Commitment Of Mesenchymal Stem Cells Research Articles

Mechanical regulation of mesenchymal stem cell differentiation

Biophysical cues play a key role in directing the lineage commitment of mesenchymal stem cells or multipotent stromal cells (MSCs), but the mechanotransductive mechanisms at play are still not fully understood. This review article first describes the roles of both substrate mechanics (e.g. stiffness and topography) and extrinsic mechanical cues (e.g. fluid flow, compression, hydrostatic pressure, tension) on the differentiation of MSCs. A specific focus is placed on the role of such factors in regulating the osteogenic, chondrogenic, myogenic and adipogenic differentiation of MSCs. Next, the article focuses on the cellular components, specifically integrins, ion channels, focal adhesions and the cytoskeleton, hypothesized to be involved in MSC mechanotransduction. This review aims to illustrate the strides that have been made in elucidating how MSCs sense and respond to their mechanical environment, and also to identify areas where further research is needed.

GEF-H1 controls focal adhesion signaling that regulates mesenchymal stem cell lineage commitment

Focal adhesions (FAs) undergo maturation that culminates in size and composition changes that modulate adhesion, cytoskeleton remodeling and differentiation. Although it is well recognized that stimuli for osteogenesis of mesenchymal stem cells (MSCs) drive FA maturation, actin organization and stress fiber polarization, the extent to which FA-mediated signals regulated by the FA protein composition specifies MSC commitment remains largely unknown. Here, we demonstrate that, upon dexamethasone (osteogenic induction) treatment, guanine nucleotide exchange factor H1 (GEF-H1, also known as Rho guanine nucleotide exchange factor 2, encoded by ARHGEF2) is significantly enriched in FAs. Perturbation of GEF-H1 inhibits FA formation, anisotropic stress fiber orientation and MSC osteogenesis in an actomyosin-contractility-independent manner. To determine the role of GEF-H1 in MSC osteogenesis, we explore the GEF-H1-modulated FA proteome that reveals non-muscle myosin-II heavy chain-B (NMIIB, also known as myosin-10, encoded by MYH10) as a target of GEF-H1 in FAs. Inhibition of targeting NMIIB into FAs suppresses FA formation, stress fiber polarization, cell stiffness and osteogenic commitments in MSCs. Our data demonstrate a role for FA signaling in specifying MSC commitment.

MicroRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal (skeletal) stem cells.

The molecular mechanisms promoting lineage-specific commitment of human mesenchymal (skeletal or stromal) stem cells (hMSCs) into adipocytes (ADs) are not fully understood. Thus, we performed global microRNA (miRNA) and gene expression profiling during adipocytic differentiation of hMSC, and utilized bioinformatics as well as functional and biochemical assays, and identified several novel miRNAs differentially expressed during adipogenesis. Among these, miR-320 family (miR-320a, 320b, 320c, 320d and 320e) were ~2.2–3.0-fold upregulated. Overexpression of miR-320c in hMSC enhanced adipocytic differentiation and accelerated formation of mature ADs in ex vivo cultures. Integrated analysis of bioinformatics and global gene expression profiling in miR-320c overexpressing cells and during adipocytic differentiation of hMSC identified several biologically relevant gene targets for miR-320c including RUNX2, MIB1 (mindbomb E3 ubiquitin protein ligase 1), PAX6 (paired box 6), YWHAH and ZWILCH. siRNA-mediated silencing of those genes enhanced adipocytic differentiation of hMSC, thus corroborating an important role for those genes in miR-320c-mediated adipogenesis. Concordant with that, lentiviral-mediated stable expression of miR-320c at physiological levels (~1.5-fold) promoted adipocytic and suppressed osteogenic differentiation of hMSC. Luciferase assay validated RUNX2 (Runt-related transcription factor 2) as a bona fide target for miR-320 family. Therefore, our data suggest miR-320 family as possible molecular switch promoting adipocytic differentiation of hMSC. Targeting miR-320 may have therapeutic potential in vivo through regulation of bone marrow adipogenesis.

