Communications between bone cells and hematopoietic stem cells

The skeletal system, while characterized by a hard tissue component, is in fact an extraordinarily dynamic system, with disparate functions ranging from structural support, movement and locomotion and soft-organ protection, to the maintenance of calcium homeostasis. Amongst these functions, it has long been known that mammalian bones house definitive hematopoiesis. In fact, several data demonstrate that the bone microenvironment provides essential regulatory cues to the hematopoietic system. In particular, interactions between the bone-forming cells, or osteoblasts, and the most primitive Hematopoietic Stem Cells (HSC) have recently been defined. This review will focus mainly on the role of osteoblasts as HSC regulatory cells, discussing the signaling mechanisms and molecules currently thought to be involved in their modulation of HSC behavior. We will then review additional cellular components of the HSC niche, including endothelial cells and osteoclasts. Finally, we will discuss the potential clinical implications of our emerging understanding of the complex HSC microenvironment.     

Clastogenicity of carbazole in mouse bone marrow cells in vivo

Clastogenicity of carbazole was evaluated by employing mouse in vivo chromosomal aberration (CA) test. Carbazole administered intraperitoneally (i.p.) at the rate of 25, 50, 100, 150 and 200 mg/kg b.w. to Swiss albino mice in vivo resulted in mitotic depression and induction of chromosomal aberrations. Dose related decrease in mitotic index (MI) and increase in the frequencies of chromosomal aberrations per cell (CAs/cell) and percent abnormal cells were recorded in bone marrow cells. However, statistically significant reduction in MI and increase in CAs/cell and percent abnormal cells were found only for the two higher doses. The results obtained indicate that carbazole or its metabolite, if any, is moderately clastogenic in the bone marrow cells of Swiss albino mice.     

骨髓间充质干细胞向神经细胞分化的研究

无论是在体外实验、还是在体内实验,MSCs都可以向中枢神经系统(CNS)神经细胞分化,但争议颇多。因为功能性神经元不仅要具有典型神经元的形态、特异性标记,还要求具有可兴奋性、能和其他神经元形成突触联系、产生突触电位等,所以对于骨髓间充质干细胞是否能诱导出真正具有功能的神经元存在很大分歧。在此对MSCs向神经细胞诱导分化研究的现况、存在的问题及发展前景给以综述。     

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.     

小型猪骨髓间充质干细胞的分离和培养

目的建立小型猪骨髓间充质干细胞（mesenchymal stem cells,MSCs）的体外分离和培养方法。方法穿刺小型猪髂后上嵴抽取骨髓,经密度梯度法离心得到骨髓单个核细胞,接种后形成单层贴壁细胞。用形态学方法鉴定培养的MSCs。结果经培养存活的MSCs原代和一代呈纺锤型、多边型或星型。至二代起呈均一纺锤型,似成纤维细胞样,长宽比例约为（2～3）？1。体外培养的原代MSCs8～10d达到融合,传代后仍具有较强的增殖能力。结论小型猪MSCs可在体外长期、稳定培养,其分离、培养体系的建立为基础研究和组织工程技术提供了一个有价值的动物模型。     

Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow

The future use of adult mesenchymal stem cells (MSCs) for human therapies depends on the establishment of preclinical studies with other mammals such as mouse. Surprisingly, purification and characterisation of murine MSCs were only poorly documented. The aim of this study was to purify mouse MSCs from adult bone marrow and to functionally characterise their abilities to differentiate along diverse lineages. Adherent cells from adult C57Bl/6J mouse bone marrow were depleted of granulo-monocytic cells and subsequently allowed to grow on fibronectin-coated dishes in presence of fetal bovine serum and growth factors. The growing fibroblastoid cell population primarily consisted of spindle- and star-shaped cells with significant renewal capacity as they were cultured until 30 passages (about 60 doubling population). We fully demonstrated the MSC phenotype of these cells by inducing them to differentiate along osteoblastic, adipocytic, and chondrocytic pathways. Mouse MSCs (mMSCs) sharing the same morphological and functional characteristics as human MSCs can be successfully isolated from adult bone marrow without previous mouse or bone marrow treatment. Therefore, mMSCs will be an important tool to study the in vivo behaviour and fate of this cell type after grafting in mouse pathology models.     

