EMF acts on rat bone marrow mesenchymal stem cells to promote differentiation to osteoblasts and to inhibit differentiation to adipocytes

The use of electromagnetic fields (EMFs) to treat nonunion fractures developed from observations in the mid‐1900s. Whether EMF directly regulates the bone marrow mesenchymal stem cells (MSCs), differentiating into osteoblasts or adipocytes, remains unknown. In the present study, we investigated the roles of sinusoidal EMF of 15 Hz, 1 mT in differentiation along these separate lineages using rat bone marrow MSCs. Our results showed that EMF promoted osteogenic differentiation of the stem cells and concurrently inhibited adipocyte formation. EMF increased alkaline phosphatase (ALP) activity and mineralized nodule formation, and stimulated osteoblast‐specific mRNA expression of RUNX2, ALP, BMP2, DLX5, and BSP. In contrast, EMF decreased adipogenesis and inhibited adipocyte‐specific mRNA expression of adipsin, AP‐2, and PPARγ2, and also inhibited protein expression of PPARγ2. These observations suggest that commitment of MSCs into osteogenic or adipogenic lineages is influenced by EMF. Bioelectromagnetics 31:277–285, 2010. © 2009 Wiley‐Liss, Inc.     

Increased adipogenesis of osteoporotic human-mesenchymal stem cells (MSCs) characterizes by impaired leptin action

The bone marrow contains mesenchymal stem cells (MSCs) that differentiate to the osteogenic and adipogenic lineages. The fact that the decrease in bone volume of age-related osteoporosis is accompanied by an increase in marrow adipose tissue implies the importance that the adipogenic process may have in bone loss. We previously observed that MSCs from control and osteoporotic women showed differences in their capacity to differentiate into the osteogenic and adipogenic pathways. In vitro studies indicate that bone marrow stromal cells are responsive to leptin, which increases their proliferation, differentiation to osteoblasts, and the number of mineralized nodules, but inhibits their differentiation to adipocytes. The aim of the present report was to study the direct effect of leptin on control and osteoporotic MSCs analyzing whether the protective effect of leptin against osteoporosis could be expressed by inhibition of adipocyte differentiation. MSCs from control, and osteoporotic donors were subjected to adipogenic conditions, in the absence or in the presence of 62.5 nM leptin. The number of adipocytes, the content of PPARgamma protein, and mRNA, and leptin mRNA were measured by flow cytometry, Western blot, and RT-PCR, respectively. Results indicate that control and osteoporotic MSCs differ in their adipogenic potential as shown by expression of active PPARgamma protein. Leptin exerted an antiadipogenic effect only on control MSCs increasing the proportion of inactive phosphorylated PPARgamma protein. Finally, results obtained during adipogenesis of osteoporotic cells suggest that this process is abnormal not only because of increased adipocyte number, but because of impaired leptin cells response.     

Osteogenic growth peptide enhances the proliferation of bone marrow mesenchymal stem cells from osteoprotegerin-deficient mice by CDK2/cyclin A

To promote bone formation is one of the fundamental strategies in osteoporosis treatment and fractures repair. As one of the stimulators on bone formation, osteogenic growth peptide (OGP) increases both proliferation and differentiation of the osteoblasts in vitro and in vivo, in which osteoprotegerin (OPG) has been suggested being involved. In this study, we evaluated the effects of OGP on bone marrow mesenchymal stem cells (MSCs) from OPG-deficient mice in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, alkaline phosphatase (ALP) activity assay, real-time polymerase chain reaction, and western blot analysis. Results showed that OGP stimulated MSC proliferation and increased the expression of CDK2 and cyclin A in MSCs both at mRNA and protein levels. However, no differentiative effect of OGP was shown as ALP activity and the expression levels of Runx2 and Osterix were not increased significantly by OGP. Our study suggested that OGP may increase the bone formation in OPG-deficient mice by stimulating MSC proliferation rather than differentiation, and probably by triggering CDK2/cyclin A pathway.     

