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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. View Publication

Teratoma formation is a critical obstacle to safe clinical translation of human embryonic stem (ES) cell-based therapies in the future. As current methods of isolation are unable to yield 100% pure population of differentiated cells from a pluripotent donor source,potential development of these tumors is a significant concern. Here we used non-invasive reporter gene imaging to investigate the relationship between human ES cell number and teratoma formation in a xenogenic model of ES cell transplantation. Human ES cells (H9 line) were stably transduced with a double fusion (DF) reporter construct containing firefly luciferase and enhanced green fluorescent protein (Fluc- eGFP) driven by a human ubiquitin promoter. Immunodeficient mice received intramyocardial (n = 35) or skeletal muscle (n = 35) injection of 1 × 102,1 × 103,1 × 104,1 × 105 or 1 × 106 DF positive ES cells suspended in saline for myocardium and Matrigel for skeletal muscle. Cell survival and proliferation were monitored via bioluminescence imaging (BLI) for an 8 week period following transplantation. Mice negative for Fluc signal after 8 weeks were followed out to day 365 to confirm tumor absence. Significantly,in this study,a minimum of 1 × 105 ES cells in the myocardium and 1 × 104 cells in the skeletal muscle was observed to be requisite for teratoma development,suggesting that human ES cell number may be a critical factor in teratoma formation. Engraftment and tumor occurrence were also observed to be highly dependent on ES cell number. We anticipate these results should yield useful insights to the safe and reliable application of human ES cell derivatives in the clinic. Keywords View Publication

Estrogens as well as some antiestrogens have been shown to prevent bone loss in postmenopausal women. These compounds seem to inhibit bone resorption,but their anabolic effects have been less explored. In this study,bone marrow cultures were used to compare the effect of 17beta-estradiol (E2),and two triphenylethylene derivatives,tamoxifen (TAM),and FC1271a,and a benzothiophene derivative raloxifene (RAL) on differentiation of osteoblasts. All enhanced osteoblastic differentiation of 21-day cultures as indicated by increased mineralization and bone nodule formation. All,except RAL,stimulated cell proliferation during the first 6 days of the culture. However,in the presence of RAL the content of total protein was increased in 13-day cultures. SDS-PAGE and autoradiography of [14C]-proline labeled proteins revealed elevated level of the newly synthesized collagen type I. The pure antiestrogen ICI 182,780 abolished the increase of the specific activity of alkaline phosphatase by E2,TAM,and FC1271a but not the effect of RAL on protein synthesis. Our results show that E2 as well as TAM,FC1271a,and RAL stimulate bone formation in vitro but the mechanism of the anabolic action of RAL in bone clearly differs from that of E2,TAM,and FC1271a. View Publication

We report here the cloning,expression,and characterization of human PDE11A1,a member of a distinct cyclic nucleotide phosphodiesterase (PDE) family. PDE11A exhibits textless/=50% amino acid identity with the catalytic domains of all other PDEs,being most similar to PDE5,and has distinct biochemical properties. The human PDE11A1 cDNA isolated contains a complete open reading frame encoding a 490-amino acid enzyme with a predicted molecular mass of 55,786 Da. At the N terminus PDE11A1 has a single GAF domain homologous to that found in other signaling molecules,including PDE2,PDE5,PDE6,and PDE10,which constitutes a potential allosteric binding site for cGMP or another small ligand. Tissue distribution studies indicate that PDE11A mRNA occurs at highest levels in skeletal muscle,prostate,kidney,liver,pituitary,and salivary glands and testis. PDE11A is expressed as at least three major transcripts of approximately 10.5,approximately 8.5,and approximately 6.0 kb,thus suggesting the existence of multiple subtypes. This possibility is further supported by the detection of three distinct proteins of approximately 78,approximately 65,and approximately 56 kDa by Western blotting of human tissues for PDE11A isoforms. Recombinant human PDE11A1 hydrolyzes both cGMP and cAMP with K(m) values of 0.52 microM and 1.04 microM,respectively,and similar V(max) values. Therefore,PDE11A represents a dual-substrate PDE that may regulate both cGMP and cAMP under physiological conditions. PDE11A is sensitive to the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) as well as zaprinast and dipyridamole,inhibitors that are generally considered relatively specific for the cGMP-selective PDEs,with IC(50) values of 49.8 microM,12.0 microM,and 0.37 microM,respectively. View Publication