Production of human osteoclasts in a three-dimensional bone marrow culture system

Osteoclasts, which are derived from hemopoietic stem cells, play important roles in bone remodeling and resorption. Osteoclast development is critically dependent on the bone microenvironment. We have developed a novel human ex vivo bone marrow model that mimics bone marrow both structurally and functionally by providing artificial scaffolding, thus obtaining a three-dimensional growth configuration with high cell density and intimate physical contact between hemopoietic and stromal cells. In this study, we utilized the 3-D culture system to produce multinucleated cells (MNCs) from human bone marrow cells cultured in the presence of Vitamin D₃ and the absence of hydrocortisone and any exogenous growth factors. These multinucleated cells had the phenotypic and functional characteristics of osteoclasts as determined by their morphology, expression of tartarate resistant acid phosphatase (TRAP), and the ability to resorb bone. Scoring of the resorption pits revealed a dose response to Vitamin D₃ stimulation with 10⁻⁷ M being the optimal concentration. Furthermore, addition of parathyroid hormone (10⁻⁸ M) resulted in an up-to three-fold enhancement of bone resorption. These findings suggest that the 3-D culture system represent a physiologically relevant model to study osteoclastogenesis and elucidate the molecular and cellular signals associated with this process.     

Non-hematopoietic bone marrow stem cells: molecular control of expansion and differentiation

The first non-hematopoietic mesenchymal stem cells (MSCs) were discovered by Friedenstein in 1976, who described clonal, plastic adherent cells from bone marrow capable of differentiating into osteoblasts, adipocytes, and chondrocytes. More recently, investigators have now demonstrated that multi-potent MSCs can be recovered from a variety of other adult tissues and differentiate into numerous tissue lineages including myoblasts, hepatocytes and possibly even neural tissue. Because MSCs are multipotent and easily expanded in culture, there has been much interest in their clinical potential for tissue repair and gene therapy and as a result, numerous studies have been carried out demonstrating the migration and multi-organ engraftment potential of MSCs in animal models and in human clinical trials. This review describes the recent advances in the understanding of MSC biology.     

Development of liver regenerative therapy using glycoside-modified bone marrow cells

Several recent studies have reported that bone marrow cells (BMCs) have the ability to generate functional hepatocytes. However, the efficiency at which BMC transplantation generates functional hepatocytes is rather low. We assumed that if BMCs accumulated directly in liver, the functional BMC-derived hepatocytes should increase efficiently. We tried to increase the accumulation of BMCs directly in liver through the interaction between hepatic asialoglycoprotein receptor and desialylated BMCs. Desialylated BMCs were produced with treatment of neuraminidase. Desialylated BMCs that expressed green fluorescent protein (GFP) were injected into Long Evans Cinnamon (LEC) rats, a human Wilson's disease model, intravenously. At 3 and 5 months after transplantation, GFP-expressing hepatocyte nodules appeared in the liver of these BMC-transplanted LEC rats. These findings suggest that the functional BMC-derived hepatocytes can be generated by the direct accumulation of BMCs and that this strategy is new BMC therapy for liver regeneration.     

Adult rat bone marrow stromal cells differentiate into Schwann cell-like cells in vitro

Bone marrow stromal cells (MSCs) have the capability of differentiating into mesenchymal and non-mesenchymal lineages. In this study, MSCs isolated from adult Sprague-Dawley rats were cultured to proliferation, followed by in vitro induction under specific conditions. The results demonstrated that MSCs were transdifferentiated into cells with the Schwann cell (SC) phenotypes according to their morphology and immunoreactivities to SC surface markers including S-100, glial fibrillary acidic protein (GFAP) and low-affinity nerve growth factor receptor (p75). Consequently, rat adult MSCs can be induced in vitro to differentiate into SC-like cells, thus developing an abundant and accessible SC reservoir to meet the requirements of constructing tissue engineered nerve grafts for peripheral nerve repair.     

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.     