Abstract 601: Essential role of LRF in mesenchymal stem cell differentiation and tumorigenesis through Dlk1 and Sox9 repression

Abstract The regulatory factors governing the initial steps of mesenchymal stem cell (MSC) commitment and differentiation are largely unknown, and the role of MSCs in the genesis of mesenchymal tumors (sarcomas) has not been fully elucidated. In recent years, several genes and pathways have been characterized as important regulators of these cells’ differentiation into adipocytes, chondrocytes and osteoblasts. Although extremely valuable, these data have been mainly generated using cell lines or mouse embryonic fibroblasts (MEFs), leaving a more reliable model, such as one based on primary multi-potent MSCs, completely unexplored. To fill this gap, we have established new conditions enabling us to manipulate in vitro primary MSCs while maintaining their stemness properties, and have taken advantage of a series of genetically engineered mouse models to unravel a new role for LRF as both a master regulator of MSC differentiation and a tumor suppressor gene in mesenchymal tumors through the repression of two major pathways: DLK1/MAPK and SOX9. Mesenchymal tumors, although rare, have an extremely high rate of mortality, due to a lack of knowledge of the molecular mechanisms responsible for their genesis and progression, and consequently a limited number of available drugs. Our recent findings, however, significantly advance our knowledge of the mechanisms underlying sarcoma pathogenesis, and identify new LRF/DLK1/MAPK and LRF/SOX9 pathways that could prove fundamental to the stratification and targeted therapy of LRF-negative sarcomas. Citation Format: Jlenia Guarnerio, Andrea Lunardi, Pier Paolo Pandolfi. Essential role of LRF in mesenchymal stem cell differentiation and tumorigenesis through Dlk1 and Sox9 repression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 601. doi:10.1158/1538-7445.AM2014-601

Osteoblast differentiation and survival: A role for A2B adenosine receptor allosteric modulators

The A2B adenosine receptor (A2B AR), activated in response to high levels of endogenous adenosine, is the major AR subtype involved in mesenchymal stem cell (MSC) differentiation to osteoblasts and bone formation. For this reason, targeting of A2B AR with selective allosteric modulators may represent a promising pharmacological approach to the treatment of bone diseases.Herein, we report the characterization of a 3-keto-indole derivative, 2-(1-benzyl-1H-indol-3-yl)-2-oxo-N-phenylacetamide (KI-7), as A2B AR positive allosteric modulator in MSCs, demonstrating that this compound is able to potentiate the effects of either adenosine and synthetic orthosteric A2B AR agonists in mediating osteoblast differentiation in vitro. In detail, we observed that MSC treatment with KI-7 determined an increase in the expression of osteoblast-related genes (Runx2 and osterix) and osteoblast marker proteins (phosphatase alkaline and osteocalcin), associated with a stimulation of osteoblast mineralization.In the early phase of differentiation programme, KI-7 significantly potentiated physiological and A2B AR agonist-mediated down-regulation of IL-6 release. Conversely, during the late stage of differentiation, when most of the cells have an osteoblast phenotype, KI-7 caused a sustained raise in IL-6 levels and an improvement in osteoblast viability. These data suggest that a positive allosteric modulation of A2B AR not only favours MSC commitment to osteoblasts, but also ensures a greater survival of mature osteoblasts. Our study paves the way for a therapeutic use of selective positive allosteric modulators of A2B AR in the control of osteoblast differentiation, bone formation and fracture repair.

Investigating the spatial distribution of integrin β₁ in patterned human mesenchymal stem cells using super-resolution imaging.

Lineage commitment of human mesenchymal stem cells (hMSCs) could be directed through micro/nanopatterning of the extracellular matrix (ECM) between cells and substrate. Integrin receptors, integrator of the ECM and cell cytoskeleton, function as molecular bridges linking cells to different biophysical cues translated from patterned ECM. Here we report the distinct recruitment of active integrin β1 (ITG-β1) in hMSCs when they were committed toward the cardiomyogenic lineage on a micropatterned surface. In addition, a systematic study of the distribution of ITG-β1 was performed on focal adhesions (FAs) using a direct stochastic optical reconstruction microscopy (dSTORM) technique, a super-resolution imaging technique to establish the relationship between types of integrin expression and its distribution pattern that are associated with cardiomyogenic differentiation of hMSCs. We ascertained that elongated FAs of ITG-β1 expressed in patterned hMSCs were more prominent than FAs expressed in unpatterned hMSCs. However, there was no significant difference observed between the widths of FAs from both experimental groups. It was found in patterned hMSCs that the direction of FA elongation coincides with cell orientation. This phenomenon was however not observed in unpatterned hMSCs. These results showed that the biophysical induction methods like FAs patterning could selectively induce hMSCs lineage commitment via integrin-material interaction.

