技术资料

Extracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T-cell lymphoma,graft-versus-host disease,and allografted organ rejection. Its clinical and experimental efficacy in cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the antigen-presenting dendritic cell (DC) differentiation pathway,within a single day,without added cytokines,as determined by enhanced expression of relevant genes. The resulting DCs are capable of processing and presentation of exogenous and endogenous antigen and are largely maturationally synchronized,as assessed by the level of expression of costimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome reveals that activation or suppression of more than 1100 genes produces a reproducible distinctive molecular signature,common to ECP-processed monocytes from normal subjects,and those from patients. Because ECP induces normal monocytes to enter the DC differentiation pathway,this phenomenon is independent of disease state. The efficiency with which ECP stimulates new functional DCs supports the possibility that these cells participate prominently in the clinical successes of the treatment. Appropriately modified by future advances,ECP may potentially offer a general source of therapeutic DCs. View Publication

Current methodology for pluripotent human embryonic stem cells (hESCs) expansion relies on murine sarcoma basement membrane substrates (Matrigel™),which precludes the use of these cells in regenerative medicine. To realize the clinical efficacy of hESCs and their derivatives,expansion of these cells in a defined system that is free of animal components is required. This study reports the successful propagation of hESCs (HES-3 and H1) for textgreater 20 passages on tissue culture-treated polystyrene plates,coated from 5 μg/mL of human plasma-purified vitronectin (VN) solution. Cells maintain expression of pluripotent markers Tra1-60 and OCT-4 and are karyotypically normal after 20 passages of continuous culture. In vitro and in vivo differentiation of hESC by embryoid body formation and teratoma yielded cells from the ecto-,endo-,and mesoderm lineages. VN immobilized on tissue culture polystyrene was characterized using a combination of X-ray photoemission spectroscopy,atomic force microscopy,and quantification of the VN surface density with a Bradford protein assay. Ponceau S staining was used to measure VN adsorption and desorption kinetics. Tuning the VN surface density,via the concentration of depositing solution,revealed a threshold surface density of 250 ng/cm²,which is required for hESCs attachment,proliferation,and differentiation. Cell attachment and proliferation assays on VN surface densities above this threshold show the substrate properties to be equally viable. View Publication

BACKGROUND: Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations,the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally,a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor,Y-27632,previously has been identified as enhancing survival of hESCs upon single-cell dissociation,as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions. METHODOLOGY/PRINCIPAL FINDINGS: HESCs were sorted using markers for SSEA-3,TRA-1-81,and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions,cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically,treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632,hESCs were further analyzed. Specifically,hESCs sorted with and without the addition of Y-27632 retained normal morphology,expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry,and maintained a stable karyotype. In addition,the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions. CONCLUSIONS/SIGNIFICANCE: The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency,and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types,identification and isolation of stem cell subpopulations,and generation of single cell clones. Finally,these results demonstrate an additional application of ROCK inhibition to hESC research. View Publication

Almost 25 centuries ago,Hippocrates,the father of medicine,proclaimed Let food be thy medicine and medicine be thy food." Exploring the association between diet and health continues today. For example� View Publication

Tumor-initiating cells (TICs) are defined by their ability to form tumors after xenotransplantation in immunodeficient mice and appear to be relatively rare in most human cancers. Recent data in melanoma indicate that the frequency of TICs increases dramatically via more permissive xenotransplantation conditions,raising the possibility that the true frequency of TICs has been greatly underestimated in most human tumors. We compared the growth of human pancreatic,non-small cell lung,and head and neck carcinomas in NOD/SCID and NSG mice. Although TIC frequency was detected up to 10-fold higher in NSG mice,it remained low (textless1 in 2500 cells) in all cases. Moreover,aldehyde dehydrogenase-positive (ALDH(+)) and CD44(+)CD24(+) cells,phenotypically distinct cells enriched in TICs,were equally tumorigenic in NOD/SCID and NSG mice. Our findings demonstrate that TICs are rare in these cancers and that the identification of TICs and their frequency in other human malignancies should be validated via primary tumors and highly permissive xenotransplantation conditions. View Publication

CRKL (CRK-like) is an adapter protein predominantly phosphorylated in cells that express the tyrosine kinase p210(BCR-ABL),the fusion product of a (9;22) chromosomal translocation causative for chronic myeloid leukemia. It has been unclear,however,whether CRKL plays a functional role in p210(BCR-ABL) transformation. Here,we show that CRKL is required for p210(BCR-ABL) to support interleukin-3-independent growth of myeloid progenitor cells and long-term outgrowth of B-lymphoid cells from fetal liver-derived hematopoietic progenitor cells. Furthermore,a synthetic phosphotyrosyl peptide that binds to the CRKL SH2 domain with high affinity blocks association of endogenous CRKL with the p210(BCR-ABL) complex and reduces c-MYC levels in K562 human leukemic cells as well as in mouse hematopoietic cells transformed by p210(BCR-ABL) or the imatinib-resistant mutant T315I. These results indicate that the function of CRKL as an adapter protein is essential for p210(BCR-ABL)-induced transformation. View Publication

