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.     

人骨髓基质细胞向成骨细胞分化过程中差异表达基因的筛选

为了探讨人骨髓基质细胞（bone marrow stromal cells,BMSCs）向成骨细胞分化过程中差异表达的基因,本实验采用体外培养人BMSCs,诱导向成骨细胞分化。分别选取培养12和21d的细胞作为驱动方（driver）和实验方（tester）,进行抑制消减杂交,构建cDNA消减文库,将挑选出的阳性克隆与GenBank人基因库中己公布的核酸序列进行同源性比较分析。结果表明,从培养21d的BMSCs中,筛查出5个差异基因,与人基因库中己知基因的同源性分别达到90％以上。有兴趣的是,核心蛋白聚糖和Bax inhibitorl在培养2ld的BMSCs中差异表达。RT-PCR检测显示,核心蛋白聚糖基因在培养21d的细胞中高表达,而在12d的细胞中未检测到表达;Bax inhibitorl基因在培养21d细胞中的表达明显高于12d的细胞。     

A dual role of cholesterol in osteogenic differentiation of bone marrow stromal cells

Osteoblasts, the chief bone-forming cells, are differentiated from mesenchymal stromal/stem cells. Disruption of this differentiation process can cause osteoporosis, a bone disease characterized by low bone mass and deteriorated bone structure. Cholesterol has been implicated in pathogenesis of osteoporosis, and was recently identified as an endogenous activator of Hedgehog (Hh) signaling. However, its pathological and physiological roles in osteoblast differentiation are still poorly understood. Moreover, it is unclear whether these potential roles played by cholesterol are related to its capability to modulate Hh pathway. In this study, we investigated the role of exogenous versus endogenous cholesterol in osteogenesis and Hh pathway activation using ST2 cells, a bone marrow stromal cell line. We found that exogenous cholesterol significantly inhibited alkaline phosphatase (ALP) activity and messenger RNA expression of osteoblast markers genes (Alpl, Sp7, and Ibsp) while modestly activating expression of Gli1 (a readout of Hh signaling) under both basal osteogenic culture condition and Wnt3a treatment. Similarly, exogenous cholesterol suppressed osteogenic response of ST2 cells to sonic Hh (Shh) or purmorphamine (Purmo) treatment, which, however, was accompanied by diminished induction of Gli1, indicating the involvement of a Hh-dependent mechanism. Interestingly, depletion of endogenous cholesterol also reduced Shh-induced ALP activity and Gli1 expression. Likewise, cholesterol depletion inhibited osteogenic response to Purmo, although it did not affect Gli1 induction. Taken together, our findings have demonstrated that cholesterol plays a dual role in osteoblast differentiation likely through both Hh-dependent and -independent mechanisms.     

Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells

Dexamethasone is capable of directing osteoblastic differentiation of bone marrow stromal cells (BMSCs) in vitro, but its effects are not lineage-specific, and sustained exposure has been shown to down-regulate collagen synthesis and induce maturation of an adipocyte subpopulation within BMSC cultures. Such side effects might be reduced if dexamethasone is applied in a regimented manner, but the discrete steps in osteoblastic maturation that are stimulated by dexamethasone are not known. To examine this, dexamethasone was added to medium to initiate differentiation of rat BMSCs cultures and then removed after a varying number of days. Cell layers were analyzed for cell number, rate of collagen synthesis, expression of osteocalcin (OC), bone sialoprotein (BSP) and lipoprotein lipase (LpL), and matrix mineralization. Withdrawal of dexamethasone at 3 and 10 days was found to enhance cell number relative to continuous exposure, but did not affect to decrease collagen synthesis slightly. Late markers of osteoblastic differentiation, BSP expression and matrix mineralization, were also sensitive to dexamethasone and increased systematically with exposure while LpL systematically decreased. These results indicate that dexamethasone acts at both early and late stages to direct proliferative osteoprogenitor cells toward terminal maturation.     

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.     

骨髓基质细胞的特征及其在细胞和基因治疗中的应用

骨髓基质细胞是一类独特的间质干细胞,可分化为多种非造血系的组织。骨髓基质细胞具有贴壁生长的特性,因而易于在体外分离和扩增;另外骨髓基质细胞可在体内外表达多种治疗性的外湖目的基因。因此,骨髓基质细胞被认为是一种理想的治疗性细胞的基因治疗中的靶细胞。本文对骨髓基质细胞的研究进展及其在细胞和基因治疗中的应用作一综述。     

Osteogenic differentiation of multipotent mesenchymal stromal cells isolated from human bone marrow and subcutaneous adipose tissue

