参考文献

Insight into the normal function of PrP(C),and how it can be subverted to produce neurotoxic effects,is provided by PrP molecules carrying deletions encompassing the conserved central region. The most neurotoxic of these mutants,Δ105-125 (called ΔCR),produces a spontaneous neurodegenerative illness when expressed in transgenic mice,and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous,or can be exerted on neighboring cells,is unknown. To investigate this question,we have utilized co-cultures of differentiated neural stem cells derived from mice expressing ΔCR or wild-type PrP. Cells from the two kinds of mice,which are marked by the presence or absence of GFP,are differentiated together to yield neurons,astrocytes,and oligodendrocytes. As a surrogate read-out of ΔCR PrP toxicity,we assayed sensitivity of the cells to the cationic antibiotic,Zeocin. In a previous study,we reported that cells expressing ΔCR PrP are hypersensitive to the toxic effects of several cationic antibiotics,an effect that is suppressed by co-expression of wild type PrP,similar to the rescue of the neurodegenerative phenotype observed in transgenic mice. Using this system,we find that while ΔCR-dependent toxicity is cell-autonomous,the rescuing activity of wild-type PrP can be exerted in trans from nearby cells. These results provide important insights into how ΔCR PrP subverts a normal physiological function of PrP(C),and the cellular mechanisms underlying the rescuing process. View Publication

The mechanisms by which human embryonic stem cells (hESC) differentiate to endodermal lineage have not been extensively studied. Mathematical models can aid in the identification of mechanistic information. In this work we use a population-based modeling approach to understand the mechanism of endoderm induction in hESC,performed experimentally with exposure to Activin A and Activin A supplemented with growth factors (basic fibroblast growth factor (FGF2) and bone morphogenetic protein 4 (BMP4)). The differentiating cell population is analyzed daily for cellular growth,cell death,and expression of the endoderm proteins Sox17 and CXCR4. The stochastic model starts with a population of undifferentiated cells,wherefrom it evolves in time by assigning each cell a propensity to proliferate,die and differentiate using certain user defined rules. Twelve alternate mechanisms which might describe the observed dynamics were simulated,and an ensemble parameter estimation was performed on each mechanism. A comparison of the quality of agreement of experimental data with simulations for several competing mechanisms led to the identification of one which adequately describes the observed dynamics under both induction conditions. The results indicate that hESC commitment to endoderm occurs through an intermediate mesendoderm germ layer which further differentiates into mesoderm and endoderm,and that during induction proliferation of the endoderm germ layer is promoted. Furthermore,our model suggests that CXCR4 is expressed in mesendoderm and endoderm,but is not expressed in mesoderm. Comparison between the two induction conditions indicates that supplementing FGF2 and BMP4 to Activin A enhances the kinetics of differentiation than Activin A alone. This mechanistic information can aid in the derivation of functional,mature cells from their progenitors. While applied to initial endoderm commitment of hESC,the model is general enough to be applicable either to a system of adult stem cells or later stages of ESC differentiation. View Publication

BACKGROUND: Previously,we enriched a subpopulation of head and neck cancer-derived tumor initiating cells (HNC-TICs) presented high tumorigenic,chemo-radioresistant,and coupled with epithelial-mesenchymal transition (EMT) properties. The purpose of this study was to investigate the therapeutic effect and molecular mechanisms of quercetin on HNC-TICs. METHOD: ALDH1 activity of head and neck cancer cells with quercetin treatment was assessed by the Aldefluor assay flow cytometry analysis. Self-renewal,invasiveness,and EMT capability of HNC-TICs with different doses of quercetin was presented. RESULTS: We first observed that the treatment of quercetin significantly downregulated the ALDH1 activity of head and neck cancer cells in a dose-dependent manner (p textless .05). Moreover,quercetin reduced self-renewal property and stemness signatures expression in head and neck cancer-derived sphere cells. The migration ability of head and neck cancer-derived sphere cells was lessened under quercetin treatment partially due to the decreased productions of Twist,N-cadherin,and vimentin. CONCLUSION: Quercetin suppressing HNC-TICs characteristics may therefore be valuable therapeutics clinically in combination with standard treatment modalities. View Publication

EGFRvIII is a tumor-specific variant of the epidermal growth factor receptor (EGFR). Although EGFRvIII is most commonly found in glioblastoma,its expression in other tumor types remains controversial. In this study,we investigated EGFRvIII expression and amplification in primary breast carcinoma. Our analyses confirmed the presence of EGFRvIII,but in the absence of amplification or rearrangement of the EGFR locus. Nested reverse transcriptase PCR and flow cytometry were used to detect a higher percentage of positive cases. EGFRvIII-positive cells showed increased expression of genes associated with self-renewal and epithelial-mesenchymal transition along with a higher percentage of stem-like cells. EGFRvIII also increased in vitro sphere formation and in vivo tumor formation. Mechanistically,EGFRvIII mediated its effects through the Wnt/β-catenin pathway,leading to increased β-catenin target gene expression. Inhibition of this pathway reversed the observed effects on cancer stem cell (CSC) phenotypes. Together,our findings show that EGFRvIII is expressed in primary breast tumors and contributes to CSC phenotypes in breast cancer cell lines through the Wnt pathway. These data suggest a novel function for EGFRvIII in breast tumorigenesis. View Publication

