible nitric oxide synthase and high proportional CD11c-positive (M1) macrophages. Conditioned medium from unstimulated monocytes promoted the osteogenic differentiation of valve interstitial cells in vitro, as evidenced by increased markers such as bone morphogenetic protein 2, osteopontin, and alkaline phosphatase. Conditioned medium from M1 macrophages further enhanced valve interstitial cells calcification. Enzyme-linked immunosorbent assay showed that M1 phenotype macrophages secreted tumor necrosis factors α and interleukin 6, and neutralizing antibodies to these 2 proinflammatory cytokines attenuated induction of osteogenic differentiation and calcification by the conditioned media. ConclusionsBoth total numbers and polarization of macrophage influence the process of calcification in human aortic valve. The shift toward M1 phenotype might promote valve interstitial cell calcification.

Effects of herbal Epimedium on the improvement of bone metabolic disorder through the induction of osteogenic differentiation from bone marrow-derived mesenchymal stem cells.

Herbal Epimedium (HE) has been commonly used as a tonic, antirheumatic agent and in the treatment of bone-associated diseases including osteoporosis. Treatment for osteoporosis is important to increase bone mass density and maintain to balance of bone remodeling. The present study was performed to investigate the effects of HE on mouse bone marrow mesenchymal stem cell (mBMMSC) proliferation and osteogenic differentiation, using MTT assays, proliferating cell nuclear antigen (PCNA) detection and apoptosis and differentiation assays. HE was demonstrated to inhibit the proliferation of mBMMSCs up to 45.43±3.33% and to decrease the level of PCNA expression compared with untreated cells. HE also induced late apoptosis at 24 and 48 h after treatment up to 71.93 and 67.03%, respectively, while only 14.93% of untreated cells exhibited apoptosis. By contrast, HE induced differentiation of mBMMSCs into an osteogenic lineage at the beginning of three weeks after commencement of treatment. This suggested that HE is a candidate as an inducer of osteogenesis from bone marrow mesenchymal stem cells, and additionally has potential for use in the treatment of bone metabolic disorders such as osteoporosis.

Establishment of 3D culture and induction of osteogenic differentiation of pre-osteoblasts using wet-collected aligned scaffolds

Aligned fibrous scaffolds have attracted much interest in bone tissue engineering, because they are supposed to induce osteogenic differentiation. For the first time, aligned silk fibroin nanofibres were loosely packed using a novel wet-collection electrospinning method. Moreover, three-dimensional (3D) culture of MC3T3-E1 pre-osteoblasts was established on these fibrous scaffolds. Physicochemical properties of the scaffolds and the behaviour of MC3T3-E1 pre-osteoblasts on the scaffolds were analysed and compared with scaffolds obtained using traditional method. Ethanol bath improved the uniformity and alignment of the fibres and increased the thickness and porosity of the scaffolds. Structures of the fibres were well maintained after immediate crosslinking in ethanol. Cells on the wet-collected scaffolds exhibited more ordered arrangement and elongated morphology as well as faster and deeper infiltration. The ordered infiltration resulted in the establishment of the 3D culture of cells, promoting proliferation and osteogenic differentiation of the pre-osteoblasts. Thus, the wet-collected aligned scaffolds with improved topographical and physicochemical properties presents significant potential application in bone regeneration.

Induction of osteogenic differentiation of osteoblast-like cells MG-63 during cultivation on fibroin microcarriers