The role of catecholamines in mesenchymal stem cell fate.

Mesenchymal stem cells (MSCs) are multipotent stem cells found in many adult tissues, especially bone marrow (BM) and are capable of differentiation into various lineage cells such as osteoblasts, adipocytes, chondrocytes and myocytes. Moreover, MSCs can be mobilized from connective tissue into circulation and from there to damaged sites to contribute to regeneration processes. MSCs commitment and differentiation are controlled by complex activities involving signal transduction through cytokines and catecholamines. There has been an increasing interest in recent years in the neural system, functioning in the support of stem cells like MSCs. Recent efforts have indicated that the catecholamine released from neural and not neural cells could be affected characteristics of MSCs. However, there have not been review studies of most aspects involved in catecholamines-mediated functions of MSCs. Thus, in this review paper, we will try to describe the current state of catecholamines in MSCs destination and discuss strategies being used for catecholamines for migration of these cells to damaged tissues. Then, the role of the nervous system in the induction of osteogenesis, adipogenesis, chondrogenesis and myogenesis from MSCs is discussed. Recent progress in studies of signaling transduction of catecholamines in determination of the final fate of MSCs is highlighted. Hence, the knowledge of interaction between MSCs with the neural system could be applied towards the development of new diagnostic and treatment alternatives for human diseases.

Up regulation of liver-enriched transcription factors HNF4a and HNF6 and liver-specific microRNA (miR-122) by inhibition of let-7b in mesenchymal stem cells.

MicroRNAs are small non-coding RNAs that regulate key processes of the stem cells. Although, microRNAs have emerged as powerful regulators of differentiation, few studies have been focused on the post-transcriptional regulation of hepatic differentiation in mesenchymal stem cells (MSCs) by microRNAs. The aim of this study was to evaluate the specific effect of let-7 microRNAs in particular let-7b in hepatic commitment of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs). The dynamic expression profile of let-7a, b, c microRNAs and two liver-enriched transcription factors (LETFs) HNF4a and HNF6 was studied during in vitro hepatic differentiation of hAT-MSCs. Let-7b was used for transient overexpression and knockdown investigations. It was shown that the expression of LETFs is inversely correlated with those of let-7 miRNAs during differentiation progress (p < 0.05). Inhibition of let-7b caused upregulation of LETFs, an increase in the expression of miR-122 (p < 0.01) emulating the features of functional hepatocytes, and accumulation of hAT-MSCs in the G0 /G1 phase of cell cycle, triggering initiation of hepatic commitment. In conclusion, transient inhibition of let-7b activates hepatic differentiation of hAT-MSCs. The findings of this work might help optimization of in vitro hepatogenic differentiation utilizing microRNAs and hAT-MSCs that could be used for therapeutic purposes.

Distal-less homeobox 2 promotes the osteogenic differentiation potential of stem cells from apical papilla

