We have isolated a cardiomyogenic cell line (CMG) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneously beating cells were repeatedly screened. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in ∼30% of the cells; they connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. They expressed atrial natriuretic peptide and brain natriuretic peptide and were stained with anti-myosin, anti-desmin, and anti-actinin antibodies. Electron microscopy revealed a cardiomyocyte-like ultrastructure, including typical sarcomeres, a centrally positioned nucleus, and atrial granules. These cells had several types of action potentials, such as sinus node-like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain, and α-actin, indicated that their muscle phenotype was similar to that of fetal ventricular cardiomyocytes. These cells expressed Nkx2.5/Csx, GATA4, TEF-1, and MEF-2C mRNA before 5-azacytidine treatment and expressed MEF-2A and MEF-2D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte differentiation.

We investigated the capacity'of a clonal osteogenic cell line MC3T3-EI, established from newborn mouse calvaria and selected on the basis of high alkaline phosphatase (ALP) activity in the confluent state, to differentiate into osteoblasts and mineralize in vitro. The cells in the growing state showed a fibroblastic morphology and grew to form multiple layers. On day 21, clusters of cells exhibiting typical osteoblastic morphology were found in osmiophilic nodular regions. Such nodules increased in number and size with incubation time and became easily identifiable with the naked eye by day 40-50. In the central part of well-developed nodules, osteocytes were embedded in heavily mineralized bone matrix. Osteoblasts were arranged at the periphery of the bone spicules and were surrounded by lysosome-rich cells and a fibroblastic cell layer. Numerous matrix vesicles were scattered around the osteoblasts and young osteocytes. Matrix vesicles and plasma membranes of osteoblasts, young osteocytes, and lysosome-rich cells showed strong reaction to cytochemical stainings for ALP activity and calcium ions. Minerals were initially localized in the matrix vesicles and then deposited on well-banded collagen fibrils. Deposited minerals consisted exclusively of calcium and phosphorus, and some of the crystals had matured into hydroxyapatite crystals. These results indicate that MC3T3-EI cells have the capacity to differentiate into osteoblasts and osteocytes and to form calcified bone tissue in vitro.Recently, we established eight cell lines from newborn mouse calvaria and selected a clone, designated MC3T3-EI, from one of these lines on the basis of its high alkaline phosphatase (ALP) activity in the resting state (l). Cells of this clonal line had very low ALP activity in the growing state, but enzyme activity increased several hundredfold after cultures reached a confluent state. The cells produced abundant fibrous intercellular substances. In addition, intercellular spaces in day-30 cultures stained positively with alizarin red S. These observations suggest that MC3T3-EI cells are an osteogenic cell line, having the capacity to differentiate into osteoblasts and deposit minerals in vitro.We undertook the studies described here to further characterize the process of differentiation of MC3T3-E1 cells and calcification in vitro. We found that calcified bone tissue was formed in MC3T3-E 1 cultures by a process closely resembling that observed in intramembranous osteogenesis in vivo. Mineral deposits were identified as hydroxyapatite by energy dispersive x-ray analysis and electron diffraction analysis. MATERIALS AND METHODSCell Culture: The MC3T3-EI cells were grown in alpha modification of Eagle's minimal essential medium (a-MEM) (Flow Laboratories, McLean, VA) supplemented with 10% newborn calf serum (Flow Lab., Stanmore, New South Wales, Australia) and 60 lag/ml of kanamycin sulfate (Meiji Seika, Tokyo, Japan). CelLs were maintained at 37°C in a fully-humidified atmosphere of 5% CO2 in air and subcultured every 3 ...

An efficient system was developed that induced the differentiation of embryonic stem (ES) cells into blood cells of erythroid, myeloid, and B cell lineages by coculture with the stromal cell line OP9. This cell line does not express functional macrophage colony-stimulating factor (M-CSF). The presence of M-CSF had inhibitory effects on the differentiation of ES cells to blood cells other than macrophages. Embryoid body formation or addition of exogenous growth factors was not required, and differentiation was highly reproducible even after the selection of ES cells with the antibiotic G418. Combined with the ability to genetically manipulate ES cells, this system will facilitate the study of molecular mechanisms involved in development and differentiation of hematopoietic cells.

