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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

Previous reports have shown that antidepressants increase neuronal cell proliferation and enhance neuroplasticity both in vivo and in vitro. This study investigated the direct effects of one such antidepressant, fluoxetine , on cell proliferation and on the production of neurotrophic factors in neuronal precursors derived from human embryonic stem cells (hESCs; H9). Fluoxetine induced the differentiation of neuronal precursors, strongly enhancing neuronal characteristics. The rate of proliferation was higher in fluoxetine -treated cells than in control cells, as determined by MTT [3(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay. The CPDL (cumulative population doubling level) of the fluoxetine-treated cells was significantly increased in comparison to that of control cells (ptextless.001). Bromodeoxyuridine incorporation and staurosporine-induced apoptosis assays were elevated in fluoxetine-treated cells. Quantitative RT-PCR analysis revealed no significant differences in the expression of neurotrophic factors, brain-derived neurotrophic factor (BDNF);glial-derived neurotrophic factor (GDNF) and cAMP-responsive element-binding protein (CREB) between cells treated with fluoxetine for two weeks and their untreated counterparts. These results may help elucidate the mechanism of action of fluoxetine as a therapeutic drug for the treatment of depression. Data presented herein provide more evidence that, in addition to having a direct chemical effect on serotonin levels, fluoxetine can influence hESC-derived neuronal cells by increasing cell proliferation, while allowing them to maintain their neuronal characteristics.

Platelet factor 4 (PF4) is a negative regulator of megakaryopoiesis, but its mechanism of action had not been addressed. Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) has been shown to mediate endothelial cell responses to PF4 and so we tested this receptor's importance in PF4's role in megakaryopoiesis. We found that LRP1 is absent from megakaryocyte-erythrocyte progenitor cells, is maximally present on large, polyploidy megakaryocytes, and near absent on platelets. Blocking LRP1 with either receptor-associated protein (RAP), an antagonist of LDL family member receptors, or specific anti-LRP1 antibodies reversed the inhibition of megakaryocyte colony growth by PF4. In addition, using shRNA to reduce LRP1 expression was able to restore megakaryocyte colony formation in bone marrow isolated from human PF4-overexpressing mice (hPF4(High)). Further, shRNA knockdown of LRP1 expression was able to limit the effects of PF4 on megakaryopoiesis. Finally, infusion of RAP into hPF4(High) mice was able to increase baseline platelet counts without affecting other lineages, suggesting that this mechanism is important in vivo. These studies extend our understanding of PF4's negative paracrine effect in megakaryopoiesis and its potential clinical implications as well as provide insights into the biology of LRP1, which is transiently expressed during megakaryopoiesis.

Development of non-invasive and accurate methods to track cell fate after delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. In this protocol, we describe the in vivo monitoring of stem cell survival, proliferation and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase and herpes simplex virus thymidine kinase (HSVtk)) reporter genes using lentiviral transduction. We used fluorescence-activated cell sorting to purify these populations in vitro, bioluminescence imaging and positron emission tomography (PET) imaging to track them in vivo and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking, such as iron particle and radionuclide labeling, reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny.

Natural killer T (NKT) cells are innate-like lymphocytes that recognize lipid antigens and have been shown to enhance B-cell activation and antibody production. B cells typically recruit T-cell help by presenting internalized antigens recognized by their surface antigen receptor. Here, we demonstrate a highly efficient means whereby human B cells present lipid antigens to NKT cells, capturing the antigen using apolipoprotein E (apoE) and the low-density lipoprotein receptor (LDL-R). ApoE dramatically enhances B-cell presentation of alpha-galactosylceramide (alphaGalCer), an exogenous CD1d presented antigen, inducing activation of NKT cells and the subsequent activation of B cells. B cells express the LDL-R on activation, and the activation of NKT cells by B cells is completely LDL-R dependent, as shown by blocking experiments and the complete lack of presentation when using apoE2, an isoform of apoE incapable of LDL-R binding. The dependence on apoE and the LDL-R is much more pronounced in B cells than we had previously seen in dendritic cells, which can apparently use alternate pathways of lipid antigen uptake. Thus, B cells use an apolipoprotein-mediated pathway of lipid antigen presentation, which constitutes a form of innate help for B cells by NKT cells.

BACKGROUND The in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required. METHODOLOGY/PRINCIPAL FINDINGS In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors, which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes, neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five gene expression signatures that correlate with successive stages in the path from ES cells to neurons. These include a gene signature for a primitive ectoderm-like stage that appears after ES cells enter differentiation, and three gene signatures for subsequent stages of neural progenitor development, from an early stage that follows neural induction to a final stage preceding terminal differentiation. CONCLUSIONS/SIGNIFICANCE Overall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing in addition novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons.

