111In oxine labelled mesenchymal stem cell SPECT after intravenous administration in myocardial infarction.

Mesenchymal stem cells (MSCs) have shown therapeutic potential if successfully delivered to the intended site of myocardial infarction. The purpose of this pilot study was to test the feasibility of 111In oxine labelling of MSCs and single photon emission computed tomography (SPECT) imaging after intravenous administration in a porcine model of myocardial infarction. Adult farm pigs (n=2) were subjected to closed chest experimental myocardial infarction. 111In oxine labelled MSCs (1 x 10(7) to 2 x 10(7) cells) were infused intravenously, and SPECT imaging was performed initially and on days 1, 2, 7 and 14. High quality SPECT images were obtained through 2 weeks of imaging. High initial MSC localization occurred in the lungs and slow progressive accumulation occurred in the liver, spleen and bone marrow. Renal activity was mild and persistent throughout imaging. No appreciable accumulation occurred in the myocardium. It is concluded that 111In oxine radiolabelling of MSCs is feasible, and in vivo imaging with SPECT provides a non-invasive method for sequentially monitoring cell trafficking with good spatial resolution. Because intravenous administration of MSCs results in significant lung activity that obscures the assessment of myocardial cell trafficking, alternative routes of administration should be investigated for this application.

Human mesenchymal stem cells maintain transgene expression during expansion and differentiation.

Human adult bone marrow contains both hematopoietic stem cells that generate cells of all hematopoietic lineages and human mesenchymal stem cells (hMSCs), which support hematopoiesis and contribute to the regeneration of multiple connective tissues. The goal of the current study was to demonstrate that transduced hMSCs maintain transgene expression after stem cell differentiation in vitro and in vivo. We have introduced genes into cultured hMSCs by retroviral vector transfer and demonstrated long-term in vitro and in vivo expression of human interleukin 3 (hIL-3) and green fluorescent protein (GFP). Protocols were developed to achieve transduction efficiencies of 80-90% in these stem cells. In vitro expression of hIL-3 averaged 350 ng/10(6)cells/24 h over 17 passages (> 6 months) and GFP expression was stable over the same time period. Transduced hMSCs were able to differentiate into osteogenic, adipogenic, and chondrogenic lineages and maintained transgene expression after differentiation. Parallel studies were performed in vivo using NOD/SCID mice. Human MSCs expressing hIL-3 were cultured on several matrices and then delivered by subcutaneous, intravenous, and intraperitoneal routes. Sampling of peripheral blood demonstrated that systemic hIL-3 expression was maintained in the range of 100-800 pg/ml over a period of 3 months. These results illustrate the ability of hMSCs to express genes of therapeutic potential and demonstrate their potential clinical utility as cellular vehicles for systemic gene delivery.

Activation of peroxisome proliferator-activated receptor-gamma pathway inhibits osteoclast differentiation.

The nuclear receptor and transcription factor, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), regulates the activity of other transcription factors in the adipogenic differentiation and inflammatory response pathways. We examined the possible function of the PPAR-gamma pathway in osteoclast (Ocl) formation from CD34(+) hematopoietic stem cells (CD34(+) HSCs), using a co-culture system comprised of human mesenchymal stem cells (hMSCs) and CD34(+) HSCs, both derived from bone marrow. Ocl formation in this co-culture system is enhanced by the addition of exogenous osteoprotegerin ligand (OPGL), an essential Ocl differentiation factor, and macrophage-colony stimulating factor (M-CSF). The data indicate that soluble OPGL (sOPGL) and M-CSF stimulate Ocl formation in the co-cultures up to 4-fold compared with CD34(+) HSCs alone treated with sOPGL and M-CSF. CD34(+) HSCs, but not hMSCs, express PPAR-gamma, and 15-deoxy-Delta(12, 14)-prostaglandin-J2 (15d-PG-J2), a PPAR-gamma agonist, completely blocked the effects of sOPGL and M-CSF on Ocl formation and activity. The inhibitory effect of 15d-PG-J2 is specific to the Ocl lineage in both human and mouse models of osteoclastogenesis. Accordingly, parallel experiments demonstrate that sOPGL activates the NF-kappaB pathway within mouse Ocl progenitors, and this effect was abolished by 15d-PG-J2. These data establish a link between PPAR-gamma and OPGL signaling within Ocl progenitors, and support a role for PPAR-gamma pathway in the modulation of osteoclastogenesis.

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.

Multilineage potential of adult human mesenchymal stem cells.

Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow.

In the adult human, mesenchymal stem cells (MSCs) resident in bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages, including cartilage. In this study, culture-expanded human MSCs (hMSCs) of 60 human donors were induced to express the morphology and gene products of chondrocytes. Chondrogenesis was induced by culturing hMSCs in micromass pellets in the presence of a defined medium that included 100 nM dexamethasone and 10 ng/ml transforming growth factor-beta(3) (TGF-beta(3)). Within 14 days, cells secreted an extracellular matrix incorporating type II collagen, aggrecan, and anionic proteoglycans. hMSCs could be further differentiated to the hypertrophic state by the addition of 50 nM thyroxine, the withdrawal of TGF-beta(3), and the reduction of dexamethasone concentration to 1 nM. Increased understanding of the induction of chondrogenic differentiation should lead to further progress in defining the mechanisms responsible for the generation of cartilaginous tissues, their maintenance, and their regeneration.