Dental tissue-derived mesenchymal stem cells (MSCs) are a reliable cell source for dental tissue regeneration. However, the molecular mechanisms underlying the directed differentiation of MSCs remain unclear; thus, their use is limited. The histone demethylase, lysine (K)-specific demethylase 4B (KDM4B), plays critical roles in the osteogenic commitment of MSCs by up-regulating distal-less homeobox 2 (DLX2) expression. The DLX2 gene is highly expressed in dental tissue-derived MSCs but the roles of DLX2 in osteogenesis are unclear. Here, we investigate DLX2 function in stem cells from apical papilla (SCAPs). We found that, in vitro, DLX2 expression was up-regulated in SCAPs by adding BMP4 and by inducing osteogenesis. The knock-down of DLX2 in SCAPs decreased alkaline phosphatase (ALP) activity and mineralization. DLX2 depletion affected the mRNA expression of ALP, bone sialoprotein (BSP) and osteocalcin (OCN) and inhibited SCAP osteogenic differentiation in vitro. Over-expression of DLX2 enhanced ALP activity, mineralization and the expression of ALP, BSP and OCN in vitro. In addition, transplant experiments in nude mice confirmed that SCAP osteogenesis was triggered when DLX2 was activated. Furthermore, DLX2 expression led to the expression of the key transcription factor, osterix (OSX) but not to the expression of runt-related transcription factor 2 (RUNX2). Taken together, these results indicate that DLX2 is stimulated by BMP signaling and enhances SCAP osteogenic differentiation by up-regulating OSX. Thus, the activation of DLX2 signaling might improve tissue regeneration mediated by MSCs of dental origin. These results provide insight into the mechanism underlying the directed differentiation of MSCs of dental origin.

Melatonin regulates mesenchymal stem cell differentiation: a review

Among the numerous functions of melatonin, the control of survival and differentiation of mesenchymal stem cells (MSCs) has been recently proposed. MSCs are a heterogeneous population of multipotent elements resident in tissues such as bone marrow, muscle, and adipose tissue, which are primarily involved in developmental and regeneration processes, gaining thus increasing interest for tissue repair and restoration therapeutic protocols. Receptor-dependent and receptor-independent responses to melatonin are suggested to occur in these cells. These involve antioxidant or redox-dependent functions of this indolamine as well as secondary effects resulting from autocrine and paracrine responses. Inflammatory cytokines and adipokines, proangiogenic/mitogenic stimuli, and other mediators that influence the differentiation processes may affect the survival and functional integrity of these mesenchymal precursor cells. In this scenario, melatonin seems to regulate signaling pathways that drive commitment and differentiation of MSC into osteogenic, chondrogenic, adipogenic, or myogenic lineages. Common pathways suggested to be involved as master regulators of these processes are the Wnt/β-catenin pathway, the MAPKs and the, TGF-β signaling. In this respect melatonin emerges a novel and potential modulator of MSC lineage commitment and adipogenic differentiation. These and other aspects of the physiological and pharmacological effects of melatonin as regulator of MSC are discussed in this review.

Bone and Stem Cells. The mechanism of osteogenic differentiation from mesenchymal stem cell

Osteoblasts and osteocytes originate from pluripotent mesenchymal stem cells. Mesenchymal stem cells commit to osteogenic lineage and differentiate into mature osteoblasts and osteocytes through osteoprogenitor cells and preosteoblasts in response to multiple stimuli. The osteoblast commitment, differentiation, and functions are governed by several transcription factors. Among these transcription factors, runt-related transcription factor 2 (Runx2) is a crucial factor in osteoblast differentiation and controls bone formation. Differentiation toward these osteogenic lineage is controlled by a multitude of cytokines including WNTs, bone morphogenetic protein (BMP) , transforming growth factor-β (TGF-β) , hedgehog, parathyroid hormone (PTH) /parathyroid hormone related protein (PTHrP) , insulin-like growth factor-1 (IGF-1) , fibroblast growth factor (FGF) , and Notch. Although regulation of Runx2 activity is a point of convergence of many of the signal transduction routes, there is also a high degree of cross-talk between these pathways. Thus, the combined action of the signal transduction pathways induced by some cytokines determines the commitment and differentiation of mesenchymal stem cells toward the osteogenic lineage.

Promyelocytic leukemia zinc-finger induction signs mesenchymal stem cell commitment: identification of a key marker for stemness maintenance?

