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Telomere length must be tightly regulated in highly proliferative tissues,such as the lymphohematopoietic system. Under steady-state conditions,the levels and functionality of hematopoietic-committed or multipotent progenitors were not affected in late-generation telomerase-deficient mice (mTerc(-/-)) with critically short telomeres. Evaluation of self-renewal potential of mTerc(-/-) day-12 spleen colony-forming units demonstrated no alteration as compared with wildtype progenitors. However,the replating ability of mTerc(-/-) granulocyte-macrophage CFUs (CFU-GMs) was greatly reduced as compared with wildtype CFU-GMs,indicating a diminished capacity of late-generation mTerc(-/-) committed progenitors when forced to proliferate. Long-term bone marrow cultures of mTerc(-/-) bone marrow (BM) cells show a reduction in proliferative capacity; this defect can be mainly attributed to the hematopoietic,not to the stromal,mTerc(-/-) cells. In serial and competitive transplantations,mTerc(-/-) BM stem cells show reduced long-term repopulating capacity,concomitant with an increase in genetic instability compared with wildtype cells. Nevertheless,in competitive transplantations late-generation mTerc(-/-) precursors can occasionally overcome this proliferative impairment and reconstitute irradiated recipients. In summary,our results demonstrate that late-generation mTerc(-/-) BM cells with short telomeres,although exhibiting reduced proliferation ability and reduced long-term repopulating capacity,can still reconstitute myeloablated animals maintaining stem cell function. View Publication

Embryonic stem (ES) cells have been established as permanent lines of undifferentiated pluripotent cells from early mouse embryos. ES cells provide a unique system for the genetic manipulation and the creation of knockout strains of mice through gene targeting. By cultivation in vitro as 3D aggregates called embryoid bodies,ES cells can differentiate into derivatives of all 3 primary germ layers,including cardiomyocytes. Protocols for the in vitro differentiation of ES cells into cardiomyocytes representing all specialized cell types of the heart,such as atrial-like,ventricular-like,sinus nodal-like,and Purkinje-like cells,have been established. During differentiation,cardiac-specific genes as well as proteins,receptors,and ion channels are expressed in a developmental continuum,which closely recapitulates the developmental pattern of early cardiogenesis. Exploitation of ES cell-derived cardiomyocytes has facilitated the analysis of early cardiac development and has permitted in vitro gain-of-function" or "loss-of-function" genetic studies. Recently� View Publication

The E2F family plays a critical role in the expression of genes required for entry into and progression through S phase. E2F-mediated transcription is repressed by the tumor suppressor retinoblastoma protein (pRb),which results in sequestration of E2F in a multiprotein complex that includes pRb. Derepression of E2F results from a series of complex phosphorylation events mediated by cyclin D/cdk4 and cyclin E/cdk2. We have employed a novel 3-substituted indolinone compound,3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516),which selectively inhibits cdk2 activity (Lane et al.,Cancer Res 2001;61:6170-7) to investigate these events. Electrophoretic mobility gel shift assays were performed on SU9516-treated and -untreated HT-29,SW480,and RKO human colon cancer cell extracts. Treatment with 5 microM SU9516 prevented dissociation of pRb from E2F1 in all cell lines (HT-29textgreaterRKOtextgreaterSW480). Treatment effects were time-dependent,demonstrating greater inhibition at 48 hr versus 24hr in HT-29 cells. Furthermore,E2F species were sequestered in complexes with p107,p130,DP-1,and cyclins A and E. After a 24-hr treatment with 5 microM SU9516,cyclin D1 and cdk2 levels decreased by 10-60%. These findings delineate a previously undescribed mechanism for SU9516-mediated cell growth arrest through down-regulation of cyclin D1,inhibition of cdk2 levels and activity,and pan-sequestration of E2F. View Publication

Here we describe the in vitro generation of a novel adherent cell fraction derived from highly enriched,mobilized CD133(+) peripheral blood cells after their culture with Flt3/Flk2 ligand and interleukin-6 for 3 to 5 weeks. These cells lack markers of hematopoietic stem cells,endothelial cells,mesenchymal cells,dendritic cells,and stromal fibroblasts. However,all adherent cells expressed the adhesion molecules VE-cadherin,CD54,and CD44. They were also positive for CD164 and CD172a (signal regulatory protein-alpha) and for a stem cell antigen defined by the recently described antibody W7C5. Adherent cells can either spontaneously or upon stimulation with stem cell factor give rise to a transplantable,nonadherent CD133(+)CD34(-) stem cell subset. These cells do not generate in vitro hematopoietic colonies. However,their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice induced substantially higher long-term multilineage engraftment compared with that of freshly isolated CD34(+) cells,suggesting that these cells are highly enriched in SCID-repopulating cells. In addition to cells of the myeloid lineage,nonadherent CD34(-) cells were able to give rise to human cells with B-,T-,and natural killer-cell phenotype. Hence,these cells possess a distinct in vivo differentiation potential compared with that of CD34(+) stem cells and may therefore provide an alternative to CD34(+) progenitor cells for transplantation. View Publication