BACKGROUND Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold,respectively,without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D,natural cytotoxicity receptors,and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred,expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion,persisted up to 4 weeks,were readily detectable in the human bone,inhibited myeloma growth and protected bone from myeloma-induced osteolysis. CONCLUSIONS These studies provide the rationale for testing expanded natural killer cells in humans. View Publication

Targeting native progenitors with small molecule pharmaceuticals that direct cell fate decisions is an attractive approach for regenerative medicine. Here,we show that 3,5-disubstituted isoxazoles (Isx),stem cell-modulator small molecules originally recovered in a P19 embryonal carcinoma cell-based screen,directed cardiac muscle gene expression in vivo in target tissues of adult transgenic reporter mice. Isx also stimulated adult mouse myocardial cell cycle activity. Narrowing our focus onto one target cardiac-resident progenitor population,Isx directed muscle transcriptional programs in vivo in multipotent Notch-activated epicardium-derived cells (NECs),generating Notch-activated adult cardiomyocyte-like precursors. Myocardial infarction (MI) preemptively differentiated NECs toward fibroblast lineages,overriding Isx's cardiogenic influence in this cell population. Isx dysregulated gene expression in vivo in Notch-activated repair fibroblasts,driving distinctive (pro-angiogenesis) gene programs,but failed to mitigate fibrosis or avert ventricular functional decline after MI. In NECs in vitro,Isx directed partial muscle differentiation,which included biosynthesis and assembly of sarcomeric α-actinin premyofibrils,beaded structures pathognomonic of early developing cardiomyocytes. Thus,although Isx small molecules have promising in vivo efficacy at the level of cardiac muscle gene expression in native multipotent progenitors and are first in class in this regard,a greater understanding of the dynamic interplay between fibrosis and cardiogenic small molecule signals will be required to pharmacologically enable regenerative repair of the heart. View Publication

The potential for human disease treatment using human pluripotent stem cells,including embryonic stem cells and induced pluripotent stem cells (iPSCs),also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies,which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study,a comparison of DNA repair pathways in pluripotent cells,as compared to those in non-pluripotent cells,demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair,we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells,while differentiated cells lacked response to this stimulus,and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition,the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype,but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together,these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines,in order to characterize their genomic stability,prior to their pre-clinical and clinical use. View Publication

Human embryonic stem cell-derived dendritic cells (hESC-DCs) may potentially provide a platform to generate off-the-shelf" therapeutic cancer vaccines. To apply hESC-DCs for cancer immunotherapy in a semiallogeneic setting� View Publication

Adult stem-cell therapy holds promise for the treatment of gastrointestinal diseases. Here we describe methods for long-term expansion of colonic stem cells positive for leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5(+) cells) in culture. To test the transplantability of these cells,we reintroduced cultured GFP(+) colon organoids into superficially damaged mouse colon. The transplanted donor cells readily integrated into the mouse colon,covering the area that lacked epithelium as a result of the introduced damage in recipient mice. At 4 weeks after transplantation,the donor-derived cells constituted a single-layered epithelium,which formed self-renewing crypts that were functionally and histologically normal. Moreover,we observed long-term (textgreater6 months) engraftment with transplantation of organoids derived from a single Lgr5(+) colon stem cell after extensive in vitro expansion. These data show the feasibility of colon stem-cell therapy based on the in vitro expansion of a single adult colonic stem cell. View Publication

Neurogenesis persists in the rodent dentate gyrus (DG) throughout adulthood but declines with age and stress. Neural progenitor cells (NPCs) residing in the subgranular zone of the DG are regulated by an array of growth factors and respond to the microenvironment,adjusting their proliferation level to determine the rate of neurogenesis. Here we report that genetic deletion of neurofibromin (Nf1),a tumor suppressor with RAS-GAP activity,in adult NPCs enhanced DG proliferation and increased generation of new neurons in mice. Nf1 loss-associated neurogenesis had the functional effect of enhancing behavioral responses to subchronic antidepressants and,over time,led to spontaneous antidepressive-like behaviors. Thus,our findings establish an important role for the Nf1-Ras pathway in regulating adult hippocampal neurogenesis,and demonstrate that activation of adult NPCs is sufficient to modulate depression- and anxiety-like behaviors. View Publication