参考文献

INTRODUCTION: Lung cancer contains a small population of cancer stem cells that contribute to its initiation and progression. We investigated the biological function and clinical significance of aldehyde dehydrogenase 1A1 (ALDH1A1) in non-small-cell lung carcinoma (NSCLC). METHODS: ALDH1A1 assay or small interfering RNA transfection was employed to isolate ALDH1A1+ cells or knock down ALDH1A1 expression in H2087 cells,respectively. Biological functions of ALDH1A1+ and ALDH1A1 silenced cells were investigated using in vitro and in vivo methods. ALDH1A1 expression was analyzed using immunohistochemistry on tissue microarrays with 179 lung cancer tissues and 26 normal lung tissues. RESULTS: The abilities of clone formation,proliferation,cell growth,and migration were increased in ALDH1A1+ and ALDH1A1 silenced cells. ALDH1A1+ lung cancer cells initiated tumors that resembled the histopathologic characteristics and heterogeneity of the parental lung cancer cells in mice. The silencing of ALDH1A1 expression in H2087 lung cancer cells inhibited cell proliferation and migration significantly. ALDH1A1 was expressed in 42% of normal lung tissues (11 of 26),with strong expression in the basal cells and globular cells of the normal bronchus and weak expression in the alveolar epithelial cells. Compared with normal lung tissues,45% of NSCLC samples (81 of 179) were read as positive for ALDH1A1. Positive ALDH1A1 expression was correlated with patients' smoking status (p = 0.022),lymph-node metastasis (p = 0.006),clinical stage (p = 0.004),and a decreased overall survival time (p textless 0.001). Positive ALDH1A1 expression in lung cancer tissues was an independent prognostic factor for NSCLC (odds ratio = 5.232,p textless 0.001). CONCLUSION: Elucidating the biological functions of ALDH1A1 could be helpful in studying lung tumorigenesis and for developing new therapeutic approaches. View Publication

Studies in the past have illuminated the potential benefit of resveratrol as an anticancer (pro-apoptosis) and life-extending (pro-survival) compound. However,these two different effects were observed at different concentration ranges. Studies of resveratrol in a wide range of concentrations on the same cell type are lacking,which is necessary to comprehend its diverse and sometimes contradictory cellular effects. In this study,we examined the effects of resveratrol on cell self-renewal and differentiation of human mesenchymal stem cells (hMSCs),a type of adult stem cells that reside in a number of tissues,at concentrations ranging from 0.1 to 10 µM after both short- and long-term exposure. Our results reveal that at 0.1 µM,resveratrol promotes cell self-renewal by inhibiting cellular senescence,whereas at 5 µM or above,resveratrol inhibits cell self-renewal by increasing senescence rate,cell doubling time and S-phase cell cycle arrest. At 1 µM,its effect on cell self-renewal is minimal but after long-term exposure it exerts an inhibitory effect,accompanied with increased senescence rate. At all concentrations,resveratrol promotes osteogenic differentiation in a dosage dependent manner,which is offset by its inhibitory effect on cell self-renewal at high concentrations. On the contrary,resveratrol suppresses adipogenic differentiation during short-term exposure but promotes this process after long-term exposure. Our study implicates that resveratrol is the most beneficial to stem cell development at 0.1 µM and caution should be taken in applying resveratrol as an anticancer therapeutic agent or nutraceutical supplement due to its dosage dependent effect on hMSCs. View Publication

Human embryonic stem cells (hESCs) can self-renew and become all three germ layers. Nodal/Activin signaling specifies developmental status in hESCs: moderate Nodal/Activin signaling maintains pluripotency,while enhancement and inhibition promote definitive endoderm (DE) and neuroectoderm (NE) development,respectively. However,how modulation of Nodal/Activin signaling influences developmental competence and commitment toward specific lineages is still unclear. Here,we showed that enhancement of Nodal/Activin signaling for 4 days was necessary and sufficient to upregulate DE markers,while it diminished the upregulation of NE markers by inhibition of Nodal/Activin signaling. This suggests that after 4 days of enhanced Nodal/Activin signaling,hESCs are committed to the DE lineage and have lost competence toward the NE lineage. In contrast,inhibition of Nodal/Activin signaling using LY364947 for 2 days was sufficient to impair competence toward the DE lineage,although cells were still able to activate LEFTY1 and NODAL,direct targets of Nodal/Activin signaling. Expression analyses indicated that the levels of pluripotency regulators NANOG and POU5F1 were significantly diminished by 2 days of LY364947 treatment,although the expression of NANOG,but not POU5F1,was restored immediately upon Activin A treatment. Thus,downregulation of POU5F1 coincided with the abrogation of DE competence caused by inhibition of Nodal/Activin signaling. View Publication

Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein-coupled APELIN receptor (APLNR). APLNR-positive cells,identified by binding of the fluoresceinated peptide ligand,APELIN (APLN),or an anti-APLNR mAb,were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs),increased the expression of hematoendothelial genes in differentiating hESCs,and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore,APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells. View Publication