The small cytokine-inducible SH2 domain-containing protein (CIS) has been implicated in the negative regulation of signaling through cytokine receptors. CIS reduces growth of erythropoietin receptor (EpoR)-dependent cell lines,but its role in proliferation,differentiation,and survival of erythroid progenitor cells has not been resolved. To dissect the function of CIS in cell lines and erythroid progenitor cells,we generated green fluorescent protein (GFP)-tagged versions of wild type CIS,a mutant harboring an inactivated SH2 domain (CIS R107K),and a mutant with a deletion of the SOCS Box (CISDeltaBox). Retroviral expression of the GFP fusion proteins in BaF3-EpoR cells revealed that both Tyr-401 in the EpoR and an intact SH2 domain within CIS are prerequisites for receptor recruitment. As a consequence,both are essential for the growth inhibitory effect of CIS,whereas the CIS SOCS box is dispensable. Accordingly,the retroviral expression of GFP-CIS but not GFP-CIS R107K impaired proliferation of erythroid progenitor cells in colony assays. Erythroid differentiation was unaffected by either protein. Interestingly,apoptosis of erythroid progenitor cells was increased upon GFP-CIS expression and this required the presence both of an intact SH2 domain and the SOCS box. Thus,CIS negatively regulates signaling at two levels,apoptosis and proliferation,and thereby sets a threshold for signal transduction. View Publication

Purmorphamine,which is a 2,6,9-trisubstituted purine compound,was discovered through cell-based high-throughput screening from a heterocycle combinatorial library. It differentiates multipotent mesenchymal progenitor cells into an osteoblast lineage. It will serve as a unique chemical tool to study the molecular mechanisms of osteogenesis of stem cells and bone development. View Publication

Caveolae,specialized flask-shaped lipid rafts on the cell surface,are composed of cholesterol,sphingolipids,and structural proteins termed caveolins; functionally,these plasma membrane microdomains have been implicated in signal transduction and transmembrane transport. In the present study,we examined the role of caveolin-1 in multiple myeloma cells. We show for the first time that caveolin-1,which is usually absent in blood cells,is expressed in multiple myeloma cells. Analysis of myeloma cell-derived plasma membrane fractions shows that caveolin-1 is co-localized with interleukin-6 receptor signal transducing chain gp130 and with insulin-like growth factor-I receptor. Cholesterol depletion by beta-cyclodextrin results in the loss of caveola structure in myeloma cells,as shown by transmission electron microscopy,and loss of caveolin-1 function. Interleukin-6 and insulin-like growth factor-I,growth and survival factors in multiple myeloma,induce caveolin-1 phosphorylation,which is abrogated by pre-treatment with beta-cyclodextrin. Importantly,inhibition of caveolin-1 phosphorylation blocks both interleukin-6-induced protein complex formation with caveolin-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. beta-Cyclodextrin also blocks insulin-like growth factor-I-induced tyrosine phosphorylation of insulin-responsive substrate-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. Therefore,cholesterol depletion by beta-cyclodextrin abrogates both interleukin-6- and insulin-like growth factor-I-triggered multiple myeloma cell survival via negative regulation of caveolin-1. Taken together,this study identifies caveolin-1 and other structural membrane components as potential new therapeutic targets in multiple myeloma. View Publication

Liver becomes the predominant site of hematopoiesis by 11.5 dpc (days after coitus) in the mouse and 15 gestational weeks in humans and stays so until the end of gestation. The reason the liver is the major hematopoietic site during fetal life is not clear. In this work,we tried to define which of the fetal liver microenvironmental cell populations would be associated with the development of hematopoiesis and found that a population of cells with mixed endodermal and mesodermal features corresponded to hematopoietic-supportive fetal liver stroma. Stromal cells generated from primary cultures or stromal lines from mouse or human fetal liver in the hematopoietic florid phase expressed both mesenchymal markers (vimentin,osteopontin,collagen I,alpha smooth muscle actin,thrombospondin-1,EDa fibronectin,calponin,Stro-1 antigens,myocyte-enhancer factor 2C) and epithelial (alpha-fetoprotein,cytokeratins 8 and 18,albumin,E-cadherin,hepatocyte nuclear factor 3 alpha) markers. Such a cell population fits with the description of cells in epithelial-to-mesenchymal transition (EMT),often observed during development,including that of the liver. The hematopoietic supportive capacity of EMT cells was lost after hepatocytic maturation,induced by oncostatin M in the cell line AFT024. EMT cells were observed in the fetal liver microenvironment during the hematopoietic phase but not in nonhematopoietic liver by the end of gestation and in the adult. EMT cells represent a novel stromal cell type that may be generated from hepatic endodermal or mesenchymal stem cells or even from circulating hematopoietic stem cells (HSCs) seeding the liver rudiment. View Publication