High affinity leptin receptors are present in human mesenchymal stem cells (MSCs) derived from control and osteoporotic donors

There are disparate observations on central and peripheral effects of leptin, but several studies consistently support its role as a link between fat and bone. Bone marrow stroma contains mesenchymal stem cells (MSCs), which differentiate into osteoblasts and adipocytes, among others. In this study we assessed the expression of leptin receptors protein in MSCs from control and osteoporotic postmenopausal donors and their change during osteogenic and adipogenic differentiation. Also, we assessed the effects of leptin on osteogenic and adipogenic differentiation of these cells. We demonstrated high affinity leptin binding (KD = 0.36 +/- 0.02 nM) in both types of cells. Binding was very low under basal, but increased significantly (2-3 times) through osteogenic and adipogenic differentiation. Osteoporotic MSCs showed lower leptin binding capacity than control cells at an early osteogenic and adipogenic differentiation time, which could restrict cell sensitivity to the protective action of leptin. In this regard, we observed that leptin significantly inhibited adipocyte differentiation in control but not in osteoporotic MSCs, while it exerted a low stimulatory effect on calcium deposition (10%-20%) in both types of MSCs cells. In summary, we report the presence of high affinity leptin receptors on control and osteoporotic MSCs, which were modified distinctly by osteogenic and adipogenic stimulation and a direct and distinct effect of leptin on both type of cells.     

Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.     

Arsenic trioxide promotes senescence and regulates the balance of adipogenic and osteogenic differentiation in human mesenchymal stem cells

Arsenic trioxide (ATO) as an anti-tumor drug could induce differentiation and apoptosis in tumor cells. Mesenchymal stem cells (MSCs) play important roles in the hematogenesis of bone marrow. Many reports have shown that the disorder of MSC adipogenic and osteogenic differentiation occurs in some diseases. However, reports about the effects of ATO on MSCs are limited. In this study, we found that 1 μM ATO promoted MSC senescence mainly through p21, although it had no effect on apoptosis at this dose. Furthermore, ATO promoted adipogenic differentiation, but inhibited osteogenic differentiation in MSCs. Our study also showed that CCAAT/enhancer-binding protein alpha C/EBPα and peroxisome proliferator-activated receptor gamma PPARγ might be involved in the regulation of adipogenic and osteogenic differentiation induced by ATO. Our results indicated that ATO may exert an anti-tumor effect by influencing bone marrow micro-environment. Moreover, it may regulate the adipogenic and osteogenic differentiation of MSCs.     

Effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and the adipocytic trans-differentiation of mouse primary osteoblasts

A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, alkaline phosphatase (ALP) activity measurement and Oil Red O stain and measurement were employed to assess the effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (MSCs) and the adipogenic trans-differentiation of mouse primary osteoblasts. The results showed that except for individual concentrations of zinc ion there was no effect on the proliferation of MSCs and osteoblasts. Zinc ion inhibited the osteogenic differentiation of MSCs at all the concentrations tested. It also inhibited adipogenic differentiation at all concentrations tested except 10(-9)mol/L. Both of the inhibition effects were attenuated with time increasing. Zinc ion depressed adipocytic trans-differentiation of osteoblasts at concentrations of 10(-11) and 10(-10)mol/L, but the effect could be reversed to promote or even be removed when concentration was increased. It suggests that the influence of zinc ion on osteogenic, adipogenic differentiation of MSCs and adipocytic trans-differentiation of osteoblasts depends on zinc ion concentrations and incubation time. The protective effects of zinc ion on bone may be mediated by modulating differentiation of MSCs away from the adipocytes and inhibiting adipocytic trans-differentiation of osteoblasts. This may in turn promote osteoblast formation and reduce secretion of cytokines which may inhibit osteoclast formation and activation. These findings may be valuable for better understanding the mechanism of the effect of zinc ion on bone.     

骨髓来源的间充质干细胞分化能力的鉴定

本文研究了人骨髓来源的间充质干细胞(MSCs)的成骨及成脂分化的潜能.通过加入诱导成骨的诱导剂,人的MSCs出现成骨分化的机箱,通过碱性磷酸酶活性测定,茜素红染色及主要调控基因BMP2和Runx2的表达,确定了MSCs具有成骨分化的潜能.对于成脂分化,通过油红O染色,及主要标志基因PPARγ的表达确定其具有成脂分化的潜能.所以,从骨髓分离的到的MSCs纯度达到标准,并且具有成骨成脂分化的多向潜能,是一种理想的实验模型细胞.     

The isolation and characterization of putative mesenchymal stem cells from the spiny mouse

The bone marrow represents the most common source from which to isolate mesenchymal stem cells (MSCs). MSCs are capable of differentiating into tissues of the three primary lineages and have the potential to enhance repair in damaged organs through the principals of regenerative medicine. Given the ease with which MSCs may be isolated from different species the aim of this study was to isolate and characterize putative bone marrow derived MSCs from the spiny mouse, Acomys cahirinus. MSCs were isolated from the spiny mouse in a traditional manner, and based on plastic adherence, morphology, colony forming unit-fibroblast assays and functional assessment (adipogenic, osteogenic and chondrogenic differentiation potential) a population of putative mesenchymal stem cells from the compact bone of the spiny mouse have been isolated and characterized. Such methodological approaches overcome the lack of species-specific antibodies for the spiny mouse and could be employed for other species where the cost of generating species-specific antibodies is not warranted.     