Alternatively spliced exons of the beta tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon.

The rat beta-tropomyosin (TM) gene expresses two isoforms via alternative RNA splicing, namely skeletal muscle beta-TM and fibroblast TM-1. The latter is also expressed in smooth muscle where it corresponds to smooth muscle beta-TM. Skeletal muscle beta-TM contains exons 7 and 10, whereas exons 6 and 11 are used in fibroblasts and smooth muscle. In order to study the properties of the alternatively spliced proteins, recombinant TMs derived from bacterial and insect cell expression systems were produced, including the normal beta gene products, fibroblast TM-1 and beta skeletal muscle TM, two carboxy-terminal chimeric TMs, TM-6/10 and TM-7/11, as well as a carboxyl-truncated version of each, TM-6Cla and TM-7Cla. The purified TM isoforms were used in actin filament association studies. The apparent TM association constants (Ka) were taken as the free concentration at half saturation and were found to be 6 microM for beta Sk TM, 8.5 for TM-6/10, 25 microM for TM-1, and 30 microM for TM-7/11 at an F-actin concentration of 42 microM. For the truncated TMs, the values determined were higher still but the binding was not carried out to full saturation. Isoforms were also produced using the baculovirus-insect cell system which produces proteins with an acetylated amino terminus as is normally found in vivo. This modification significantly enhanced the F-actin association of TM-1 but not the beta skeletal TM or the other isoforms. Fibroblast TM-2 or TM-3, both products of the alpha gene, enhanced the affinity of TM-1 for F-actin, demonstrating different isoforms can act cooperatively on binding to actin. This effect was not detected with the other expressed beta gene products. The presence of 83 kDa nonmuscle caldesmon was found to enhance the binding of TM-1 for F-actin. This effect was dependent on the presence of both exons 6 and 11, as caldesmon had little effect on the other beta gene products. Collectively these results demonstrate TMs differ in their affinity for F-actin, which can be altered by other TMs or actin-binding proteins. The beta tropomyosin isoforms were fluorescently-tagged and microinjected into cultured cells to study their in vivo localization where it was found that each of the full-length TMs bound to microfilaments but, at the light microscopy level, the isoforms were not differentially localized in these fibroblasts.

Functional properties of non-muscle tropomyosin isoforms.

Tropomyosins are a family of actin filament binding proteins. They have been identified in many organisms, including yeast, nematodes, Drosophila, birds and mammals. In metazoans, different forms of tropomyosin are characteristic of specific cell types. Most non-muscle cells, such as fibroblasts, express five to eight isoforms of tropomyosins. The various isoforms exhibit distinct biochemical properties that appear to be required for specific cellular functions.

In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts.

Most cell types express several tropomyosin isoforms, the individual functions of which are poorly understood. In rat fibroblasts there are at least six isoforms; TM-1, TM-2, TM-3, TM-4, TM-5a, and TM-5b. TM-1 is the product of the beta gene. TM-4 is produced from the TM-4 gene, and TMs 2, 3, 5a, and 5b are the products of the alpha gene. To begin to study the localization and function of the isoforms in fibroblasts, cDNAs for TM isoforms 2, 3, 5a, and 5b were placed into bacterial expression vectors and used to produce TM isoforms. The bacterially produced TMs were determined to be full length by sequencing the amino- and carboxy termini. These TMs were found to bind to F-actin in vitro, with properties similar to that of skeletal muscle TM. In addition, competition experiments demonstrated that TM-5b was better than TM-5a in displacing other TM isoforms from F-actin in vitro. To investigate the intracellular localization of these fibroblast isoforms, each was derivatized with a fluorescent chromophore and microinjected into rat fibroblasts. TM-2, TM-3, TM-5a, and TM-5b were each found to associate along actin filaments. There was no preferred cellular location or subset of actin filaments for these isoforms. Furthermore, co-injection of two isoforms labeled with different fluorochromes showed identical staining. At the level of the light microscope, these isoforms from the alpha gene do not appear to achieve different functions by binding to particular subsets of actin filaments or locations in cells. Some alternative possibilities are discussed. The results show that bacterially produced TMs can be used to study in vitro and in vivo properties of the isoforms.

Retention of autoregulatory control of tubulin synthesis in cytoplasts: demonstration of a cytoplasmic mechanism that regulates the level of tubulin expression.

