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It is well established that mitogens inhibit differentiation of skeletal muscle cells,but the insulin-like growth factors (IGFs),acting through a single receptor,stimulate both proliferation and differentiation of myoblasts. Although the IGF-I mitogenic signaling pathway has been extensively studied in other cell types,little is known about the signaling pathway leading to differentiation in skeletal muscle. By using specific inhibitors of the IGF signal transduction pathway,we have begun to define the signaling intermediates mediating the two responses to IGFs. We found that PD098059,an inhibitor of mitogen-activated protein (MAP) kinase kinase activation,inhibited IGF-stimulated proliferation of L6A1 myoblasts and the events associated with it,such as phosphorylation of the MAP kinases and elevation of c-fos mRNA and cyclin D protein. Surprisingly,PD098059 caused a dramatic enhancement of differentiation,evident both at a morphological (fusion of myoblasts into myotubes) and biochemical level (elevation of myogenin and p21 cyclin-dependent kinase inhibitor expression,as well as creatine kinase activity). In sharp contrast,LY294002,an inhibitor of phosphatidylinositol 3-kinase,and rapamycin,an inhibitor of the activation of p70 S6 kinase (p70(S6k)),completely abolished IGF stimulation of L6A1 differentiation. We found that p70(S6k) activity increased substantially during differentiation,and this increase was further enhanced by PD098059. Our results demonstrate that the MAP kinase pathway plays a primary role in the mitogenic response and is inhibitory to the myogenic response in L6A1 myoblasts,while activation of the phosphatidylinositol 3-kinase/p70(S6k) pathway is essential for IGF-stimulated differentiation. Thus,it appears that signaling from the IGF-I receptor utilizes two distinct pathways leading either to proliferation or differentiation. View Publication

The Ly-49 family consists of at least nine members,of which Ly-49A and C have been found to be NK-cell inhibitory receptors specific for class I MHC. The functions of other Ly-49 molecules are still unclear. Further analysis of Ly-49 is complicated by the cross-reactivities of some anti-Ly-49 antibodies initially thought to be specific for individual Ly-49 molecules. Studies on the role of Ly-49 in hybrid resistance as well as on allelic exclusion are also complicated by our recent finding that a novel Ly-49CB6 gene is the likely allelic form of Ly-49CBALB as opposed to a previously reported highly related but distinct gene in B6 mice. In cell-cell binding assays,only Ly-49A and C show significant binding to class I MHC. Ly-49A and C also bind some polysaccharides,and carbohydrates on class I MHC seem to be important for its binding to Ly-49. However,this interaction involves not only the carbohydrate recognition domain of Ly-49 but also a part of the stalk region,suggesting that both carbohydrates and peptide backbone of class I MHC may be recognized by Ly-49. It is likely that additional Ly-49 molecules yet to be identified function as NK-inhibitory receptors specific for class I MHC. View Publication

The immunosuppressive effects of microcolin A,a lipopeptide extracted from the marine blue green alga Lyngbya majuscula were investigated. Microcolin A suppressed concanavalin A (IC50 = 5.8 nM),phytohemagglutinin (IC50 = 12.5 nM) and lipopolysaccharide (IC50 = 8.0 nM) induced proliferation of murine splenocytes. Mixed lymphocyte reaction (IC50 = 5.0 nM),anti-IgM (mu-chain specific) (IC50 = 10.0 nM),and phorbol 12-myristate 13-acetate plus ionomycin (IC50 = 5.8 nM) stimulation of murine splenocytes were all similarly suppressed by microcolin A. The inhibitory activity of microcolin A was time-dependent and reversible and was not associated with a reduction in cell viability. Moreover,microcolin A not only inhibited IL-2 production and IL-2 receptor expression by concanavalin A activated splenocytes,but also suppressed in vitro antibody responsiveness to keyhole limpet hemocyanin. These results indicate that microcolin A is a potent immunosuppressive and antiproliferative agent. View Publication