IntroductionMesenchymal stem cells (MSCs) are an attractive cell source for cartilage and bone tissue engineering given their ability to differentiate into chondrocytes and osteoblasts. However, the common origin of these two specialized cell types raised the question about the identification of regulatory pathways determining the differentiation fate of MSCs into chondrocyte or osteoblast.MethodsChondrogenesis, osteoblastogenesis, and adipogenesis of human and mouse MSC were induced by using specific inductive culture conditions. Expression of promyelocytic leukemia zinc-finger (PLZF) or differentiation markers in MSCs was determined by RT-qPCR. PLZF-expressing MSC were implanted in a mouse osteochondral defect model and the neotissue was analyzed by routine histology and microcomputed tomography.ResultsWe found out that PLZF is not expressed in MSCs and its expression at early stages of MSC differentiation is the mark of their commitment toward the three main lineages. PLZF acts as an upstream regulator of both Sox9 and Runx2, and its overexpression in MSC enhances chondrogenesis and osteogenesis while it inhibits adipogenesis. In vivo, implantation of PLZF-expressing MSC in mice with full-thickness osteochondral defects resulted in the formation of a reparative tissue resembling cartilage and bone.ConclusionsOur findings demonstrate that absence of PLZF is required for stemness maintenance and its expression is an early event at the onset of MSC commitment during the differentiation processes of the three main lineages.

Directed differentiation of human mesenchymal stem cells toward a cardiomyogenic fate commitment through formation of cell aggregates

Cell morphology is known to modulate the multipotential lineage commitment of stem cells. We provide a new strategy to induce the early lineage commitment of human mesenchymal stem cells (hMSCs) toward a cardiomyogenic fate through the formation of cell aggregates. A surface-immobilized polyamidoamine dendrimer with fifth generation of dendron structure was used during the culturing of hMSCs. These hMSCs cultured on the G5 surface formed aggregates through active migration and division. More than 22% of cardiac troponin-T (cTnT)-positive (cTnT+) cells in aggregates formed on the dendrimer surface; the population formed on the dendrimer surface was higher than that in conventional culture vessel. When cell aggregate was reseeded onto a fresh G5 surface, single cells migrated out of the aggregates, proliferated, and formed new aggregates. This passage method, accompanied with repetitive aggregate dispersion and formation, was applied to cultures over 40 days. The proportion of cTnT+ cells increased to 62% by the end of third passage. Our results suggest that culturing hMSCs on G5 surface results in directed commitment of the hMSCs toward a cardiomyocyte-like fate.

GEF-H1 controls focal adhesion signaling that regulates mesenchymal stem cell lineage commitment.

ABSTRACTFocal adhesions (FAs) undergo maturation that culminates in size and composition changes that modulate adhesion, cytoskeleton remodeling and differentiation. Although it is well recognized that stimuli for osteogenesis of mesenchymal stem cells (MSCs) drive FA maturation, actin organization and stress fiber polarization, the extent to which FA-mediated signals regulated by the FA protein composition specifies MSC commitment remains largely unknown. Here, we demonstrate that, upon dexamethasone (osteogenic induction) treatment, guanine nucleotide exchange factor H1 (GEF-H1, also known as Rho guanine nucleotide exchange factor 2, encoded by ARHGEF2) is significantly enriched in FAs. Perturbation of GEF-H1 inhibits FA formation, anisotropic stress fiber orientation and MSC osteogenesis in an actomyosin-contractility-independent manner. To determine the role of GEF-H1 in MSC osteogenesis, we explore the GEF-H1-modulated FA proteome that reveals non-muscle myosin-II heavy chain-B (NMIIB, also known as myosin-10, encoded by MYH10) as a target of GEF-H1 in FAs. Inhibition of targeting NMIIB into FAs suppresses FA formation, stress fiber polarization, cell stiffness and osteogenic commitments in MSCs. Our data demonstrate a role for FA signaling in specifying MSC commitment.

Quantitative, Label-Free Characterization of Stem Cell Differentiation at the Single-Cell Level by Broadband Coherent Anti-Stokes Raman Scattering Microscopy

We use broadband coherent anti-Stokes Raman scattering (BCARS) microscopy to characterize lineage commitment of individual human mesenchymal stem cells cultured in adipogenic, osteogenic, and basal culture media. We treat hyperspectral images obtained by BCARS in two independent ways, obtaining robust metrics for differentiation. In one approach, pixel counts corresponding to functional markers, lipids, and minerals, are used to classify individual cells as belonging to one of the three lineage groups: adipocytes, osteoblasts, and undifferentiated stem cells. In the second approach, we use multivariate analysis of Raman spectra averaged exclusively over cytosol regions of individual cells to classify the cells into the same three groups, with consistent results. The exceptionally high speed of spectral imaging with BCARS allows us to chemically map a large number of cells with high spatial resolution, revealing not only the phenotype of individual cells, but also population heterogeneity in the degree of phenotype commitment.