Virtually all animal cells rapidly and specifically depress synthesis of new alpha- and beta-tubulin polypeptides in response to microtubule inhibitors that increase the pool of depolymerized subunits, or in response to direct elevation of the cellular tubulin subunit content through microinjection of exogenous tubulin subunits. Collectively, these previous findings have documented the presence of an apparent eucaryotic, autoregulatory control mechanism that specifies the level of expression of tubulin in cultured animal cells. Mechanistically, this regulation of tubulin synthesis is achieved through modulation of tubulin mRNA levels. To dissect further the molecular pathway that underlies this autoregulatory phenomenon, we have now investigated whether enucleated cells still retain the requisite regulatory machinery with which to alter tubulin synthetic levels in response to fluctuations in the pool size of unpolymerized tubulin subunits. Using two-dimensional gel electrophoresis to analyze the patterns of new polypeptide synthesis, we have determined that such cytoplasts can indeed respond to drug-induced microtubule depolymerization by specific repression of new beta-tubulin synthesis. Moreover, the response of cytoplasts is, if anything, greater in magnitude than that of whole cells. We conclude that autoregulatory control of beta-tubulin gene expression must derive principally, if not exclusively, from a cytoplasmic control mechanism that modulates beta-tubulin mRNA stability. For alpha-tubulin, although the response of cytoplasts after drug-induced microtubule depolymerization is quantitatively less dramatic than that of whole cells, at least part of the regulatory machinery must also be activated through a cytoplasmic regulatory event.

Reconstruction of appropriate tubulin and actin gene regulation after transient transfection of cloned beta-tubulin and beta-actin genes.

Most animal cells rapidly depress the synthesis of new alpha- and beta-tubulin polypeptides in response to microtubule inhibitors that increase the pool of depolymerized subunits. This apparent autoregulatory control of tubulin synthesis is achieved through the modulation of tubulin mRNA levels. To begin to analyze the molecular mechanism responsible for such regulation, we have introduced exogenous beta-tubulin gene sequences into cultured mouse cells by DEAE-dextran-mediated DNA transfection. We find that the heterologous tubulin genes are expressed and that their RNA transcripts are accurately processed to mature mRNAs. Moreover, after drug-induced microtubule depolymerization, the expression of unintegrated tubulin gene sequences is regulated coordinately with the endogenous mouse alpha- and beta-tubulin RNA transcripts. Such regulation appears to be specific for transfected tubulin genes, since similar down-regulation is not observed in a contransfected beta-actin gene. Curiously, in response to microtubule depolymerization, the amount of RNA transcripts from a transfected beta-actin gene increases twofold, which qualitatively and quantitatively parallels that seen by the RNAs encoded by the endogenous actin genes. Thus, the transient DNA transfection approach may permit the unambiguous elucidation of regulatory sequences involved in establishing the proper level of expression of these two important cytoskeletal gene families.

Elevation of tubulin levels by microinjection suppresses new tubulin synthesis.

Most eukaryotic cells rapidly and specifically depress synthesis of alpha- and beta-tubulin polypeptides in response to microtubule inhibitors which cause microtubule depolymerization and presumably increase the intracellular concentration of free subunits. Other drugs which interfere with microtubule function but which lead to a decrease in the subunit pool size have little effect on the rate of new tubulin synthesis. These findings have previously been interpreted to indicate that cultured cells synthesize tubulin constitutively unless the subunit pool rises above a specified level. At this point an autoregulatory control mechanism is triggered which suppresses new tubulin synthesis through specific loss of tubulin mRNAs. That tubulin RNA levels are dramatically lowered by microtubule depolymerizing drugs is unquestionably correct; that fluctuations in the depolymerized tubulin pool size are responsible for altered RNA levels rests, however, entirely on the presumptive effects of different microtubule drugs. This caveat is not trivial, as these drugs induce gross morphological alterations, and the specificities and detailed mechanisms of action of such drugs remain poorly understood. To investigate the effect of altered levels of tubulin subunits on the rate of new tubulin synthesis in mammalian cells, we have microinjected purified tubulin subunits into cells in culture and analysed the synthesized proteins. We report here that tubulin synthesis is rapidly and specifically suppressed by injection of an amount of tubulin roughly equivalent to 25-50% of the amount initially present in the cell, thus indicating the presence of an eukaryotic, autoregulatory control mechanism which specifies tubulin content in a cultured mammalian cell line.

Subcellular sorting of isoactins: selective association of gamma actin with skeletal muscle mitochondria.

We describe two subpopulations of actin antibodies isolated by affinity chromatography from a polyclonal antibody to chicken gizzard actin. One subpopulation recognizes gamma actins from smooth muscle and nonmuscle cells, but does not recognize alpha actin from skeletal muscle. The other subpopulation recognizes determinants that are common to alpha actin from skeletal muscle and the two gamma actin isotypes. Neither antibody recognizes cytoplasmic beta actin. Both antibodies recognize only actins or molecules with determinants that are also present in actins. By immunofluorescence we found that the anti-gamma actin colocalizes with mitochondria in fibers of mouse diaphragm, and that it does not bind detectably to the 1 bands of sarcomeres. The antibody that recognizes both alpha and gamma actins stains 1 bands intensely, as expected. We interpret these observations as preliminary evidence for selective association of gamma actin with skeletal muscle mitochondria and, more broadly, as evidence for subcellular sorting of isoactins.