PURPOSE: Prostate cancer cells include a small population of cancer stem-like/cancer initiating cells,which have roles in cancer initiation and progression. Recently aldehyde dehydrogenase activity was used to isolate stem cells of various cancer and normal cells. We evaluated the aldehyde dehydrogenase activity of the human prostate cancer cell line 22Rv1 (ATCC®) with the ALDEFLUOR® assay and determined its potency as prostate cancer stem-like/cancer initiating cells. MATERIALS AND METHODS: The human prostate cancer cell line 22Rv1 was labeled with ALDEFLUOR reagent and analyzed by flow cytometry. ALDH1(high) and ALDH1(low) cells were isolated and tumorigenicity was evaluated by xenograft transplantation into NOD/SCID mice. Tumor sphere forming ability was evaluated by culturing in a floating condition. Invasion capability was evaluated by the Matrigel™ invasion assay. Gene expression profiling was assessed by microarrays and reverse transcriptase-polymerase chain reaction. RESULTS: ALDH1(high) cells were detected in 6.8% of 22Rv1 cells,which showed significantly higher tumorigenicity than ALDH1(low) cells in NOD/SCID mice (p textless 0.05). Gene expression profiling revealed higher expression of the stem cell related genes PROM1 and NKX3-1 in ALDH1(high) cells than in ALDH1(low) cells. ALDH1(high) cells also showed higher invasive capability and sphere forming capability than ALDH1(low) cells. CONCLUSIONS: Results indicate that cancer stem-like/cancer initiating cells are enriched in the ALDH1(high) population of the prostate cancer cell line 22Rv1. This approach may provide a breakthrough to further clarify prostate cancer stem-like/cancer initiating cells. To our knowledge this is the first report of cancer stem-like/cancer initiating cells of 22Rv1 using the aldehyde dehydrogenase activity assay. View Publication

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy,in vitro modeling of human disease,and drug screening. To date,most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively,transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This unit describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector,which includes the coding sequences of human OCT4,SOX2,KLF4,and cMYC linked with picornaviral 2A plasmids. Moreover,after reprogramming has been achieved,this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. View Publication

PURPOSE: There are no effective therapies for patients with poorly differentiated papillary thyroid cancer (PTC) or anaplastic thyroid cancer (ATC),and metastasis to the bone represents a significantly worse prognosis. Src family kinases (SFKs) are overexpressed and activated in numerous tumor types and have emerged as a promising therapeutic target,especially in relation to metastasis. We recently showed that Src is overexpressed and activated in thyroid cancer. We therefore tested whether inhibition of Src with dasatinib (BMS-354825) blocks thyroid cancer growth and metastasis. EXPERIMENTAL DESIGN: The effects of dasatinib on thyroid cancer growth,signaling,cell cycle,and apoptosis were evaluated in vitro. The therapeutic efficacy of dasatinib was further tested in vivo using an orthotopic and a novel experimental metastasis model. Expression and activation of SFKs in thyroid cancer cells was characterized,and selectivity of dasatinib was determined using an Src gatekeeper mutant. RESULTS: Dasatinib treatment inhibited Src signaling,decreased growth,and induced cell-cycle arrest and apoptosis in a subset of thyroid cancer cells. Immunoblotting showed that c-Src and Lyn are expressed in thyroid cancer cells and that c-Src is the predominant SFK activated. Treatment with dasatinib blocked PTC tumor growth in an orthotopic model by more than 90% (P = 0.0014). Adjuvant and posttreatment approaches with dasatinib significantly inhibited metastasis (P = 0.016 and P = 0.004,respectively). CONCLUSION: These data provide the first evidence that Src is a central mediator of thyroid cancer growth and metastasis,indicating that Src inhibitors may have a higher therapeutic efficacy in thyroid cancer,as both antitumor and antimetastatic agents. View Publication

The 2009 H1N1 influenza pandemic was a major international public health crisis which caused considerable morbidity and mortality worldwide. The goal of this study was to produce anti-H1 monoclonal antibodies (MAbs) for improving diagnostic immunological assays and to develop potential immunotherapeutics. Nine MAbs were produced after immunizing mice with recombinant hemagglutinin (HA) protein from A/California/06/09. Two spleenocyte myeloma fusions yielded 1588 hybridoma cultures. After screening the hybridoma culture supernatants for antibody reactivity to rHA,nine clones were selected for further characterization. Cross-reactivity studies of the anti-rHA antibodies against a panel of influenza viruses (H1-H16) revealed eight out of nine MAbs were specific to the pandemic H1 subtype,except for MAb F256G2sc1 which also cross-reacted with H5 subtype virus. All MAbs were of the IgG1κ isotype,except F256G2sc1 which was IgG2aκ. The anti-rHA MAbs had binding affinities to rHA that ranged from a K(D) (disassociation constant) of 1.34×10(-9)M (F255G7sc1) to the weakest affinity of 4.60×10(-8)M (F255G4sc1). Interestingly,in a plaque reduction neutralization assay,all MAbs except F255G3sc1 demonstrated neutralizing ability. Furthermore,all MAbs except F255G3sc1 and F255G9sc1 exhibited anti-hemagglutinin activity against pandemic H1N1 viruses,but not against classical North American swine influenza viruses of the same subtype. Immunofluorescence assay (IFA) demonstrated that all MAbs except F255G1sc1 and F255G3sc1 were able to detect 2009 pandemic H1N1 (2009) virus- infected MDCK cells. The MAbs were also evaluated for potential use in competitive ELISA (cELISA),and with the exception of F255G3sc1,all MAbs showed competitive activity with serum collected from pigs infected with pandemic H1N1 virus (2009). The developed MAbs have demonstrated utility as immunodiagnostic and research reagents,and their neutralizing capabilities also hold potential for designing antiviral drugs against pandemic influenza. View Publication

Exercise has been shown to positively augment adult hippocampal neurogenesis; however,the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here,we used transgenic Csf1r-GFP mice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely,microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX(3)CL1,a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX(3)CL1 receptor,CX(3)CR1,but not control IgG,dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX(3)CL1 was observed following running,reduced levels of this chemokine were found in the aged brain. Lower levels of CX(3)CL1 with advancing age correlated with the natural decline in neural precursor cell activity,a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus,and that signaling through the CX(3)CL1-CX(3)CR1 axis critically contributes toward this process. View Publication

Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression,reproducibility,length,and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation,embryoid body (EB) differentiation,and direct neuronal differentiation. Here,we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage. View Publication