We have developed microcarriers made from silk fibroin. Microcarriers can be used as a substrate for cell cultivation and cell delivery during cell-based therapy and for the construction of bioengineered tissue. Fibroin microcarriers were mineralized, which led to the appearance of calcium phosphate crystals on their surface. The ability of mineralized and nonmineralized microcarriers to support osteogenic differentiation of the osteoblast-like cell line MG-63 was estimated by alkaline phosphatase activity, an early marker of bone formation. The experiment showed cells actively proliferating on the surface of both mineralized and nonmodified microcarriers. Culturing MG-63 on the surface of fibroin microcarriers resulted in an increase of alkaline phosphatase activity indicative of osteogenic differentiation of MG-63 cells in the absence of inductors. The level of alkaline phosphatase was higher when mineralized microcarriers were used. Alkaline phosphatase activity of MG-63 cells cultivated using traditional two-dimensional approaches were close to zero. As opposed to conventional monolayer culturing, microcarrier culture cells are in a three-dimensional environment that is closer to physiological conditions. This can have a significant impact on their morphology and functional properties. During this study, we also characterized mechanical properties of porous scaffolds used for microcarriers.

A newly identified mechanism involved in regulation of human mesenchymal stem cells by fibrous substrate stiffness

Stiffness of biomaterial substrates plays a critical role in regulation of cell behavior. Although the effect of substrate stiffness on cell behavior has been extensively studied, molecular mechanisms of regulation rather than those involving cytoskeletal activities still remain elusive. In this study, we fabricated aligned ultrafine fibers and treated the fiber with different annealing temperatures to produce fibrous substrates with different stiffness. Human mesenchymal stem cells (hMSCs) were then cultured on these fibrous substrates. Our results showed that annealing treatment did not change the diameter of electrospun fibers but increased their polymer crystallinity and mechanical properties. The mRNA expression of RUNX2 was upregulated while the mRNA expression of scleraxis was downregulated in response to an increase in substrate stiffness, suggesting that increased stiffness favorably drives hMSCs into the osteogenic lineage. With subsequent induction of osteogenic differentiation, osteogenesis of hMSCs on stiffer substrates was increased compared to that of the cells on control substrates. Cells on stiffer substrates increasingly activated AKT and YAP and upregulated transcript expression of YAP target genes compared to those on control substrates, and inhibition of AKT led to decreased expression of YAP and RUNX2. Furthermore, macrophage migration inhibitory factor (MIF) was increasingly produced by the cell on stiffer substrates, and knocking down MIF by siRNA resulted in decreased AKT phosphorylation. Taken together, we hereby demonstrate that simply using the annealing approach can manipulate stiffness of an aligned fibrous substrate without altering the material chemistry, and substrate stiffness dictates hMSC differentiation through the MIF-mediated AKT/YAP/RUNX2 pathway. Statement of SignificanceStiffness of biomaterial substrates plays a critical role in regulation of cell behavior. Although the effect of substrate stiffness on cell behavior has been extensively studied, molecular mechanisms of regulation rather than those involving cytoskeletal activities still remain elusive. In this manuscript, we report our new findings that simply using the annealing approach can manipulate stiffness of an aligned fibrous substrate without altering the material chemistry, and substrate stiffness dictates human mesenchymal stem cell (hMSC) differentiation through the macrophage migration inhibitory factor-mediated AKT/YAP/RUNX2 pathway. The findings are novel and interesting because we have identified a new mechanism rather than those involving cytoskeleton activity, by which substrate stiffness regulates hMSC behavior.

Polymer mesh scaffold combined with cell-derived ECM for osteogenesis of human mesenchymal stem cells.

BackgroundTissue-engineered scaffold should mimic the structure and biological function of the extracellular matrix and have mechanically supportive properties for tissue regeneration. In this study, we utilized a PLGA/PLA mesh scaffold, coated with cell-derived extracellular matrix (CDM) and assessed its potential as an osteogenic microenvironment for human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). CDM was obtained by decellularization of in vitro-cultured type I collagen overexpressing (Col I -293 T-DK) cells. Test groups are mesh itself (control), fibronectin-coated (FN-mesh), and CDM-coated mesh scaffold (CDM-mesh). CDM was then solubilized and used for scaffold coating.ResultsCDM was successfully collected and applied to mesh scaffolds. The presence of CDM was confirmed via SEM and FN immunofluorescence. After then, UCB-MSCs were seeded into the scaffolds and subjected to the induction of osteogenic differentiation for 21 days in vitro. We found that the seeded cells were viable and have better proliferation activity on CDM-mesh scaffold. In addition, when osteogenic differentiation of UCB-MSCs was examined for up to 21 days, alkaline phosphatase (ALP) activity and osteogenic marker (COL I, ALP, osteocalcin, bone sialoprotein) expression were significantly improved with UCB-MSCs when cultured in the CDM-mesh scaffold compared to the control and FN-mesh.ConclusionPolymer mesh scaffold incorporated with CDM can provide UCB-MSCs with a better microenvironment for osteogenesis in vitro.