Circulating CD14+ monocytes are precursors of phagocytes, such as macrophages and dendritic cells. Here we report primitive cells with a fibroblast-like morphology derived from human peripheral blood CD14+ monocytes that can differentiate into several distinct mesenchymal cell lineages. We named this cell population monocyte-derived mesenchymal progenitor (MOMP). MOMPs were obtained in vitro from human peripheral blood mononuclear cells cultured on fibronectin in the presence of fetal bovine serum alone as a source of growth factors. MOMPs had a unique molecular phenotype-CD14+CD45+CD34+type I collagen+-and showed mixed morphologic and molecular features of monocytes and endothelial and mesenchymal cells. MOMPs were found to be derived from a subset of circulating CD14+ monocytes, and their differentiation required that they bind fibronectin and be exposed to one or more soluble factors derived from peripheral blood CD14- cells. MOMPs could be expanded in culture without losing their original phenotype for up to five passages. The induction of MOMPs to differentiate along multiple limb-bud mesodermal lineages resulted in the expression of genes and proteins specific for osteoblasts, skeletal myoblasts, chondrocytes, and adipocytes. Our findings represent the first evidence that human circulating CD14+ monocytes are a source of progenitors that exhibit mesenchymal cell differentiation.

We demonstrated previously that a single injection of recombinant human macrophage colony-stimulating factor (rhM-CSF) is sufficient for osteoclast recruitment and survival in osteopetrotic (op/op) mice with a deficiency in osteoclasts resulting from a mutation in M-CSF gene. In this study, we show that a single injection of recombinant human vascular endothelial growth factor (rhVEGF) can similarly induce osteoclast recruitment in op/op mice. Osteoclasts predominantly expressed VEGF receptor 1 (VEGFR-1), and activity of recombinant human placenta growth factor 1 on osteoclast recruitment was comparable to that of rhVEGF, showing that the VEGF signal is mediated through VEGFR-1. The rhM-CSF–induced osteoclasts died after injections of VEGFR-1/Fc chimeric protein, and its effect was abrogated by concomitant injections of rhM-CSF. Osteoclasts supported by rhM-CSF or endogenous VEGF showed no significant difference in the bone-resorbing activity. op/op mice undergo an age-related resolution of osteopetrosis accompanied by an increase in osteoclast number. Most of the osteoclasts disappeared after injections of anti-VEGF antibody, demonstrating that endogenously produced VEGF is responsible for the appearance of osteoclasts in the mutant mice. In addition, rhVEGF replaced rhM-CSF in the support of in vitro osteoclast differentiation. These results demonstrate that M-CSF and VEGF have overlapping functions in the support of osteoclastic bone resorption.

The chloroplast membrane of higher plants contains an unusually high concentration of trienoic fatty acids. Plants grown in colder temperatures have a higher content of trienoic fatty acids. Transgenic tobacco plants in which the gene encoding chloroplast omega-3 fatty acid desaturase, which synthesizes trienoic fatty acids, was silenced contained a lower level of trienoic fatty acids than wild-type plants and were better able to acclimate to higher temperatures.

After our previous report that osteoclast-like multinucleated cells (MNCs) were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] in cocultures of mouse spleen cells and osteoblast-rich populations freshly isolated from fetal mouse calvariae, we examined whether such primary osteoblast-like cells can be replaced by established cell lines in inducing osteoclast-like cell formation. We first used two clonal cell lines simultaneously established from newborn mouse calvariae. One was the osteoblastic cell line MC3T3-E1, and the other was the preadipose cell line MC3T3-G2/PA6. Tartrate-resistant acid phosphatase (TRACP; a marker enzyme of osteoclasts)-positive mononuclear cells and MNCs were formed in the cocultures of spleen cells and MC3T3-G2/PA6 cells in the presence of 1 alpha,25-(OH)2D3. Dexamethasone greatly potentiated TRACP-positive MNC formation induced by 1 alpha,25-(OH)2D3, whereas the glucocorticoid alone had no effect on it. In contrast, osteoblastic MC3T3-E1 cells failed to induce TRACP-positive cells in the cocultures. Another bone marrow-derived stromal cell line ST2 also induced TRACP-positive MNC formation in the cocultures in response to 1 alpha,25-(OH)2D3 and dexamethasone. Salmon calcitonin enhanced cAMP production in the cocultures only when TRACP-positive cells were formed. Autoradiographic studies demonstrated that [125I]calcitonin specifically bound to TRACP-positive cells formed in the cocultures. When spleen cells and either MC3T3-G2/PA6 or ST2 cells were cocultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 and dexamethasone, numerous resorption lacunae were formed. These results show that the two bone marrow-derived stromal cell lines can support osteoclast-like cell differentiation in cocultures with spleen cells.

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