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.     

Osteogenic growth peptide C-terminal pentapeptide [OGP(10-14)] acts on rat bone marrow mesenchymal stem cells to promote differentiation to osteoblasts and to inhibit differentiation to adipocytes

Cumulative evidence indicates that bone marrow mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating to osteogenic and adipogenic lineages when stimulated under appropriate conditions. Whether OGP(10-14) directly regulates the progenitor cells differentiating into osteoblasts or adipocytes remains unknown. In the present study, we investigated the roles of OGP(10-14) in differentiation along these separate lineages using rat bone marrow MSCs. Our results showed that OGP(10-14) promoted osteogenic differentiation of the stem cells and concurrently inhibited adipocyte formation. OGP(10-14) increased alkaline phosphatase (ALP) activity and mineralized nodule formation, and stimulated osteoblast-specific mRNA expression of core-binding factor 1 (cbfa1). In contrast, OGP(10-14) decreased adipocyte numbers and inhibited adipocyte-specific mRNA expression of peroxisome proliferator-activated receptor-gamma 2 (PPARgamma2). These observations suggest that commitment of MSCs into osteogenic or adipogenic lineages is regulated by OGP(10-14).     

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.     

A Lox/CHOP‐10 crosstalk governs osteogenic and adipogenic cell fate by MSCs

Accelerated marrow adipogenesis has been associated with ageing and osteoporosis and is thought to be because of an imbalance between adipogenic and osteogenic differentiation of mesenchymal stem cell (MSCs). We have previously found that lysyl oxidase (Lox) inhibition disrupts BMP4‐induced adipocytic lineage commitment and differentiation of MSCs. In this study, we found that lox inhibition dramatically up‐regulates BMP4‐induced expression of CCAAT/enhancer binding protein (C/EBP) homologous protein 10 (CHOP‐10), which then promotes BMP4‐induced osteogenesis of MSCs both in vitro and in vivo. Specifically, Lox inhibition or CHOP‐10 up‐regulation activated Wnt/β‐catenin signalling to enhance BMP4‐induced osteogenesis, with pro‐adipogenic p38 MAPK and Smad signalling suppressed. Together, we demonstrate that Lox/CHOP‐10 crosstalk regulates BMP4‐induced osteogenic and adipogenic fate determination of MSCs, presenting a promising therapeutic target for osteoporosis and other bone diseases.     

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.     

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.     

Coexpression of osteogenic and adipogenic differentiation markers in selected subpopulations of primary human mesenchymal progenitor cells

Knowledge of the basic mechanisms controlling osteogenesis and adipogenesis might provide new insights into the prevention of osteoporosis and age-related osteopenia. With the help of magnetic cell sorting and fluorescence activated cell sorting (FACS), osteoblastic subpopulations of mesenchymal progenitor cells were characterized. Alkaline phosphatase (AP) negative cells expressed low levels of osteoblastic and adipocytic markers. AP positive cells expressed adipocytic markers more strongly than the AP negative cell populations, thus suggesting that committed osteoblasts exhibit a greater adipogenic potential. AP negative cells differentiated to the mature osteoblastic phenotype, as demonstrated by increased AP-activity and osteocalcin secretion under standard osteogenic culture conditions. Surprisingly, this was accompanied by increased expression of adipocytic gene markers such as peroxisome proliferator-activated receptor-gamma2, lipoprotein lipase and fatty acid binding protein. The induction of adipogenic markers was suppressed by transforming growth factor-beta1 (TGF-beta1) and promoted by bone morphogenetic protein 2 (BMP-2). Osteogenic culture conditions including BMP-2 induced both the formation of mineralized nodules and cytoplasmic lipid vacuoles. Upon immunogold electron microscopic analysis, osteoblastic and adipogenic marker proteins were detectable in the same cell. Our results suggest that osteogenic and adipogenic differentiation in human mesenchymal progenitor cells might not be exclusively reciprocal, but rather, a parallel event until late during osteoblast development.     

Effects of strontium on proliferation and differentiation of rat bone marrow mesenchymal stem cells

Strontium ranelate (SrR) was an effective anti-osteoporotic drug to increase bone formation and decrease bone resorption. However, reports about the effect of SR on osteoblastic and adipocytic differentiation from bone marrow mesenchymal stem cells (BMMSCs) are limited. The purpose of this study is to evaluate whether SrR affects the ability of BMMSCs to differentiate into osteoblasts or adipocytes. Rat BMMSCs were identified by flow cytometry and exposed to SR (0.1 and 1.0mMSr(2+)) under osteogenic or adipogenic medium for 1 and 2weeks. The proliferation and differentiation of BMMSCs were analyzed by MTT, alkaline phosphatase (ALP), Oil red O staining, quantitative real-time RT-PCR and Western blot assays. SrR significantly inhibited the proliferation, increased osteoblastic but decreased adipocytic differentiation of rat BMMSCs dose-dependently. In osteogenic medium, SrR increased the expression of ALP, the mRNA levels of Cbfa1/Runx2, bone sialoprotein, and osteocalcin by RT-PCR, and the protein levels of Cbfa1/Runx2 by Western blot. In adipogenic medium, SrR decreased the mRNA levels of PPARγ2, adipocyte lipid-binding protein 2 (aP2/ALBP), and lipoprotein lipase (LPL) by RT-PCR, and the protein expression of PPARγ in Western blot analysis. These results indicated that the effects of SrR to promote osteoblastic but inhibit adipocytic differentiation of BMMSCs might contribute to its effect on osteoporosis treatment.     