PPARγ; and Wnt Signaling in Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) arise from a variety of tissues, including bone marrow and adipose tissue and, accordingly, have the potential to differentiate into multiple cell types, including osteoblasts and adipocytes. Research on MSCs to date has demonstrated that a large number of transcription factors and ectocytic or intrastitial signaling pathways regulate adipogenic and osteogenic differentiation. A theoretical inverse relationship exists in adipogenic and osteogenic lineage commitment and differentiation, such that signaling pathways induce adipogenesis at the expense of osteogenesis and vice versa. For example, peroxisome proliferator-activated receptor γ(PPARγ), which belongs to the nuclear hormone receptor superfamily of ligand-activated transcription factors, is known to function as a master transcriptional regulator of adipocyte differentiation, and inhibit osteoblast differentiation. Moreover, recent studies have demonstrated that inducers of osteogenic differentiation, such as bone morphogenetic protein (BMP) and Wnt, inhibit the function of PPARγ transactivation during MSC differentiation towards adipocytes through a variety of mechanisms. To illustrate this, the canonical Wnt/β-catenin pathway represses expression of PPARγ mRNA, whereas the noncanonical Wnt pathway activates histone methyltransferases that inhibit PPARγ transactivation via histone H3 lysine 9 (H3K9) methylation of its target genes. The role of microRNAs (miRNAs) in adipogenesis and osteoblastogenesis is garnering increased attention, and studies in this area have shed light on the integration of miRNAs with Wnt signaling and transcription factors such as Runx2 and PPARγ. This review summarizes our current understanding of the mechanistic basis of these signaling pathways, and indicates future clinical applications for stem cell-based cell transplantation and regenerative therapy.

Role of TAZ in genistein induced osteoblastogenic differentiation of mouse bone marrow-derived mesenchymal stem cells

Objective To investigate the role of transcriptional-coactivator with PDZ-binding motif(TAZ) in genistein-induced osteoblastogenic differentiation of mouse bone marrow-derived mesenchymal stem cells(BMSCs). Methods Mouse BMSCs were cultured in phenol red-free α-MEM containing osteogenic supplements for inducing osteogenic differentiation. BMSCs were transfected with siRNA-TAZ and treated with genistein. The temporal sequence of osteoblastic differentiation in BMSCs cultures was assayed by measuring alkaline phosphatase activity(ALP) and calcium deposition. The mRNA expression of bone sialoprotein(BSP) and osteocalcin(OC) were detected by reverse transcription-polymerase chain reaction(RT-PCR). The binding interaction between TAZ and cbfa1 was identified by co-immunoprecipitation. Results TAZ expression was detected during the induction of osteogenic differentiation, the ALP activity and calcium deposition were significantly decreased in BMSCs which were transfected with siRNA-TAZ. Genistein(0.01-1 μmol/L) exhibited a dose-dependent effect on TAZ expression in mouse BMSCs cultures. Treatment with genistein(1 μmol/L) resulted in increased ALP avtivity and calcium deposition of BMSC cultures as function of time. Genistein(1 μmol/L) also promoted the nuclear localization of TAZ and augmented the interaction between TAZ and cbfa1, and by which upregulated cbfa1-mediated gene expression such as BSP and OC. However, the ALP avtivity and calcium deposition, as well as the expression of BSP and OC were not promoted by genistein in BMSCs transfected with siRNA-TAZ. Conclusion These data suggest that the TAZ plays an important role in genistein-induced osteoblastic differentiation of mouse BMSCs cultures. (Chin J Endocrinol Metab, 2016, 32: 133-138) Key words: Genistein; Bone marrow-derived mesenchymal stem cells; Osteoblastic differentiation; Transcriptional-coactivator with PDZ-binding motif