The small heat-shock protein HSP25 is expressed in the heart early during development,and although multiple roles for HSP25 have been proposed,its specific role during development and differentiation is not known. P19 is an embryonal carcinoma cell line which can be induced to differentiate in vitro into either cardiomyocytes or neurons. We have used P19 to examine the role of HSP25 in differentiation. We found that HSP25 expression is strongly increased in P19 cardiomyocytes. Antisense HSP25 expression reduced the extent of cardiomyocyte differentiation and resulted in reduced expression of cardiac actin and the intermediate filament desmin and reduced level of cardiac mRNAs. Thus,HSP25 is necessary for differentiation of P19 into cardiomyocytes. In contrast,P19 neurons did not express HSP25 and antisense HSP25 expression had no effect on neuronal differentiation. The phosphorylation of HSP25 by the p38/SAPK2 pathway is known to be important for certain of its functions. Inhibition of this pathway by the specific inhibitor SB203580 prevented cardiomyocyte differentiation of P19 cells. In contrast,PD90589,which inhibits the ERK1/2 pathway,had no effect. Surprisingly,cardiogenesis was only sensitive to SB203580 during the first 2 days of differentiation,before HSP25 expression increases. In contrast to the effect of antisense HSP25,SB203580 reduced the level of expression of the mesodermal marker Brachyury-T during differentiation. Therefore,we propose that the p38 pathway acts on an essential target during early cardiogenesis. Once this initial step is complete,HSP25 is necessary for the functional differentiation of P19 cardiomyocytes,but its phosphorylation by p38/SAPK2 is not required. View Publication

Retinoic acid derivatives (retinoids) exert their pleiotropic effects on cell development through specific nuclear receptors,the retinoic acid receptors and retinoid X receptors. Despite recent progress in understanding the cellular and molecular mechanisms of retinoid activity,it is unknown which of the retinoid receptor pathways are involved in the specific processes of sebocyte growth and development. In this study,we investigated the roles of specific retinoid receptors in sebocyte growth and differentiation,by testing the effects of selective retinoic acid receptor and retinoid X receptor ligands at concentrations between 10-10 M and 10-6 M in a primary rat preputial cell monolayer culture system. Cell growth was determined by number of cells and colonies,and cell differentiation by analysis of lipid-forming colonies. All-trans retinoic acid and selective retinoic acid receptor agonists (CD271 = adapalene,an RAR-beta,gamma agonist; CD2043 = retinoic acid receptor pan-agonist; and CD336 = Am580,an RAR-alpha agonist) caused significant decreases in numbers of cells,colonies,and lipid-forming colonies,but with an exception at high doses of all-trans retinoic acid (10-6 M),with which only a small number of colonies grew but they became twice as differentiated as controls (42.2 +/- 4.0% vs 22.6 +/- 2.7%,mean +/- SEM,lipid-forming colonies,p textless 0.01). Furthermore,the RAR-beta,gamma antagonist CD2665 antagonized the suppressive effects of all-trans retinoic acid,adapalene,and CD2043 on both cell growth and differentiation. In contrast,the retinoid X receptor agonist CD2809 increased cell growth slightly and lipid-forming colonies dramatically in a clear dose-related manner to a maximum of 73.7% +/- 6.7% at 10-6 M (p textless 0. 001). Our data suggest that retinoic acid receptors and retinoid X receptors differ in their roles in sebocyte growth and differentiation: (i) retinoic acid receptors,especially the beta and/or gamma subtypes,mediate both the antiproliferative and antidifferentiative effects of retinoids; (ii) retinoid X receptors mediate prominent differentiative and weak proliferative effects; (iii) the antiproliferative and antidifferentiative effects of all-trans retinoic acid are probably mediated by retinoic acid receptors,whereas its differentiative effect at high dose may be mediated by retinoid X receptors via all-trans retinoic acid metabolism to 9-cis retinoic acid,the natural ligand of retinoid X receptors. View Publication