Resveratrol mimics insulin activity in the adipogenic commitment of human bone marrow mesenchymal stromal cells

Bone marrow mesenchymal stromal cells (BM-MSCs) are multipotent cells capable of differentiating toward osteoblatic and adipocytic phenotypes. BM-MSCs play several key roles including bone remodeling, establishment of hematopoietic niche and immune tolerance induction. Here, we investigated the effect of resveratrol (RSV), a therapeutically promising natural polyphenol, on the commitment of human BM-MSCs primary cultures. Cell differentiation was evaluated by means of morphological analysis, specific staining and expression of osteogenic and adipocytic master genes (Runx-2, PPARγ). To maintain BM-MSC multipotency, all experiments were performed on cells at very early passages. At any concentration RSV, added to standard medium, did not affect the phenotype of confluent BM-MSCs, while, when added to osteogenic or adipogenic medium, 1 μM RSV enhances the differentiation toward osteoblasts or adipocytes, respectively. Conversely, the addition of higher RSV concentration (25 μM) to both differentiation media resulted exclusively in BM-MSCs adipogenesis. Surprisingly, the analysis of RSV molecular effects demonstrated that the compound completely substitutes insulin, a key component of adipogenic medium. We also observed that RSV treatment is associated to enhanced phosphorylation of CREB, a critical effector of insulin adipogenic activity. Finally, our observations contribute to the mechanistic elucidation of the well-known RSV positive effect on insulin sensitivity and type 2 diabetes mellitus.     

Cellular retinol-binding protein 1 (CRBP-1) regulates osteogenenesis and adipogenesis of mesenchymal stem cells through inhibiting RXRα-induced β-catenin degradation

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that can differentiate into osteoblasts, chondrocytes and adipocytes, providing a potential source for musculoskeletal tissue engineering. Retinoid signaling plays very important roles in skeletal development. CRBP1 (cellular retinol binding protein 1), a key component of retinoid signaling pathway, is known to take part in vitamin A metabolism and intracellular transporting of retinoids. However, the role of CRBP1 in MSCs remains still obscure. In this study, we investigated the cellular effects of CRBP1 on osteogenic and adipogenic differentiation of bone marrow derived MSCs in vitro and in vivo. Our results showed that CRBP1 overexpression promoted osteogenic differentiation of bone marrow derived MSCs, while inhibited their adipogenic differentiation. We also demonstrated that the possible underlying mechanism for CRBP1 promoting osteogenic differentiation of MSCs was by inhibiting RXRα-induced β-catenin degradation, maintaining β-catenin and pERK1/2 at higher levels. These findings reveal a potential role of CRBP1 in the regulation of β-catenin turnover which can greatly affect the process of osteogenesis and adipogenesis of MSCs.     

Serum‐free medium with osteogenic supplements induces adipogenesis in rat bone marrow stromal cells

Adult BMSCs (bone marrow stromal cells) contain MSCs (mesenchymal stem cells). MSCs can differentiate into osteoblasts, chondrocytes, adipocytes and myoblasts and are thus considered useful in tissue engineering for therapeutic and clinical purposes. FCS (fetal calf serum) is usually included in the differentiation medium, but the clinical application of FCS may pose a problem for some patients. To improve the efficiency and safety of BMSC cultivation, the effect of SF (serum‐free) conditions on the osteogenic differentiation of rat BMSCs was examined. In the presence of 10% FCS and osteogenic supplements, the cells formed mineralized von Kossa‐positive deposits. Under SF conditions with osteogenic supplementation, however, the cells possessed cytosolic lipid vacuoles that could be stained with Oil Red O. The mRNA expression of PPARγ (peroxisome proliferator‐activated receptor γ), an adipogenic marker, increased under the SF condition with osteogenic supplements. These data indicate that rat BMSCs differentiate into adipocytes under SF conditions even with osteogenic supplementation and that FCS is needed to induce proper osteogenic differentiation in rat BMSCs.     

Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue

Mesenchymal stem cells (MSCs) have been investigated as promising candidates for use in new cell-based therapeutic strategies such as mesenchyme-derived tissue repair. MSCs are easily isolated from adult tissues and are not ethically restricted. MSC-related literature, however, is conflicting in relation to MSC differentiation potential and molecular markers. Here we compared MSCs isolated from bone marrow (BM), umbilical cord blood (UCB), and adipose tissue (AT). The isolation efficiency for both BM and AT was 100%, but that from UCB was only 30%. MSCs from these tissues are morphologically and immunophenotypically similar although their differentiation diverges. Differentiation to osteoblasts and chondroblasts was similar among MSCs from all sources, as analyzed by cytochemistry. Adipogenic differentiation showed that UCB-derived MSCs produced few and small lipid vacuoles in contrast to those of BM-derived MSCs and AT-derived stem cells (ADSCs) (arbitrary differentiation values of 245.57 +/- 943 and 243.89 +/- 145.52 mum(2) per nucleus, respectively). The mean area occupied by individual lipid droplets was 7.37 mum(2) for BM-derived MSCs and 2.36 mum(2) for ADSCs, a finding indicating more mature adipocytes in BM-derived MSCs than in treated cultures of ADSCs. We analyzed FAPB4, ALP, and type II collagen gene expression by quantitative polymerase chain reaction to confirm adipogenic, osteogenic, and chondrogenic differentiation, respectively. Results showed that all three sources presented a similar capacity for chondrogenic and osteogenic differentiation and they differed in their adipogenic potential. Therefore, it may be crucial to predetermine the most appropriate MSC source for future clinical applications.     

miR-17-5p and miR-106a are involved in the balance between osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into several distinct cell types, including osteoblasts and adipocytes. The balance between osteogenic and adipogenic differentiation is disrupted in several osteogenic-related disorders, such as osteoporosis. So far, little is known about the molecular mechanisms that drive final lineage commitment of MSCs. In this study, we revealed that miR-17-5p and miR-106a have dual functions in the modulation of human adipose-derived mesenchymal stem cells (hADSCs) commitment by gain- and loss-of-function assays. They could promote adipogenesis and inhibit osteogenesis. Luciferase reporter assay, western blot and ELISA suggested BMP2 was a direct target of miR-17-5p and miR-106a. Downregulation of endogeneous BMP2 by RNA interference suppressed osteogenesis and increased adipogenesis, similar to the effect of miR-17-5p and miR-106a upregulation. Moreover, the inhibitory effects of miR-17-5p on osteogenic and adipogenic differentiation of hADSCs could be reversed by BMP2 RNA interference. In conclusion, miR-17-5p and miR-106a regulate osteogenic and adipogenic lineage commitment of hADSCs by directly targeting BMP2, and subsequently decreased osteogenic TAZ, MSX2 and Runx2, and increased adipogenic C/EBPα and PPARγ.     

去卵巢骨质疏松山羊骨髓基质细胞成骨分化能力的研究

目的：研究在构建的去卵巢骨质疏松山羊动物模型中,骨髓基质细胞（MSCs）的生物学特性以及其成骨能力。方法：建立去卵巢骨质疏松山羊动物模型,使用全骨髓法获取去卵巢骨质疏松山羊（实验组）和正常山羊（对照组）MSCs,流式细胞仪检测实验组和对照组细胞周期及增殖指数（PI）;地塞米松诱导21d时油红O染色,观察成脂分化比例;成骨诱导液诱导14d,碱性磷酸酶（ALP）染色、检测ALP表达量。结果：对照组PI高于实验组;地塞米松诱导后实验组脂肪细胞比例明显高于对照组;成骨诱导第7d,对照组ALP的表达量明显高于实验组。结论：去卵巢骨质疏松山羊的MSCs增殖和成骨分化能力都降低,可能与骨质疏松症的发病机理有关。     

Effects of Zinc Transporter on Differentiation of Bone Marrow Mesenchymal Stem Cells to Osteoblasts

The differentiation of bone marrow mesenchymal stem cells (MSCs) into osteoblasts is a crucial step during bone formation. However, the exact mechanisms regulating the early stages of osteogenic differentiation remain unknown. In the present study, we found that ZnT7, a member of the zinc transporter family SLC30A(ZnTs), was downregulated during dexamethasone-induced differentiation of rat MSCs into osteoblasts. Dexamethasone treatment resulted in significantly lower levels of ZnT7 compared with cocultured cells without dexamethasone. Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity and staining for ALP, von Kossa, collagen type I, and osteocalcin. Overexpression of ZnT7 decreased the expression of the osteoblast alkaline phosphatase, type I collagen, as well as calcium deposition in mesenchymal cells. In contrast, knockdown of ZnT7 using siRNA promoted gene expression associated with osteoblast differentiation and matrix mineralization in vitro. Moreover, according to the ZnT7 inhibition or activation experiments, Wnt and ERK signaling pathways were found to be important signal transduction pathways in mediating the osteogenic effect of MSCs, and this effect is intensified by a decrease in the level of ZnT7 induced by dexamethasone. These findings suggest that ZnT7 is involved in the switch from the undifferentiated state of MSC to an osteogenic program, and marking the expression level of ZnT7 may be useful in the detection of early osteogenic differentiation.