Skeletal muscle differentiation potential of human adult bone marrow cells

Murine bone marrow (BM) cells have been shown to undergo myogenic differentiation and participate in muscle repair in different muscle regeneration models. In the present paper, we report on a subset of cells (CD45+/desmin+) with myogenic potential being present at very low frequencies in human adult BM. By a simple culture method, we were able to obtain in vitro multinucleated myotubes in up to 20% of the cultures. Myotubes were generated using both BM flushed from rib fragments obtained during thoracotomy and BM derived from iliac crest aspirates. Cells of the different adherent and non-adherent fractions expressed numerous muscle specific markers by immunocytochemistry, real-time RT-PCR, flow cytometry, and Western blot analyses. Moreover, direct injection of whole BM into the right tibialis anterior muscle of immunodeficient mice (NOD/RAG) that had previously been treated with cardiotoxin to induce muscle degeneration, showed a variable but significant level of human cell engraftment (from 0.06 to 0.26% Dys+/FISH+ fibers). These data suggest that cells with skeletal muscle differentiation potential are present in adult human BM can differentiate in vitro and give rise to myogenic cells in vivo in immunodeficient mice after muscle damage. Further improvements might allow new approaches to cell-mediated therapies for muscular diseases.     

Vitreous cryopreservation of tissue engineered bone composed of bone marrow mesenchymal stem cells and partially demineralized bone matrix

Cryopreservation of tissue engineered products by maintaining their structure and function is a prerequisite for large-scale clinical applications. In this study, we examined the feasibility of cryopreservation of tissue engineered bone (TEB) composed of osteo-induced canine bone marrow mesenchymal stem cells (cBMSCs) and partially demineralized bone matrix (pDBM) scaffold by vitrification. A novel vitreous solution named as VS442 containing 40% dimethyl-sulfoxide (DMSO), 40% EuroCollins (EC) solution and 20% basic culture medium (BCM) was developed. After being cultured in vitro for 8 days, cell/scaffold complex in VS442 was subjected to vitreous preservation for 7 days and 3 months, respectively. Cell viability, proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons being made with those cryopreserved in VS55 vitreous solution. Compared with that cryopreserved in VS55, cell viability and subsequent proliferative ability of TEB in VS442 after being rewarmed were significantly higher as detected by live/dead staining and DNA assay. The level of alkaline phosphatase (ALP) expression and osteocalcin (OCN) deposition in VS442 preserved TEB was also higher than those in the VS55 group since 3 days post-rewarm. Both cell viability and osteogenic capability of the VS55 group were found to be declined to a negligible level within 15 days post-rewarm. Furthermore, it was observed that extending the preservation of TEB in VS442 to 3 months did not render any significant effect on its survival and osteogenic potential. Thus, the newly developed VS442 vitreous solution was demonstrated to be more efficient in maintaining cellular viability and osteogenic function for vitreous cryopreservation of TEB over VS55.     

Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells

Bone marrow mesenchymal stem cells have been shown to transdifferentiate into cardiomyocytes after 5-azacytidine treatment or co-culturing with rodent cardiomyocytes. We investigate if adult human bone marrow stem cells can be differentiated ex vivo into cardiomyocyte-like cells (CLCs) independent of cytotoxic agents or co-culturing technique. Sternal bone marrow was collected from 16 patients undergoing coronary artery bypass surgery. Mesenchymal stem cells were differentiated in a cardiomyogenic differentiation medium containing insulin, dexamethasone, and ascorbic acid. Differentiation towards CLCs was determined by induced expression of cardiomyocyte-specific proteins. Differentiated CLCs expressed multiple structural and contractile proteins that are associated with cardiomyocytes. Thin filament associated myofibrillar proteins were detected early in the cells, with cardiac troponin I, sarcomeric tropomyosin, and cardiac titin among the first expressed. Some CLCs were found to develop into a nascent cardiomyocyte phenotype with cross-striated myofibrils characterized by alpha-actinin-positive Z bands after 4-5 passages in differentiated culture. These lineage-defined CLCs may be potentially useful for repairing damaged myocardium.     