We studied the suitability of collagen-based semisolid medium for assay of endogenous erythroid colony formation performed in myeloproliferative disorders. Bone marrow (BM) mononuclear cells (MNC) from 103 patients suspected of having polycythemia vera (PV,76 patients) or essential thrombocythemia (ET,27 patients) were grown in collagen-based,serum-free,cytokine-free semisolid medium. Colony analysis at day 8 or 10 showed that this collagen assay is specific,as endogenous growth of erythroid colonies was never observed in cultures of 16 healthy donors and 6 chronic myelogenous leukemia (CML) patients. Endogenous erythroid colony formation was observed in 53.3% of patients suspected of PV,with only 15.4% of positive cultures for patients with 1 minor PV criterion and 72% (p = 0.009) of positive cultures for patients with textgreater or =2 minor or 1 major PV criterion. Similarly,endogenous growth of erythroid colonies was found in 44.4% of patients suspected of ET,with 31.6% of positive cultures for patients with 1 ET criterion versus 75% for patients with textgreater or =2 ET criteria. In addition,we found that in collagen gels,tests of erythropoietin (EPO) hypersensitivity in the presence of 0.01 or 0.05 U/ml of EPO and tests of endogenous colony-forming units-megakaryocyte (CFU-MK) formation cannot be used to detect PV or ET,as these tests were positive for,respectively,21.4% and 50% of healthy donors and 83% and 50% of CML patients. A retrospective analysis suggests that collagen assays are more sensitive than methylcellulose assays to assess endogenous growth of erythroid colonies. In summary,serum-free collagen-based colony assays are simple and reliable assays of endogenous growth of erythroid colonies in myeloproliferative diseases. They also appear to be more sensitive than methylcellulose-based assays. View Publication

The Dishevelled (Dvl) gene family encodes cytoplasmic proteins that are necessary for Wnt signal transduction. Utilizing the yeast two-hybrid system,we identified protein phosphatase 2Calpha (PP2C) as a Dvl-PDZ domain-interacting protein. PP2C exists in a complex with Dvl,beta-catenin,and Axin,a negative regulator of Wnt signaling. In a Wnt-responsive LEF-1 reporter gene assay,expression of PP2C activates transcription and also elicits a synergistic response with beta-catenin and Wnt-1. In addition,PP2C expression relieves Axin-mediated repression of LEF-1-dependent transcription. PP2C utilizes Axin as a substrate both in vitro and in vivo and decreases its half-life. These results indicate that PP2C is a positive regulator of Wnt signal transduction and mediates its effects through the dephosphorylation of Axin. View Publication

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1),EP(2),EP(3),EP(4),DP,FP,IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed,for the first time,an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should,therefore,result in a greater understanding of prostanoid receptor biology. View Publication

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells,up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking,however,decreased to 0.1% or lower within 2 weeks. In contrast,EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks,the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis,and EGFP expression was observed in CD4(+),CD8(+),CD20(+),and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452) View Publication

The molecular and cellular requirements for the development of different populations of human dendritic cells (DC) were studied. Conditions were defined that support DC production from lymphoid progenitors but that fail to induce DC formation from peripheral monocytes. The production of these lymphoid-related DC was severely blocked when hematopoietic progenitors overexpressed Ik7,a mutant dominant-negative Ikaros protein. In contrast,Ik7 did not block the formation of DC in conditions supporting the development of monocyte-derived DC. Furthermore,Ik7 did not block the formation of monocyte/macrophages and enhanced granulopoiesis. One of the molecular mechanisms mediated by Ik7 appears to be down-regulation of the flt3-receptor mRNA. Thus,distinct signals control the formation of DC demonstrating that some aspects of DC diversity are determined in part by distinct molecular cues at the hematopoietic level. (Blood. 2000;95:128-137) View Publication

Dietary flavonoid intake has been reported to be inversely related to mortality from coronary heart disease,and the anti-atherosclerotic effect of flavonoids is considered to be due probably to their antioxidant properties. Oxidation of low density lipoprotein (LDL) has been reported to be induced by the constituent cells of the arterial wall. Accordingly,we examined the effect of pretreatment with tea flavonoids,such as theaflavin digallate,on the ability of cells to oxidize LDL. Theaflavin digallate pretreatment of macrophages or endothelial cells reduced cell-mediated LDL oxidation in a concentration- (0-400 microM) and time- (0-4 hr) dependent manner. This inhibitory effect of flavonoids on cell-mediated LDL oxidation was in the order of theaflavin digallate textgreater theaflavin textgreater or = epigallocatechin gallate textgreater epigallocatechin textgreater gallic acid. Further,we investigated the mechanisms by which flavonoids inhibited cell-mediated LDL oxidation using macrophages and theaflavin digallate. Theaflavin digallate pretreatment decreased superoxide production of macrophages and chelated iron ions significantly. These results suggest that tea flavonoids attenuate the ability of the cell to oxidize LDL,probably by reducing superoxide production in cells and chelating iron ions. View Publication