Different mechanisms of bone formation of preosteoblast on metals in vitro

Different mechanisms of bone formation of preosteoblast on metals in vitro

Differentiation and Molecular Properties of Mesenchymal Stem Cells Derived from Murine Induced Pluripotent Stem Cells Derived on Gelatin or Collagen.

The generation of induced-pluripotential stem cells- (iPSCs-) derived mesenchymal stem cells (iMSCs) is an attractive and promising approach for preparing large, uniform batches of applicable MSCs that can serve as an alternative cell source of primary MSCs. Appropriate culture surfaces may influence their growth and differentiation potentials during iMSC derivation. The present study compared molecular properties and differentiation potential of derived mouse iPS-MSCs by deriving on gelatin or collagen-coated surfaces. The cells were derived by a one-step method and expressed CD73 and CD90, but CD105 was downregulated in iMSCs cultured only on gelatin-coated plates with increasing numbers of passages. A pairwise scatter analysis revealed similar expression of MSC-specific genes in iMSCs derived on gelatin and on collagen surfaces as well as in primary mouse bone marrow MSCs. Deriving iMSCs on gelatin and collagen dictated their osteogenic and adipose differentiation potentials, respectively. Derived iMSCs on gelatin upregulated Bmp2 and Lif prior to induction of osteogenic or adipose differentiation, while PPARγ was upregulated by deriving on collagen. Our results suggest that extracellular matrix components such as gelatin biases generated iMSC differentiation potential towards adipose or bone tissue in their derivation process via up- or downregulation of these master genes.

Histone Deacetylase Inhibitors can Repress Proliferation and Induce Apoptosis in Gingiva Progenitor Cells from Idiopathic Gingival Fibromatosis

Objective: Idiopathic gingival fibromatosis (IGF), which is characterized by diffuse enlargement of the gingiva due to expansion and accumulation of connective tissue, is a relatively rare hereditary condition that has no specific cause. The aim of this study was to investigate whether the anti-tumor drugs trichostatin A (TSA; inhibits histone deacetylase) and panobinostat (LBH589; a non-selective inhibitor of histone deacetylase) inhibit the proliferation of cells derived from gingiva in patients with IGF. Materials and methods: After osteogenic differentiation and adipogenic induction, the differences between normal gingival mesenchymal stem cells ( N-GMSCs ) and hyperplastic gingival mesenchymal stem ( H-GMSCs, theI cells from IGF ) were determined in terms of alkaline phosphatase staining and activity, alizarin red staining, calcium-nodule formation and, lipid droplets,. The effects of TSA and LBH589 were investigated via the MTT assay, flow cytometry, and reverse transcription polymerase chain reaction (RT-PCR). Results: Alkaline phosphatase staining and activity, alizarin red staining, lipid droplets detected the differentiative capacity in normal and IGF cells. These assays indicated that IGF cells possess multipotent differentiation properties similar to those of normal cells. RT-PCR showed that mRNA levels of the gene encoding p21Waf/Cip1, a cyclin-dependent kinase inhibitor and an essential regulator of growth inhibition, were lower in H-GMSCs than in N-GMSCs. After exposure to 1000 nM TSA or 1000 nM LBH589 for 48 h, p21Waf/Cip1 mRNA levels increased in H-GMSCs. Conclusions: TSA and LBH589 can repress the growth and proliferation of hyperplastic IGF cells by regulating p21Waf/Cip1 mRNA levels.