BACKGROUND: Despite the promising therapeutic potential of regulatory T cells (Treg) in animal studies of graft-versus-host disease (GVHD), little is known about their effect on human GVHD. Whether Treg are capable of ameliorating GVHD tissue damage has never been demonstrated in humans. It is also unknown whether Treg modulation of GVH histopathologic damage relies on their presence during effector T-cell priming, or whether allogeneic Treg are safe to use clinically. METHODS: To address these questions, we used an in vitro human skin explant GVHD model, which mimics the physiopathology of GVHD. First, donor"-derived CD8 T cells were stimulated with human leukocyte antigen-unmatched "recipient" dendritic cells (priming phase)�

Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5alpha and APOBEC3G, which target HIV-1 capsid and viral infectivity factor (Vif), respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV), including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (chiHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34(+) hematopoietic stem cells (HSCs), the chiHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34(+) HSCs, the chiHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques, the chiHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus, 15 to 30% versus 1 to 5%; second rhesus, 7 to 15% versus 0.5 to 2%, respectively) 3 to 7 months postinfusion. In summary, we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application.

Serotonergic axons from the raphe nuclei in the brainstem project to every region of the brain, where they make connections through their extensive terminal arborizations. This serotonergic innervation contributes to various normal behaviors and psychiatric disorders. The protocadherin-alpha (Pcdha) family of clustered protocadherins consists of 14 cadherin-related molecules generated from a single gene cluster. We found that the Pcdhas were strongly expressed in the serotonergic neurons. To elucidate their roles, we examined serotonergic fibers in a mouse mutant (Pcdha(Delta CR/Delta CR)) lacking the Pcdha cytoplasmic region-encoding exons, which are common to the gene cluster. In the first week after birth, the distribution pattern of serotonergic fibers in Pcdha(Delta CR/Delta CR) mice was similar to wild-type, but by 3 weeks of age, when the serotonergic axonal termini complete their arborizations, the distribution of the projections was abnormal. In some target regions, notably the globus pallidus and substantia nigra, the normally even distribution of serotonin axonal terminals was, in the mutants, dense at the periphery of each region, but sparse in the center. In the stratum lacunosum-molecular of the hippocampus, the mutants showed denser serotonergic innervation than in wild-type, and in the dentate gyrus of the hippocampus and the caudate-putamen, the innervation was sparser. Together, the abnormalities suggested that Pcdha proteins are important in the late-stage maturation of serotonergic projections. Further examination of alternatively spliced exons encoding the cytoplasmic tail showed that the A-type (but not the B-type) cytoplasmic tail was essential for the normal development of serotonergic projections.

Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and remains a major cause of mortality in children with recurrent disease and in adults. Despite observed graft-versus-leukemia effects after stem cell transplantation, successful immune therapies for ALL have proven elusive. We previously reported immunostimulatory oligodeoxynucleotides containing CpG motifs (CpG ODN) enhance allogeneic T(h)1 responses and reduce leukemic burden of primary human ALL xenografts. To further the development of CpG ODN as a novel ALL therapy, we investigated the antileukemia activity induced by CpG ODN in a transplantable syngeneic pre-B ALL model. CpG ODN induced early killing of leukemia by innate immune effectors both in vitro and in vivo. Mice were treated with CpG ODN starting 7 days after injection with leukemia to mimic a minimal residual disease state and achieved T cell-dependent remissions of more than 6 months. In addition, mice in remission after CpG ODN treatment were protected from leukemia rechallenge, and adoptive transfer of T cells from mice in remission conferred protection against leukemia growth. To our knowledge, this is the first demonstration that CpG ODN induce a durable remission and ongoing immune-mediated protection in ALL, suggesting this treatment may have clinical utility in patients with minimal residual disease.

The polycomb repressive complex (PRC) 2 contains 3 core proteins, EZH2, SUZ12, and EED, in which the SET (suppressor of variegation-enhancer of zeste-trithorax) domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation of lysine 27 on histone H3, regulates the expression of HOX genes, and promotes proliferation and aggressiveness of neoplastic cells. In this study, we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) depletes EZH2 levels, and inhibits trimethylation of lysine 27 on histone H3 in the cultured human acute myeloid leukemia (AML) HL-60 and OCI-AML3 cells and in primary AML cells. DZNep treatment induced p16, p21, p27, and FBXO32 while depleting cyclin E and HOXA9 levels. Similar findings were observed after treatment with small interfering RNA to EZH2. In addition, DZNep treatment induced apoptosis in cultured and primary AML cells. Furthermore, compared with treatment with each agent alone, cotreatment with DZNep and the pan-histone deacetylase inhibitor panobinostat caused more depletion of EZH2, induced more apoptosis of AML, but not normal CD34(+) bone marrow progenitor cells, and significantly improved survival of nonobese diabetic/severe combined immunodeficiency mice with HL-60 leukemia. These findings indicate that the combination of DZNep and panobinostat is effective and relatively selective epigenetic therapy against AML cells.