Human progenitors of the megakaryocyte (Mk) lineage were detected by their ability to generate colonies-containing from 3 to textgreater 100 Mk,detectable as glycoprotein IIb/IIIa+ cells in APAAP-stained whole mount agarose cultures. Optimal growth conditions were achieved through the use of a defined serum substitute and a suitable cocktail of recombinant cytokines. Under these culture conditions,the smallest Mk-containing colonies (CFC-Mk) were detectable within a week followed by colonies containing larger numbers of Mk over the ensuing 2 weeks. The total number of CFC-Mk at 18-21 d was linearly related to the number of cells plated. Variation in the cytokines added showed that thrombopoietin (TPO) or IL-3 alone would support the formation of large numbers of CFC-Mk. However,optimal yields of colonies containing cells of both Mk and non-Mk lineages required the addition of other growth factors,of which a combination of IL-3,IL-6,GM-CSF and Steel factor (SF) +/- TPO was the best of those tested. The further addition of erythropoietin to this combination reduced the number of large pure' Mk colonies seen and in their place a corresponding number of mixed erythroid-Mk colonies became detectable. Flt3-ligand alone was unable to support the growth of CFC-Mk nor did it enhance their growth when combined with other factors. Plating of FACS-sorted sub-populations of CD34+ marrow cells in both serum-free agarose and methylcellulose assays demonstrated that most CFC-Mk are generated from CD34+ cells that are CD45RA- and CD71+� View Publication

Previous studies have shown that primitive human hematopoietic cells detectable as long-term culture-initiating cells (LTC-ICs) and colony-forming cells (CFCs) can be amplified when CD34(+) CD38(-) marrow cells are cultured for 10 days in serum-free medium containing flt3 ligand (FL),Steel factor (SF),interleukin (IL)-3,IL-6,and granulocyte colony-stimulating factor. We now show that the generation of these two cell types in such cultures is differentially affected at the single cell level by changes in the concentrations of these cytokines. Thus,maximal expansion of LTC-ICs (60-fold) was obtained in the presence of 30 times more FL,SF,IL-3,IL-6,and granulocyte colony-stimulating factor than could concomitantly stimulate the near-maximal (280-fold) amplification of CFCs. Furthermore,the reduced ability of suboptimal cytokine concentrations to support the production of LTC-ICs could be ascribed to a differential response of the stimulated cells since this was not accompanied by a change in the number of input CD34(+) CD38(-) cells that proliferated. Reduced LTC-IC amplification in the absence of a significant effect on CFC generation also occurred when the concentrations of FL and SF were decreased but the concentration of IL-3 was high (as compared with cultures containing high levels of all three cytokines). To our knowledge,these findings provide the first evidence suggesting that extrinsically acting cytokines can alter the self-renewal behavior of primary human hematopoietic stem cells independent of effects on their viability or proliferation. View Publication

Prostaglandin F2alpha inhibits adipose differentiation of primary culture of adipocyte precursors and of the adipogenic cell line 1246 in defined medium. In the present paper,we investigated the effect of FP receptor agonists cloprostenol and fluprostenol on the differentiation of newborn rat adipocyte precursors in primary culture. The results show that cloprostenol and fluprostenol are very potent inhibitors of adipose differentiation. Dose response studies indicate that both agonists are more potent than PGF2alpha in inhibiting adipocyte precursors differentiation. 50% inhibition of adipose differentiation was observed at a concentration of 3 x 10(-12) M for cloprostenol and 3 to 10 x 10(-11) M for fluprostenol respectively whereas the PGF2alpha concentration required to elicit the same effect was 10(-8) M. In contrast compounds structurally related to PGE2 such as 17-phenyl trinor PGE2 had no effect on adipose differentiation except when added at a 10,000-fold higher concentration. View Publication