Cellular populations with phenotypes similar to multipotent mesenchymal stromal cells were isolated from two different sources, including human bone marrow (BM) and subcutaneous adipose tissue (SAT). Comparative analysis of the efficiency of differentiation in the direction of osteogenesis has revealed morphological changes confirmed by staining with Alizarin red and von Kossa in bone marrow cells at the 14th day and in adipose tissue cells at the 28th day of cultivation in the medium with inductors. Analysis of expression of the osteopontin, osteocalcin, and bone sialoprotein genes in RT-PCR reactions has detected essential differences in the potential of these cells to differentiate into bone tissue cells. Cells isolated from BM of both the control and experimental groups were positive for octeopontin (OP) on the 14th day. Unlike these cells, in cells isolated from SAT in medium without an inductor, no product of OP gene expression was identified. In the cells subjected to differentiation, OP appeared at day 14. In the BM cells, octeocalcin (OC) was found at the 14th day, while the bone sialoprotein (BS) was found at the 21st day of cultivation in induction medium. In cells isolated from SAT, OC, and BS were not detected, even at the 28th day after the beginning of induction.     

Antagonistic and synergistic effects of bone morphogenetic protein 2/7 and all‐trans retinoic acid on the osteogenic differentiation of rat bone marrow stromal cells

The osteogenesis of bone marrow stromal cells (BMSCs) is of paramount importance for the repair of large‐size bone defects, which may be compromised by the dietary‐accumulated all‐trans retinoic acid (ATRA). We have shown that heterodimeric bone morphogenetic protein 2/7 (BMP2/7) could induce bone regeneration in a significantly higher dose‐efficiency in comparison with homodimeric BMPs. In this study, we evaluated the effects of ATRA and BMP2/7 on the proliferation, differentiation, mineralization and osteogenic genes. ATRA and BMP2/7 exhibited both antagonistic and synergistic effects on the osteogenesis of BMSCs. ATRA significantly inhibited proliferation and expression of osteocalcin but enhanced the activity of alkaline phosphatase of BMSCs. On day 21, 50 ng/mL BMP2/7 could antagonize the inhibitive effects of ATRA and significantly enhance osteogenesis of BMSCs. These findings suggested a promising application potential of heterodimeric BMP2/7 in clinic to promote bone regeneration for the cases with dietary accumulated ATRA.     

Protein expression profile in the differentiation of rat bone marrow stromal cells into Schwann cell-like cells

During the last decade, increasing evidence suggested that bone marrow stromal cells (MSCs) have the potential to differentiate into neural lineages. Many studies have reported that MSCs showed morphological changes and expressed a limited number of neural proteins under experimental conditions. However, no proteomic studies on MSCs differentiated into Schwann cell-like cells have been reported. In this study, we isolated MSCs from adult Sprague-Dawley rat femur and tibia bone marrows and induced the cells in vitro under specific conditions. By using two-dimensional gel electrophoresis (2-DE), we compared the protein profiles of MSCs before and after induced differentiation. We obtained 792 protein spots in the protein profile by 2-DE, and found that 74 spots changed significantly before and after the differentiation using PDQuest software, with 43 up-regulated and 31 down-regulated. We analyzed these 74 spots by a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and by database searching, and found that they could be grouped into various classes, including cytoskeleton and structure proteins, growth factors, metabolic proteins, chaperone proteins, receptor proteins, cell cycle proteins, calcium binding proteins, and other proteins. These proteins also include neural and glial proteins, such as BDNF, CNTF and GFAP. The results may provide valuable proteomic information about the differentiation of MSCs into Schwann cell-like cells. Supported by National High-Tech Research and Development Program of China (Grant No. 2006AA02A128) and National Natural Science Foundation of China (Grant No. 30670667).     

Inflammatory T cells rapidly induce differentiation of human bone marrow stromal cells into mature osteoblasts

Activated T cells secrete multiple osteoclastogenic cytokines which play a major role in the bone destruction associated with rheumatoid arthritis. While the role of T cells in osteoclastogenesis has received much attention recently, the effect of T cells on osteoblast formation and activity is poorly defined. In this study, we investigated the hypothesis that in chronic inflammation activated T cells contribute to enhanced bone turnover by promoting osteoblastic differentiation. We show that T cells produce soluble factors that induce alkaline phosphatase activity in bone marrow stromal cells and elevated expression of mRNA for Runx2 and osteocalcin. This data indicate that T cell derived factors have the capacity to stimulate the differentiation of bone marrow stromal cells into the osteoblast phenotype. RANKL mRNA was undetectable under any conditions in highly purified bone marrow stromal cells. In contrast, RANKL was constitutively expressed in primary osteoblasts and only moderately up-regulated by activated T cell conditioned medium. Interestingly, both bone marrow stromal cells and osteoblasts expressed mRNA for RANK, which was strongly up-regulated in both cell types by activated T cell conditioned medium. Although, mRNA for the RANKL decoy receptor, osteoprotegerin, was also up-regulated by activated T cell conditioned medium, it's inhibitory effects may be mitigated by a simultaneous rise in the osteoprotegerin competitor TNF-related apoptosis-inducing ligand. Based on our data we propose that during chronic inflammation, T cells regulate bone loss by a dual mechanism involving both direct stimulation of osteoclastogenesis, by production of osteoclastogenic cytokines, and indirectly by induction of osteoblast differentiation and up-regulation of bone turnover via coupling.     