Role of p38, ERK1/2, focal adhesion kinase, RhoA/ROCK and cytoskeleton in the adipogenesis of human mesenchymal stem cells

Adipogenesis is important to health and is thought occurring in the two stages of mesenchymal stem cell commitment to a preadipocyte fate and terminal differentiation of the preadipocyte. However, the mechanism of adipogenesis is still not clear. In this study, the roles of p38, extracellular regulated protein kinases 1/2 (ERK1/2), focal adhesion kinase (FAK), RhoA/ROCK, and cytoskeleton in both of the two stages of adipogenesis were assayed. Our results showed that the treatments of SB203580 (the inhibitor of p38) and U0126 (the inhibitor of ERK1/2) suppressed the adipogenesis induced by differentiation medium, and the treatments of PF573228 (a specific inhibitor of FAK), Y27632 (a specific inhibitor of RhoA/ROCK) and cytochalasin D (an inhibitor of cytoskeletal organization) promoted the adipogenesis. The treatments of SB203580 and U0126 significantly inhibited the adipogenic differentiation of hMSCs cultured in differentiation medium in the presence of PF573228, Y27632 or cytochalasin D. Moreover, the treatments of PF573228, Y27632 and cytochalasin D promoted p38 and ERK1/2 phosphorylations, and the treatments of U0126 and SB203580 decreased p38 and ERK1/2 phosphorylations, respectively. These results demonstrated that p38 and ERK1/2 played crucial positive roles in adipogenesis, and FAK, RhoA/ROCK and cytoskeleton played negative roles. Furthermore, FAK, RhoA/ROCK and cytoskeleton affected adipogenesis by regulating the activities of p38 and ERK1/2 which interacted with each other in the process of adipogenesis.

Impaired of a non-DNA dependent methylation status decides the fat decision of bone marrow-derived C3H10T1/2 stem cell

A decrease in the lineage commitment of multipotent Mesenchymal stem cells (MSC) to the bone forming osteoblast lineage and an increase in the commitment to the fat forming adipocyte lineage is more common in bone marrow of elderly persons. A link between methylation status and MSC differentiation remains unclear. Therefore, we hypothesize that hypomethylation may decide the fate decisions of MSC. In the current study, murine bone marrow derived-C3H10T1/2 stem cell was used to examine the role of methylation mechanism on the differentiation potential of stem cells into osteoblasts or adipocytes. C3H10T1/2 cells were treated with Periodate oxidized adenosine (Adox), an inhibitor of S-adenosylhomocysteine-dependent hydrolase (SAHH), which in turn block the non-DNA methylation pathway. The effect of hypomethylation on C3H10T1/2 stem cell differentiation was determined by measuring the alkaline phosphates activity and the degree of mineralization as well as Oil-red O staining and lipid content. The ratio of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) was determined as a metabolic indicator of cellular methylation potential. It was clearly observed that hypomethylation significantly (P < 0.05) reduces SAM: SAH ratio, alkaline phosphates activity, calcification and thereby, osteoblast differentiation. Conversely, adipocyte differentiation was stimulated by hypomethylation. Altogether, our data suggest that non-DNA hypomethylation changes the differentiation potential of C3H10T1/2 stem cells for less osteogenic and more adipogenic.