We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay,the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 x 10(5) cells. This was significantly higher than the frequency of 1 SRC in 3.0 x 10(6) adult BM cells or 1 in 6.0 x 10(6) mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus,the findings reported here will have important clinical as well as biologic implications. View Publication

We have recently shown that more than 90% of long-term culture initiating cells (LTC-IC) mobilized in the peripheral blood (PB) of normal individuals express HLA-DR and CD38 antigens and can sustain hematopoiesis for only 5 weeks. However,10% of LTC-IC in mobilized PB are CD34+ HLA-DR- and CD34+ CD38- and can sustain hematopoiesis for at least 8 weeks. We now examine the ex vivo expansion potential of CD34+ HLA-DR+ cells (rich in mature LTC-IC) and CD34+ HLA-DR- cells (rich in primitive LTC-IC) in granulocyte colony-stimulating factor (G-CSF) mobilized PB progenitor cells (PBPC). Cells were cultured in contact with M2-10B4 cells (contact) or in transwells above M2-10B4 (noncontact) without and with interleukin-3 (IL-3) and macrophage inflammatory protein (MIP-1alpha) for 2 and 5 weeks. Progeny were evaluated for the presence of colony-forming cells (CFC) and LTC-IC. When CD34+ HLA-DR+ PB cells were cultured in contact cultures without cytokines,a threefold expansion of CFC was seen at 2 weeks,but an 80% decrease in CFC was seen at week 5. Further,the recovery of LTC-IC at week 2 was only 17% and 1% at week 5. This confirms our previous observation that although CD34+ HLA-DR+ mobilized PB cells can initiate long-term cultures,they are relatively mature and cannot sustain long-term hematopoiesis. In contrast,when CD34+ HLA-DR- mobilized PB cells were cultured in contact cultures without cytokines,CFC expansion persisted until week 5 and 49% and 11% of LTC-IC were recovered at week 2 and 5,respectively. As we have shown for steady state bone marrow (BM) progenitors,recovery of LTC-IC was threefold higher when CD34+ HLA-DR- PBPC were cultured in noncontact rather than contact cultures,and improved further when IL-3 and MIP-1alpha were added to noncontact cultures (96 +/- 2% maintained at week 5). We conclude that although G-CSF mobilizes a large population of mature" CD34+ HLA-DR+ LTC-IC with a limited proliferative capacity� View Publication

One objective of clinical gene marking trials in multiple myeloma (MM) is to determine the extent to which relapse after stem cell transplant is attributable to contamination of the autograft with myeloma cells. A requirement in these studies is ex vivo genetic marking of malignant cells present in autografts which are derived from patients exposed to significant prior chemotherapy. We evaluated gene marking of cloonogenic myeloma cells in marrow aspirates from 14 patients with MM. To effect gene transfer we utilized a long-term marrow culture (LTMC) system previously shown to facilitate gene transfer into a spectrum of hematopoietic progenitor and stem cells. Transduction of cells in LTMC was performed by multiple supernatant exposure. At LTMC initiation and after 21 days of culture malignant cells were assessed by morphology,flow cytometry,and polymerase chain reaction (PCR). The mean number of day 21 LTMC adherent layer-derived granulocyte/macrophage progenitors as a percentage of the original inoculum was within the normal range for this technique. The efficiency of transduction of normal hematopoietic progenitors as determined by the number of colonies positive for proviral DNA by PCR,G418 resistance,and X-gal staining was also within the expected range; 65%,44% and 23%,respectively. Thus,there was no evidence that prior chemotherapy exposure or malignant cell contamination compromised cell survival or gene transfer efficiency in LTMC. All patients retained plasma cells in LTMCs for the duration of the 21-day culture period. Molecular analysis confirmed the persistence of clonal IgVH gene rearrangements in day 21 LTMC-derived DNA from 6 of 12 informative patients (50%). PCR using allele-specific primers when available confirmed the specificity of IgVH rearrangements for the myeloma clone. In 2 of the 14 patients,expansion of clonogenic cells was demonstrated in LTMC. In both cases there was strong evidence for transfer of reporter genes (neo and LacZ) into the myeloma clone: morphologically abnormal G418-resistant colonies demonstrated intense staining for beta-galactosidase,and cytospin preparations showed 100% plasma cells with monoclonal heavy and light chain restriction. In one patient,individual colonies positive for beta-galactosidase bore a cytogenetic abnormality characteristic of the patient's myeloma clone. PCR of DNA from pooled plasma cell colonies using tumor-specific CDR3 primers was positive. Our results demonstrate the maintenance of myeloma cells in vitro for up to 21 days in LTMC. They further illustrate that these cells can be genetically marked using transduction protocols currently being tested in clinical trials of hematopoietic cell gene transfer. View Publication

