Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential

While traditional cell culture methods have relied on growing cells as monolayers, three-dimensional (3D) culture systems can provide a convenient in vitro model for the study of complex cell–cell and cell–matrix interactions in the absence of exogenous substrates and may benefit the development of regenerative medicine strategies. In this study, mesenchymal stem cell (MSC) spheroids, or “mesenspheres”, of different sizes, were formed using a forced aggregation technique and maintained in suspension culture for extended periods of time thereafter. Cell proliferation and differentiation potential within mesenspheres and dissociated cells retrieved from spheroids were compared to conventional adherent monolayer cultures. Mesenspheres maintained in growth medium exhibited no evidence of cell necrosis or differentiation, while mesenspheres in differentiation media exhibited differentiation similar to conventional 2D culture methods based on histological markers of osteogenic and adipogenic commitment. Furthermore, when plated onto tissue culture plates, cells that had been cultured within mesenspheres in growth medium recovered morphology typical of cells cultured continuously in adherent monolayers and retained their capacity for multi-lineage differentiation potential. In fact, more robust matrix mineralization and lipid vacuole content were evident in recovered MSCs when compared to monolayers, suggesting enhanced differentiation by cells cultured as 3D spheroids. Thus, this study demonstrates the development of a 3D culture system for mesenchymal stem cells that may circumvent limitations associated with conventional monolayer cultures and enhance the differentiation potential of multipotent cells.     

Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells

The roles of various soluble factors in promoting the osteogenic differentiation of adult mesenchymal stem cells (MSCs) have been widely studied, but little is known about how the extracellular matrix (ECM) instructs the phenotypic transition between growth and differentiation. To investigate this question, we cultured MSCs on purified vitronectin or type-I collagen, motivated by our earlier tissue engineering work demonstrating that MSC adhesion to polymer scaffolds is primarily mediated by the passive adsorption of these two ECM ligands from serum. Using alkaline phosphatase activity and matrix mineralization as indicators of the early and late stages of osteogenesis, respectively, we report here that both substrates supported differentiation, but the mechanism was substrate dependent. Specifically, osteogenesis on vitronectin correlated with enhanced focal adhesion formation, the activation of focal adhesion kinase (FAK) and paxillin, and the diminished activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) pathways. By contrast, MSCs on type-I collagen exhibited reduced focal adhesion formation, reduced activation of FAK and paxillin, and increased activation of ERK and PI3K. Inhibition of ERK and FAK blocked mineral deposition on both substrates, suggesting that the observed differences in signaling pathways ultimately converge to the same cell fate. Understanding these mechanistic differences is essential to predictably control the osteogenic differentiation of MSCs and widen their use in regenerative medicine.     

Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in craniofacial defects through activation of p38 MAPK

Mesenchymal stem cells (MSCs) are multipotent cells that can be differentiated into osteoblasts and provide an excellent cell source for bone regeneration and repair. Recently, the canonical Wnt/beta-catenin signaling pathway has been found to play a critical role in skeletal development and osteogenesis, implying that Wnts can be utilized to improve de novo bone formation mediated by MSCs. However, it is unknown whether noncanonical Wnt signaling regulates osteogenic differentiation. Here, we find that Wnt-4 enhanced in vitro osteogenic differentiation of MSCs isolated from human adult craniofacial tissues and promoted bone formation in vivo. Whereas Wnt-4 did not stabilize beta-catenin, it activated p38 MAPK in a novel noncanonical signaling pathway. The activation of p38 was dependent on Axin and was required for the enhancement of MSC differentiation by Wnt-4. Moreover, using two different models of craniofacial bone injury, we found that MSCs genetically engineered to express Wnt-4 enhanced osteogenesis and improved the repair of craniofacial defects in vivo. Taken together, our results reveal that noncanonical Wnt signaling could also play a role in osteogenic differentiation. Wnt-4 may have a potential use in improving bone regeneration and repair of craniofacial defects.     

Role of N-cadherin in bone formation

Cell-cell adhesion mediated by cadherins is essential for the function of bone forming cells during osteogenesis. Here, the evidence that N-cadherin is an important regulator of osteoblast differentiation and osteogenesis is reviewed. Osteoblasts express a limited number of cadherins, including the classic N-cadherin. The expression profile of N-cadherin in osteoblasts during bone formation in vivo and in vitro suggests a role of this molecule in osteogenesis. Functional studies using neutralizing antibodies or antisense oligonucleotides indicate that N-cadherin is involved in the control the expression of osteoblast marker gene expression and differentiation. Cleavage of N-cadherin during osteoblast apoptosis also suggests a role of N-cadherin-mediated-cell-cell adhesion in osteoblast survival. Hormonal and local factors that regulate osteoblast function also regulate N-cadherin expression and subsequent cell-cell adhesion associated with osteoblast differentiation or survival. Signaling mechanisms involved in N-cadherin-mediated cell-cell adhesion and osteoblast gene expression have also been identified. Alterations of N-cadherin expression are associated with abnormal osteoblast differentiation and osteogenesis in pathological conditions. These findings indicate that N-cadherin plays a role in normal and pathological bone formation and provide some insight into the process involved in N-cadherin-mediated cell-cell adhesion and differentiation in osteoblasts.     