Guggulipid is an extract of the guggul tree Commiphora mukul and has been widely used to treat hyperlipidemia in humans. The plant sterol guggulsterone (GS) is the active agent in this extract. Recent studies have shown that GS can act as an antagonist ligand for farnesoid X receptor (FXR) and decrease expression of bile acid-activated genes. Here we show that GS,although an FXR antagonist in coactivator association assays,enhances FXR agonist-induced transcription of bile salt export pump (BSEP),a major hepatic bile acid transporter. In HepG2 cells,in the presence of an FXR agonist such as chenodeoxycholate or GW4064,GS enhanced endogenous BSEP expression with a maximum induction of 400-500{\%} that induced by an FXR agonist alone. This enhancement was also readily observed in FXR-dependent BSEP promoter activation using a luciferase reporter construct. In addition,GS alone slightly increased BSEP promoter activation in the absence of an FXR agonist. Consistent with the results in HepG2,guggulipid treatment in Fisher rats increased BSEP mRNA. Interestingly,in these animals expression of the orphan nuclear receptor SHP (small heterodimer partner),a known FXR target,was also significantly increased,whereas expression of other FXR targets including cholesterol 7alpha-hydroxylase (Cyp 7a1),sterol 12alpha-hydroxylase (Cyp 8b1),and the intestinal bile acid-binding protein (I-BABP),remained unchanged. Thus,we propose that GS is a selective bile acid receptor modulator that regulates expression of a subset of FXR targets. Guggulipid treatment in rats lowered serum triglyceride and raised serum high density lipoprotein levels. Taken together,these data suggest that guggulsterone defines a novel class of FXR ligands characterized by antagonist activities in coactivator association assays but with the ability to enhance the action of agonists on BSEP expression in vivo. View Publication

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however,the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects,with a median of 14 individual epitopic regions targeted per person (range,2 to 42),and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median,4,245) among all study participants. However,the number of epitopic regions targeted,the protein subunits recognized,and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals,with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid,sensitive,specific,and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response,even if a comprehensive pan-genome screening approach is applied. View Publication

Human immunodeficiency virus type 1 (HIV-1) impacts the activation state of multiple lineages of hematopoietic cells. Chronic HIV-1 infection among individuals with progressive disease can be associated with increased levels of activated signal transducers and activators of transcription (STATs) in peripheral blood mononuclear cells. To investigate interactions between HIV-1 and CD4(+) cells,activated,phosphorylated STAT proteins in nuclear extracts from lymphocytic and promonocytic cell lines as well as primary monocyte-derived macrophages were measured. Levels of activated STATs increased six- to tenfold in HUT78 and U937 cells within 2 h following exposure to virions. The response to virus was dose-dependent,but kinetics of activation was delayed relative to interleukin-2 or interferon-gamma. Activation of STAT1,STAT3,and STAT5 occurred with diverse viral envelope proteins,independent of coreceptor use or viral replication. Envelope-deficient virions had no effect on STAT activation. Monoclonal antibody engagement of CD4 identified a novel role for CD4 as a mediator in the activation of multiple STATs. Results provide a model for HIV-1 pathogenesis in infected and noninfected hematopoietic cells. View Publication

OBJECTIVE: We examined if cellular elements or adhesive ligands were able to alter asymmetric divisions of CD34(+)/CD38(-) cells in contrast to soluble factors at a single cell level. MATERIALS AND METHODS: After single cell deposition onto 96-well plates,cells were cocultured for 10 days with the stem cell supporting cell line AFT024,fibronectin (FN),or bovine serum albumin (BSA). The divisional history was monitored with time-lapse microscopy. Subsequent function for the most primitive cells was assessed using the myeloid-lymphoid-initiating cell (ML-IC) assay. Committed progenitors were measured using colony-forming cells (CFC). RESULTS: Only contact with AFT024 recruited significant numbers of CD34(+)/CD38(-) cells into cell cycle and increased asymmetric divisions. Although most ML-IC were still identified among cells that have divided fewer than 3 times,a significant number of ML-IC shifted into the fast-dividing fraction after exposure to AFT024. The increase in ML-IC frequency was predominantly due to recruitment of quiescent and slow-dividing cells from the starting population. Increase in CFC activity induced by AFT024 was found only among rapidly dividing cells. CONCLUSIONS: For the first time,we have demonstrated that asymmetric divisions can be altered upon exposure with a stem cell-supporting microenvironment. For the primitive subset of cells (ML-IC),this was predominantly due to recruitment into cell cycle and increased rounds of cycling without loss of function. Exposure to AFT024 cells also increased proliferation and asymmetric divisions of committed CFC. Hence direct communication between hematopoietic progenitors with stroma cells is required for maintaining self-renewal potential. View Publication