Tumor angiogenesis is essential for malignant growth and metastasis. Bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute to angiogenesis-mediated tumor growth. EPC ablation can reduce tumor growth; however,the lack of a marker that can track EPCs from the BM to tumor neovasculature has impeded progress in understanding the molecular mechanisms underlying EPC biology. Here,we report the use of transgenic mouse and lentiviral models to monitor the BM-derived compartment of the tumor stroma; this approach exploits the selectivity of the transcription factor inhibitor of DNA binding 1 (Id1) for EPCs to track EPCs in the BM,blood,and tumor stroma,as well as mature EPCs. Acute ablation of BM-derived EPCs using Id1-directed delivery of a suicide gene reduced circulating EPCs and yielded significant defects in angiogenesis-mediated tumor growth. Additionally,use of the Id1 proximal promoter to express microRNA-30-based short hairpin RNA inhibited the expression of critical EPC-intrinsic factors,confirming that signaling through vascular endothelial growth factor receptor 2 is required for EPC-mediated tumor biology. By exploiting the selectivity of Id1 gene expression in EPCs,our results establish a strategy to track and target EPCs in vivo,clarifying the significant role that EPCs play in BM-mediated tumor angiogenesis. View Publication

It has recently been shown that nucleosome distribution,histone modifications and RNA polymerase II (Pol II) occupancy show preferential association with exons (exon-intron marking")� View Publication

BACKGROUND The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus,and to show protection in a lethal animal challenge model. METHODOLOGY/PRINCIPAL FINDINGS For this purpose,the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus--a safe poxviral live vector--resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines,together with an inactivated whole virus vaccine,were assessed in a lung infection model using immune competent Balb/c mice,and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain,while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-gamma-secreting (IFN-gamma) CD4- and CD8 T-cells in lungs and spleens. In the lungs,a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus,which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition,passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus. CONCLUSIONS/SIGNIFICANCE The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza. View Publication

Retinoids are important molecules involved in the development and homeostasis of the nervous system. As such,various retinoid derivatives are often found in culture media and supplement formulations to support the growth and maintenance of neural cells. However,all-trans-retinoic acid (ATRA) and its associated derivatives are light sensitive and are highly susceptible to isomerisation. This can lead to variability in retinoid concentrations and the nature of the retinoid species present in culture solutions which in turn can influence biological activity and introduce inconsistency. We have previously described the development of the synthetic retinoid derivative,EC23,as a chemically and light stable alternative that does not degrade and has biological activity similar to ATRA. In this study we demonstrate that the addition of exogenous retinoid can significantly enhance neuronal differentiation of both human neuroprogenitor and human embryonic stem cells. In the former,both ATRA and EC23 induced increased maturation and stabilisation of the axonal cytoskeleton. However,EC23 was particularly potent at lower nanomolar concentrations resulting in significantly greater neurogenesis than ATRA. In ES cells enhanced motor neuron marker expression was also detected in response to both retinoids when incorporated into an established protocol for neuronal differentiation. We propose that synthetic retinoid EC23 represents a valuable addition to the formulation of new and existing culture supplements to enhance neuronal differentiation whilst enabling improved consistency. View Publication

INTRODUCTION: The physiological signals that direct the division and differentiation of the zygote to form a blastocyst,and subsequent embryonic stem cell division and differentiation during early embryogenesis,are unknown. Although a number of growth factors,including the pregnancy-associated hormone human chorionic gonadotropin (hCG) are secreted by trophoblasts that lie adjacent to the embryoblast in the blastocyst,it is not known whether these growth factors directly signal human embryonic stem cells (hESCs).backslashnbackslashnMETHODS: Here we used hESCs as a model of inner cell mass differentiation to examine the hormonal requirements for the formation of embryoid bodies (EB's; akin to blastulation) and neuroectodermal rosettes (akin to neurulation).backslashnbackslashnRESULTS: We found that hCG promotes the division of hESCs and their differentiation into EB's and neuroectodermal rosettes. Inhibition of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) signaling suppresses hESC proliferation,an effect that is reversed by treatment with hCG. hCG treatment rapidly upregulates steroidogenic acute regulatory protein (StAR)-mediated cholesterol transport and the synthesis of progesterone (P4). hESCs express P4 receptor A,and treatment of hESC colonies with P4 induces neurulation,as demonstrated by the expression of nestin and the formation of columnar neuroectodermal cells that organize into neural tubelike rosettes. Suppression of P4 signaling by withdrawing P4 or treating with the P4-receptor antagonist RU-486 inhibits the differentiation of hESC colonies into EB's and rosettes.backslashnbackslashnCONCLUSIONS: Our findings indicate that hCG signaling via LHCGR on hESC promotes proliferation and differentiation during blastulation and neurulation. These findings suggest that trophoblastic hCG secretion and signaling to the adjacent embryoblast could be the commencement of trophic support by placental tissues in the growth and development of the human embryo. View Publication

The Sox6 transcription factor plays critical roles in various cell types,including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells,we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar,evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo,and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied,in erythroid cells,by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation,mediated by the double Sox6 binding site within its own promoter,may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation. View Publication