Development of a surface plasmon resonance biosensor for real-time detection of osteogenic differentiation in live mesenchymal stem cells

Surface plasmon resonance (SPR) biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs). OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis.     

The organization of adherens junctions in mouse osteoblast-like cells (MC3T3-E1) and their modulation by triiodothyronine and 1,25-dihydroxyvitamin D3

Cadherin-mediated cell-cell adhesion is essential for the development and survival of multicellular tissues. Thus it is hypothesized that these molecules also play a fundamental role for the development and maintenance of bone by mediating cellular crosstalk between osteogenic cells and by providing targets for the sorting and migration of osteogenic precursors toward the bone surface. We describe the localization of cadherin-11 and N-cadherin along the cell margins of mouse osteoblast-like cells, the colocalization of "pancadherin" with alpha-catenin, beta-catenin, p120, and vinculin, and the association of these complexes with the actin microfilaments. Furthermore, we measured the influence of cell confluency and the effects of the osteogenic hormones triiodothyronine (T3) and 1,25-dihydroxyvitamin D3 (D3) on these parameters. By mRNA studies we found the abundantly expressed cadherin-11 being unaffected during T3- and D3-induced osteoblastic differentiation. However, protein levels of N-cadherin and "pancadherin" were strongly suppressed by D3. We also observed a clear distinction in cadherin immunolocalization when comparing confluent control and confluent hormone-treated cultures. Immunoprecipitation experiments indicated that vinculin is part of the junctional complex, and that the association of "pancadherin"/beta-catenin is strongly increased after treatment with T3 which might influence the functional competence of cell-cell contacts. Thus, this study demonstrates the molecular organization of adherens junctions in mouse osteoblastic MC3T3-E1 cells and their sensitivity to the osteogenic factors T3 and D3 in confluent cultures.     

Adipose-derived stem cells: An appropriate selection for osteogenic differentiation

Mesenchymal stem cells (MSCs) are a major component of various forms of tissue engineering. MSCs have self-renewal and multidifferential potential. Osteogenic differentiation of MSCs is an area of attention in bone regeneration. One form of MSCs are adipose-derived stem cells (ASCs), which can be simply harvested and differentiated into several cell lineages, such as chondrocytes, adipocytes, or osteoblasts. Due to special properties, ASCs are frequently used in vitro and in vivo bone regeneration. Identifying factors involved in osteogenic differentiation of ASCs is important for better understanding the mechanism of osteogenic differentiation. Different methods are used to stimulate osteogenesis of ASCs in literature, including common osteogenic media, growth factors, hormones, hypoxia, mechanical and chemical stimuli, genetic modification, and nanotechnology. This review article provides an overview describing the isolation procedure, characterization, properties, current methods for osteogenic differentiation of ASCs, and their basic biological mechanism.     

Osteogenic differentiation of miniature pig mesenchymal stem cells in 2D and 3D environment

Mesenchymal stem cells (MSCs) have been repeatedly shown to be able to repair bone defects. The aim of this study was to characterize the osteogenic differentiation of miniature pig MSCs and markers of this differentiation in vitro. Flow-cytometrically characterized MSCs were seeded on cultivation plastic (collagen I and vitronectin coated/uncoated) or plasma clot (PC)/plasma-alginate clot (PAC) scaffolds and differentiated in osteogenic medium. During three weeks of differentiation, the formation of nodules and deposition of calcium were visualized by Alizarin Red Staining. In addition, the production of alkaline phosphatase (ALP) activity was quantitatively detected by fluorescence. The expression of osteopontin, osteonectin and osteocalcin were assayed by immunohistochemistry and Western Blot analysis. We revealed a decrease of osteopontin expression in 2D and 3D environment during differentiation. The weak initial osteonectin signal, culminating on 7(th) or 14(th) day of differentiation, depends on collagen I and vitronectin coating in 2D system. The highest activity of ALP was detected on 21(th) day of osteogenic differentiation. The PC scaffolds provided better conditions for osteogenic differentiation of MSCs than PAC scaffolds in vitro. We also observed expected effects of collagen I and vitronectin on the acceleration of osteogenic differentiation of miniature pig MSC. Our results indicate similar ability of miniature pig MSCs osteogenic differentiation in 2D and 3D environment, but the expression of osteogenic markers in scaffolds and ECM coated monolayers started earlier than in the monolayers without ECM.     