Derivation,characterization and gene modification of cynomolgus monkey mesenchymal stem cells

Mesenchymal stem cells (MSCs) have received considerable attention in recent years. Particularly exciting is the prospect that MSCs could be differentiated into specialized cells of interest, which could then be used for cell therapy and tissue engineering. MSCs derived from nonhuman primates could be a powerful tool for investigating the differentiation potential in vitro and in vivo for preclinical research. The purpose of this study was to isolate cynomolgus mesenchymal stem cells (cMSCs) from adult bone marrow and characterize their growth properties and multipotency. Mononuclear cells were isolated from cynomolgus monkey bone marrow by density-gradient centrifugation, and adherent fibroblast-like cells grew well in the complete growth medium with 10 μM Tenofovir. cMSCs expressed mesenchymal markers, such as CD29, CD105, CD166 and were negative for hematopoietic markers such as CD34, CD45. Furthermore, the cells were capable of differentiating into osteogenic, chondrogenic, and adipogenic lineages under certain conditions, maintaining normal karyotype throughout extended culture. We also compared different methods (lipofection, nucleofection and lentivirus) for genetic modification of cMSCs and found lentivirus proved to be the most effective method with transduction efficiency of up to 44.6% and lowest level of cell death. The cells after transduction stably expressed green fluorescence protein (GFP) and maintained the abilities to differentiate down osteogenic and adipogenic lineages. In conclusion, these data showed that cMSCs isolated from cynomolgus bone marrow shared similar characteristics with human MSCs and might provide an attractive cell type for cell-based therapy in higher-order mammalian species disorder models.     

Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells

The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-β gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.     

Modulation of the proliferation and differentiation of human mesenchymal stem cells by copper

Copper plays important functional roles in bone metabolism and turnover. It is known that it is essential for normal growth and development of the skeleton in humans and in animals. Although at present the exact role that copper plays in bone metabolism is unknown, bone abnormalities are a feature of severe copper deficiency. Osteoblasts are derived from mesenchymal stem cells (MSCs) present in bone marrow stroma, which are able to differentiate into bone, adipocytes, and other cell phenotypes. Excess adipogenesis in postmenopausal women may occur at the expense of osteogenesis and, therefore, may be an important factor in the fragility of postmenopausal bone. The purpose of this study was to evaluate whether an increase of the extracellular concentration of copper affects the ability of MSCs to differentiate into osteoblasts or adipocytes. The results showed that copper modified both the differentiation and the proliferative activity of MSCs obtained from postmenopausal women. Copper (50 microM) diminished the proliferation rate of MSCs, increasing their ability to differentiate into the osteogenic and the adipogenic lineages. Copper induced a 2-fold increase in osteogenic differentiation of MSCs, measured as a increase in calcium deposition. Copper (5 and 50 microM) diminished the expression of alkaline phosphatase (50 and 80%, respectively), but induced a shift in the expression of this enzyme to earlier times during culture. Copper also induced a 1.3-fold increase in the adipogenic differentiation of MSCs. It is concluded that copper stimulates MSC differentiation, and that this is preferentially towards the osteogenic lineage.     

Hey1 basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We previously demonstrated that bone morphogenetic protein (BMP) 9 is one of the most potent and yet least characterized BMPs that are able to induce osteogenic differentiation of MSCs both in vitro and in vivo. Here, we conducted gene expression-profiling analysis and identified that Hey1 of the hairy/Enhancer of split-related repressor protein basic helix-loop-helix family was among the most significantly up-regulated early targets in BMP9-stimulated MSCs. We demonstrated that Hey1 expression was up-regulated at the immediate early stage of BMP9-induced osteogenic differentiation. Chromatin immunoprecipitation analysis indicated that Hey1 may be a direct target of the BMP9-induced Smad signaling pathway. Silencing Hey1 expression diminished BMP9-induced osteogenic differentiation both in vitro and in vivo and led to chondrogenic differentiation. Likewise, constitutive Hey1 expression augmented BMP9-mediated bone matrix mineralization. Hey1 and Runx2 were shown to act synergistically in BMP9-induced osteogenic differentiation, and Runx2 expression significantly decreased in the absence of Hey1, suggesting that Runx2 may function downstream of Hey1. Accordingly, the defective osteogenic differentiation caused by Hey1 knockdown was rescued by exogenous Runx2 expression. Thus, our findings suggest that Hey1, through its interplay with Runx2, may play an important role in regulating BMP9-induced osteoblast lineage differentiation of MSCs.