GM-CSF accelerates proliferation of endothelial progenitor cells from murine bone marrow mononuclear cells in vitro

Objective: To test whether the GM-CSF accelerates the proliferation of bone marrow endothelial progenitor cells (BM EPCs). Methods: BM EPCs were induced by endothelial cell conditioned medium (EC-CM). The effect of different concentrations of GM-CSF on the proliferation of BM EPCs was evaluated by the formation of EC-cols, MTT assay, and cell cycle assay. The single progenitor cell growth curves were quantified. Results: The data indicated that GM-CSF accelerated the proliferation of BM EPCs both in colony numbers and colony size. MTT confirmed the effect of GM-CSF on accelerating the proliferation of BM EPCs. The single colony experiments showed that EC-cols expressed different proliferation capacity, suggesting that the EC-cols with different proliferation potentials might have been derived from different levels of immature progenitors. The cell cycle assay showed that the rate of cells entering into S phase was 9.3% in the group treated with GM-CSF and 2.1% in the controls. Furthermore, these cells displayed the specific endothelial cell markers and formed capillary-like structures. Conclusions: GM-CSF accelerates proliferation of BM EPCs. The potential beneficial of GM-CSF in the application of treating vascular ischemic patients is promising.     

Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone

Summary Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm² show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation.     

cxcr2基因修饰小鼠骨髓间充质干细胞的构建与鉴定

目的构建小鼠CXC型趋化因子受体2(CXCR2)基因cxcr2过表达的骨髓间充质干细胞(Bone marrow mes-enchymal stem cell,BMSC)并进行鉴定。方法全骨髓贴壁法分离培养小鼠BMSC,采用流式细胞术检测干细胞抗原1(stem cell antigen-1,SCA-1)、CD44、CD43、CD45、IA/IE表达率,并诱导成骨分化。以含有小鼠cxcr2的质粒为模版进行PCR扩增,将获得的cxcr2克隆到慢病毒载体,命名为p Lenti-cxcr2-GZ;将其与慢病毒包装质粒共转染HEK-293T细胞,收获慢病毒后,通过离心法感染BMSC,经过1μg/mL zeocin压力选择建立了稳定表达CXCR2的小鼠BMSC(CXCR2-BMSC)。采用流式细胞术和RT-PCR分别检测其CXCR2蛋白和m RNA表达水平,Transwell趋化实验检测其迁移能力。结果 90%以上的第3代BMSC表达CD44、SCA-1,几乎不表达IA/IE、CD34、CD45,且成功诱导成骨分化。菌液PCR、质粒双酶切后,琼脂糖凝胶电泳鉴定结果得到特异、大小正确的条带及测序鉴定正确,表明成功构建了p Lenti-cxcr2-GZ表达质粒。流式细胞术和RT-PCR结果显示,CXCR2-BMSC的CXCR2蛋白和m RNA表达水平均明显高于对照组BMSC,差异有统计学意义(P<0.001)。Transwell结果显示,CXCR2-BMSC迁移能力高于对照组BMSC,差异有统计学意义(P<0.01)。结论利用慢病毒系统成功构建了稳定表达CXCR2的BM-SC,cxcr2基因修饰BMSC后可明显增加BMSC的迁移能力。     

Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction

Bone marrow comprises heterogeneous cell populations, of which certain progenitors have demonstrated the ability to differentiate into multiple mesenchymal cell lineages. This study demonstrates the bone marrow stromal cells (BMSCs) with intrinsic plasticity to differentiate into hepatocyte-like phenotypes under in vitro induction of hepatocyte growth factor (HGF). BMSCs isolated from rat femurs and tibias were cultured and passaged 3-4 times in the presence of HGF. Cells were harvested on days 0, 10, and 20 and subjected to examination of any hepatocyte characteristics by flow cytometry, RT-PCR, Western blot, and immunocytochemistry. Expression of albumin and alpha-fetoprotein at both mRNA and protein levels was detectable on day 10. By contrast, c-Met mRNA was significantly decreased in BMSC in the course of HGF induction. Here BMSC was shown to differentiate into hepatocyte-like phenotypes given the HGF induction, as an alternative source for adult stem cell transplantation in liver repair.