Down-regulation of Noggin and miR-138 coordinately promote osteogenesis of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate to osteocytes under suitable conditions. In recent years, micro-nucleotides have been progressively used to modulate gene expression in cells due to the consideration of safety. Our present study aimed to investigate whether co-delivery of Noggin-siRNA and antimiR-138 enhances the osteogenic effect of MSCs. Using a murine MSC line, C3H/10T1/2 cells, the delivery efficiency of Noggin-siRNA and antimiR-138 into MSCs was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Cell phenotype and proliferation capacity was assessed by flow cytometry and MTT assay respectively. The osteogenesis of MSCs was tested by Alkaline Phosphatase (ALP) staining, qRT-PCR, and western blot analyses. Our results demonstrated that the expression of Noggin and miR-138 were significantly silenced in MSCs by Noggin-siRNA and/or antimiR-138 delivery, while the phenotype and proliferation capacity of MSCs were not affected. Down-regulation of Noggin and miR-138 cooperatively promoted osteogenic differentiation of MSCs. The ALP positive cells reached about 83.57?±?10.18%. Compared with single delivery, the expression of osteogenic related genes, such as Alp, Col-1, Bmp2, Ocn and Runx2, were the highest in cells with co-delivery of the two oligonucleotides. Moreover, the protein level of RUNX2, and the ratios of pSMAD1/5/SMAD1/5 and pERK1/2/ERK1/2 were significantly increased. The activation of Smad, Erk signaling may constitute the underlying mechanism of the enhanced osteogenesis process. Taken together, our study provides a safe strategy for the clinical rehabilitation application of MSCs in skeletal deficiency.     

Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 via Wnt/β-catenin and MAPK pathway

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts and chondrocytes. In vitro osteogenic differentiation is critical but the molecular mechanism has yet to be further clarified. The role of TGF-β activated kinase 1 (TAK1) in MSCs osteogenesis differentiation has not been reported. By adding si-TAK1 and rhTAK1, the osteogenic differentiation of MSCs was measured. Expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs were checked. As well as molecules involved in BMP and Wnt/β-catenin signaling pathways. The phosphorylation of p38 and JNK was also checked. TAK1 is essential for mineralization of MSCs at low concentration, but excessive rhTAK1 inhibits mineralization of MSCs. It up regulates the expression levels of bone sialoprotein (BSP), osteocalcin (OSC), Alkaline phosphatase (ALP), and RUNX2 during osteogenic differentiation of MSCs. It can also promote TGF-β/BMP-2 gene expression and β-catenin expression, and down regulate GSK-3β expression. Meanwhile, TAK1 promotes the phosphorylation of p38 and JNK. Additionally, TAK1 up regulates the expression of BMP-2 at all concentration under the inhibition of p38 and JNK. Our results suggested that TAK1 is essential in MSCs osteogenesis differentiation, and functions as a double-edged sword, probably through regulation of β-catenin and p38/JNK.     

Sortilin is upregulated during osteoblastic differentiation of mesenchymal stem cells and promotes extracellular matrix mineralization

Osteoblasts and adipocytes are derived from a common precursor in bone marrow, the mesenchymal stem cell (MSC). Factors driving human MSCs (hMSCs) to differentiate down the two lineages play important roles in determining bone density because it has been shown that bone volume loss associated with osteoporosis and aging is accompanied by reduced osteoblastic bone formation and increased marrow adipose tissue. The genes upregulated in hMSCs during osteogenic differentiation were screened using cDNA microarrays and were semi-quantitated by real-time RT-PCR. One of the genes identified was sortilin, which was upregulated one day after osteogenic induction and remained upregulated for a week. The overexpression of sortilin in hMSCs using an adenovirus vector resulted in the acceleration of mineralization during osteogenic differentiation without affecting alkaline phosphatase activity. Lipoprotein lipase (LPL), produced by adipocytes, is bound by sortilin, which may mediate its endocytosis. By adding LPL to osteogenic induction medium, osteoblastic mineralization was inhibited in a dose-dependent manner. Interestingly, sortilin overexpression abolished the LPL-mediated suppression of osteogenic differentiation. hMSCs exist in marrow where LPL-producing adipose cells are abundant and where osteogenesis is negatively regulated by LPL. Sortilin has a counter effect of promoting osteogenesis by acting as a scavenger of LPL.     