BMP9 and COX-2 form an important regulatory loop in BMP9-induced osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can self-renew and differentiate into osteogenic, chondrogenic, adipogenic and myogenic lineages. It's reported that bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic BMPs to initiate the commitment of MSCs to osteoblast lineage. Cyclooxygenase-2 (COX-2) is critical for bone fracture healing and osteogenic differentiation in MSCs. However, the relationship between COX-2 and BMP9 in osteogenesis remains unknown. Herein, we investigate the role of COX-2 in BMP9-induced osteogenesis in MSCs. We demonstrate that COX-2 is up-regulated as a target of BMP9 in MSCs. Both COX-2 inhibitor (NS-398) and COX-2 knockdown siRNAs can effectively decrease alkaline phosphatase (ALP) activities induced by BMP9 in MSCs. NS-398 also down-regulates BMP9-induced expression of osteopontin and osteocalcin, so does the matrix mineralization. The in vivo studies indicate that knockdown of COX-2 attenuates BMP9-induced ectopic bone formation. In perinatal limb culture assay, NS-398 is shown to reduce the hypertropic chondrocyte zone and ossification induced by BMP9. Mechanistically, knockdown of COX-2 significantly inhibits the BMP9 up-regulated expression of Runx2 and Dlx-5 in MSCs, which can be rescued by exogenous expression of COX-2. Furthermore, knockdown of COX-2 apparently reduces BMP9 induced BMPR-Smad reporter activity, the phosphorylation of Smad1/5/8, and the expression of Smad6 and Smad7 in MSCs. NS-398 blocks the expression of BMP9 mediated by BMP9 recombinant adenovirus. Taken together, our findings suggest that COX-2 plays an important role in BMP9 induced osteogenic differentiation in MSCs; BMP9 and COX-2 may form an important regulatory loop to orchestrate the osteogenic differentiation in MSCs.

PPARγ silencing enhances osteogenic differentiation of human adipose‐derived mesenchymal stem cells

Peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis, and has been indicated as a potential therapeutic target to promote osteoblast differentiation. However, recent studies suggest that suppression of PPARγ inhibits adipogenesis, but does not promote osteogenic differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs). It was reasoned that the osteogenic effect of PPARγ suppression may be masked by the strong osteogenesis-inducing condition commonly used, resulting in a high degree of matrix mineralization in both control and experimental groups. This study investigates the role of PPARγ in the lineage commitment of human adipose-derived mesenchymal stem cells (hADSCs) by interfering with the function of PPARγ mRNA through small interfering RNAs (siRNAs) specific for PPARγ2. By applying an osteogenic induction condition less potent than that used conventionally, we found that PPARγ silencing led to retardation of adipogenesis and stimulated a higher level of matrix mineralization. The mRNA level of PPARγ decreased to 47% of control 2 days after treatment with 50 nmol/l PPARγ2 siRNA, while its protein expression was 60% of mock control. In the meantime, osteogenic marker genes, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OC), were up-regulated under PPARγ silencing. Our results suggest that transient suppression of PPARγ promotes the onset of osteogenesis, and may be considered a new strategy to stimulate bone formation in bone tissue engineering using hADSCs.

Vibration induced osteogenic commitment of mesenchymal stem cells is enhanced by cytoskeletal remodeling but not fluid shear

Consistent across studies in humans, animals and cells, the application of vibrations can be anabolic and/or anti-catabolic to bone. The physical mechanisms modulating the vibration-induced response have not been identified. Recently, we developed an in vitro model in which candidate parameters including acceleration magnitude and fluid shear can be controlled independently during vibrations. Here, we hypothesized that vibration induced fluid shear does not modulate mesenchymal stem cell (MSC) proliferation and mineralization and that cell's sensitivity to vibrations can be promoted via actin stress fiber formation. Adipose derived human MSCs were subjected to vibration frequencies and acceleration magnitudes that induced fluid shear stress ranging from 0.04Pa to 5Pa. Vibrations were applied at magnitudes of 0.15g, 1g, and 2g using frequencies of both 100Hz and 30Hz. After 14d and under low fluid shear conditions associated with 100Hz oscillations, mineralization was greater in all vibrated groups than in controls. Greater levels of fluid shear produced by 30Hz vibrations enhanced mineralization only in the 2g group. Over 3d, vibrations led to the greatest increase in total cell number with the frequency/acceleration combination that induced the smallest level of fluid shear. Acute experiments showed that actin remodeling was necessary for early mechanical up-regulation of RUNX-2 mRNA levels. During osteogenic differentiation, mechanically induced up-regulation of actin remodeling genes including Wiskott–Aldrich syndrome (WAS) protein, a critical regulator of Arp2/3 complex, was related to the magnitude of the applied acceleration but not to fluid shear. These data demonstrate that fluid shear does not regulate vibration induced proliferation and mineralization and that cytoskeletal remodeling activity may play a role in MSC mechanosensitivity.