Cholinergic neuronal differentiation of bone marrow mesenchymal stem cells in rhesus monkeys

The purpose of the present study was to determine the best cholinergic neuronal differentiation method of rhesus monkey bone marrow mesenchymal stem cells(BMSCs).Four methods were used to induce differentiation,and the groups were assigned accordingly:basal inducing group(culture media,bFGF,and forskolin);SHH inducing group(SHH,inducing group);RA inducing group(RA,basal inducing group);and SHH+RA inducing group(SHH,RA,and basal inducing group).All groups displayed neuronal morphology and increased expressio...     

The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts

We investigated the effects of different implant biomaterials on cultured canine bone marrow stromal cells (BMSC) undergoing differentiation into osteoblasts (dBMSC). BMSC were isolated from canine humerus by marrow aspiration, cultured and differentiated on calcium phosphate scaffold (CPS), hydroxyapatite, hydroxyapatite in gel form and titanium mesh. We used the MTT method to determine the effects of osteogenic media on proliferation. The characteristics of dBMSC were assessed using alizarin red (AR), immunocytochemistry and osteoblastic markers including alkaline phosphatase/von Kossa (ALP/VK), osteocalcin (OC) and osteonectin (ON), and ELISA. The morphology of dBMSC on the biomaterials was investigated using inverted phase contrast microscopy and scanning electron microscopy. We detected expression of ALP/VK, AR, OC and ON by day 7 of culture; expression increased from day 14 until day 21. CPS supported the best adhesion, cell spreading, proliferation and differentiation of BMSCs. The effects of the biomaterials depended on their surface properties. Expression of osteoblastic markers showed that canine dBMSCs became functional osteoblasts. Tissue engineered stem cells can be useful clinically for autologous implants for treating bone wounds.     

Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells

To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p<0.05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p<0.05). Lastly, we found that simvastatin induces high expression of BMP(2) in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP(2) expression in BMSCs.     

骨髓基质干细胞的分离纯化及培养

目的 建立骨髓基质干细胞(MSCs)良好的分离纯化和培养方法。方法 将小鼠骨髓基质干细胞自殷骨中分离,应用贴壁选择法结合细胞克隆挑选法进行分离纯化,应用细胞生长因子(EGF和PDGF-BB)刺激法进行MSCs的体外培养和传代,倒置显微镜下观察分离培养的细胞并照像记录。结果,培养获得了纯化的呈梭形成纤雏样细胞的骨髓基质干细胞。在生长因子EGF和PDGF-BB的共同作用下,传代MSCs生长旺盛,形态均一。结论 该方法是简便高效的骨髓基质干细胞的分离纯化和培养方法。     

Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O(2), bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G(2)/S/M phase cells increased evidently under 8% O(2) condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O(2) condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl(2)) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.     

The reduced trabecular bone mass of adult ARKO male mice results from the decreased osteogenic differentiation of bone marrow stroma cells

Male mice with androgen receptor knock-out (ARKO) show significant bone loss at a young age. However, the lasting effect of AR inactivation on bone in aging male mice remains unclear. We designed this study to evaluate the effect of AR on bone quality in aging male mice and to find the possible causes of AR inactivation contributing to the bone loss. The mice were grouped according to their ages and AR status and their trabecular bones were examined by micro-CT analysis at 6, 12, 18, and 30 weeks old. We found that bone mass consistently decreased and the bone microarchitectures continuously deteriorated in male ARKO mice at designated time points. To determine the cause of the bone loss in ARKO mice, we further examined the role of AR in bone cell fate decision and differentiation and we conducted experiments on bone marrow stromal cells (BMSC) obtained from wild type (WT) and AR knockout (KO) mice. We found that ARKO mice had higher numbers of colony formation unit-fibroblast (CFU-F), and CD44 and CD34 positive cells in bone marrow than WT mice. Our Q-RT-PCR results showed lower expression of genes linked to osteogenesis in BMSCs isolated from ARKO mice. In conclusion, AR nullification disrupted bone microarchitecture and caused trabecular bone mass loss in male ARKO mice. And the fate of BMSCs was impacted by the loss of AR. Therefore, these findings suggest that AR may accelerate the use of progenitor cells and direct them into osteogenic differentiation to affect bone metabolism.