Mitochondria transfer reverses the inhibitory effects of low stiffness on osteogenic differentiation of human mesenchymal stem cells

Microenvironment biophysical factors such as matrix stiffness can noticeably affect the differentiation of mesenchymal stem cells (MSCs). In this mechanobiology transduction process, mitochondria are shown to be an active participant. The present study aims to systematically elucidate the phenotypic and functional changes of mitochondria during the stiffness-mediated osteogenic differentiation. Additionally, the effect of mitochondria transfer on the osteogenesis of impaired MSCs caused by stiffness was investigated. Human periodontal ligament stem cells (PDLSCs) were used as model cells in the current study. Low stiffness restrained the cell spreading and significantly inhibited the proliferation and osteogenic differentiation of PDLSCs. Mitochondria of PDLSCs cultured on low stiffness exhibited shorter length, rounded shape, fusion/fission imbalance, ROS and mitophagy level increase, and ATP production reduction. The inhibited mitochondria function and osteogenic differentiation capacity were recovered to near-normal levels after transferring the mitochondria of PDLSCs cultured on the high stiffness. This study indicated that low matrix stiffness altered the mitochondrial morphology and induced systematical mitochondrial dysfunction during the osteogenic differentiation of MSCs. Mitochondria transfer was proved to be a feasible technique for maintaining MSCs function in vitro by reversing the osteogenesis ability.     

Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation

Human MSCs have been studied to define the mechanisms involved in normal bone remodeling and the regulation of osteogenesis. During osteogenic differentiation, MSCs change from their characteristic fibroblast-like phenotype to near spherical shape. In this study, we analyzed the correlation between the organization of cytoskeleton of MSCs, changes in cell morphology, and the expression of specific markers (alkaline phosphatase activity and calcium deposition) of osteogenic differentiation. For osteoblastic differentiation, cells were cultured in a culture medium supplemented with 100 nM dexamethasone, 10 mM beta- glycerophosphate, and 50 microg/ml ascorbic acid. The organization of microfilaments and microtubules was examined by inmunofluorescence using Alexa fluor 594 phalloidin and anti alpha-tubulin monoclonal antibody. Cytochalasin D and nocodazole were used to alter reversibly the cytoskeleton dynamic. A remarkable change in cytoskeleton organization was observed in human MSCs during osteogenic differentiation. Actin cytoskeleton changed from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few thick actin filament bundles located at the outermost periphery in differentiated cells. Under osteogenic culture conditions, a reversible reorganization of microfilaments induced by an initial treatment with cytochalasin D but not with nocodazole reduced the expression of differentiation markers, without affecting the final morphology of the cells. The results indicate that changes in the assembly and disassembly kinetics of microfilaments dynamic of actin network formation may be critical in supporting the osteogenic differentiation of human MSCs; also indicated that the organization of microtubules appears to have a regulatory role on the kinetic of this process.     

Vibration loading promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells via p38 MAPK signaling pathway

Low magnitude high frequency vibration (LMHFV) exhibits effectively anabolic effects on the bone tissue, and can promote osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. The role of p38 MAPK signaling in LMHFV-induced osteogenesis remains unclear. In this current study, LMHFV loading was applied to BMSCs in vitro, and cell proliferation, alkaline phosphatase (ALP), matrix mineralization, as well as osteogenic genes expression were assayed. The mechanism of mechanical signal transduction was analysed using PCR array, qRT-PCR and Western blot. LMHFV increased cell proliferation in the growth medium, while inhibited proliferation in the osteogenic medium. ALP activity, matrix mineralization and osteogenic genes expression of Runx2, Col-I, ALP, OPN and OC were increased by LMHFV. p38 and MKK6 genes expression, and p38 phosphorylation were promoted in LMHFV-induced osteogenesis. Inhibition of p38 MAPK with SB203580 and targeted p38 siRNA blunted the increased ALP activity and osteogenic genes expression by LMHFV. These findings suggest that LMHFV promotes osteogenic differentiation of BMSCs, and p38 MAPK signaling shows an important function in LMHFV-induced osteogenesis.     

Effect of monocytes/macrophages on the osteogenic differentiation of adipose-derived mesenchymal stromal cells in 3D co-culture spheroids

This study aimed to investigate the distinctive roles of the monocytes and macrophages on osteogenic differentiation of adipose-derived mesenchymal stromal cells (ADMSCs) in 3D spheroid co-cultures. We hypothesized that monocytes or macrophages (subtypes pro-inflammatory M1 and pro-wound healing M2) would affect the osteogenic differentiation of ADMSCs in 3D spheroids and that cell–cell interactions between monocytes/macrophages and ADMSCs play an important role in the osteogenic differentiation process of ADMSCs. The obtained results indicated that the osteogenic differentiation of ADMSCs was inhibited by monocytes and both macrophage subtypes in 3D spheroids. Monocytes and M2 macrophages had a stronger inhibiting effect than M1 macrophages. Cell-cell interactions mediated by N-cadherin likely played a role in the inhibiting effect of monocytes/macrophages on the osteogenic differentiation of ADMSCs.