Embryonic stem cells,derived from the inner cell mass of murine blastocysts,can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes,such as peroxisome proliferator-activated receptors gamma and delta and CCAAT/enhancer binding protein beta,during differentiation of retinoic acid-treated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes. View Publication

Pluripotent embryonic stem (ES) cells spontaneously differentiate via embryo-like aggregates into cardiomyocytes of pacemaker-,atrium- and ventricle-like type,which can be distinguished by their specific patterns of action potentials. It has been shown that retinoic acid (RA) treatment during ES cell differentiation increases the number of cardiomyocytes in a time- and concentration-dependent manner. In order to test the effect of RA on cardiomyocyte differentiation and specialization into ventricle-like cardiomyocytes,we studied gene expression of beta-galactosidase driven by the ventricular myosin light chain-2 (MLC-2v) promoter as an indicator for ventricular differentiation. Clones containing the stably integrated expression vector pGNA/MLC-2.1 were selected,which revealed an increase of beta-galactosidase activity in cardiomyocytes of embryoid bodies at day 7 + 16. RA,both,in the all-trans and in the 9-cis configuration resulted in a significant acceleration of cardiomyocyte differentiation and a transient increase of beta-galactosidase activity. To test whether this acceleration of cardiac differentiation and RA-induced increase of the MLC-2v promotor/beta-galactosidase activity reflects an increase of cardiac- and ventricle-specific gene expression,a semi-quantitative RT-PCR analysis was performed for alpha-cardiac myosin heavy chain (alpha-MHC) and MLC-2v genes. It was shown that both 10(-8) M and 10(-9) M RA resulted in an increased level of alpha-cardiac MHC and MLC-2v mRNA in embryoid bodies in early,but not in terminal developmental stages. This led us to the conclusion that the RA-induced accelerated expression of cardiac-specific genes results in an enhanced development of ventricular cardiomyocytes. An increased number of ventricle-like cells after RA treatment was also found by patch-clamp analysis. The number of cardiomyocytes with Purkinje- and ventricle-like properties was shown to be increased by RA,whereas the number of pacemaker- and atrium-like cells was reduced and early pacemaker cells were not quantitatively affected. View Publication

Ceramide has emerged as an important lipid messenger for many cellular processes triggered via surface receptors. In the present study,inflammatory activation of P388D1 macrophages with bacterial lipopolysaccharide (LPS) and platelet-activating factor (PAF) stimulated a transient accumulation of ceramide. Moreover,cell-permeable ceramide mimicked LPS/PAF in triggering arachidonate mobilization in these cells. LPS/PAF-induced ceramide synthesis did not result from sphingomyelinase activation but from increased de novo synthesis. Participation of this pathway in arachidonate signaling was detected since fumonisin B1,an inhibitor of de novo ceramide synthesis,was able to inhibit the LPS/PAF-induced response. These studies have uncovered a new role for sphingolipid metabolism in cellular signaling and constitute evidence that products of the sphingomyelin biosynthetic pathway may serve a specific role in signal transduction by influencing the activity of the novel Group V secretory phospholipase A2. View Publication