技术资料

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of motor neurons. Currently,there is no effective therapy for ALS. Stem cell transplantation is a potential therapeutic strategy for ALS,and the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) represents a novel cell source. In this study,we isolated a specific neural stem cell (NSC) population from human iPSCs based on high aldehyde dehydrogenase activity,low side scatter and integrin VLA4 positivity. We assessed the therapeutic effects of these NSCs on the phenotype of ALS mice after intrathecal or intravenous injections. Transplanted NSCs migrated and engrafted into the central nervous system via both routes of injection. Compared with control ALS,treated ALS mice exhibited improved neuromuscular function and motor unit pathology and significantly increased life span,in particular with the systemic administration of NSCs (15%). These positive effects are linked to multiple mechanisms,including production of neurotrophic factors and reduction of micro- and macrogliosis. NSCs induced a decrease in astrocyte number through the activation of the vanilloid receptor TRPV1. We conclude that minimally invasive injections of iPSC-derived NSCs can exert a therapeutic effect in ALS. This study contributes to advancements in iPSC-mediated approaches for treating ALS and other neurodegenerative diseases. View Publication

Down syndrome (DS) is among the most frequent genetic causes of intellectual disability,and ameliorating this deficit is a major goal in support of people with trisomy 21. The Ts65Dn mouse recapitulates some major brain structural and behavioral phenotypes of DS,including reduced size and cellularity of the cerebellum and learning deficits associated with the hippocampus. We show that a single treatment of newborn mice with the Sonic hedgehog pathway agonist SAG 1.1 (SAG) results in normal cerebellar morphology in adults. Further,SAG treatment at birth rescued phenotypes associated with hippocampal deficits that occur in untreated adult Ts65Dn mice. This treatment resulted in behavioral improvements and normalized performance in the Morris water maze task for learning and memory. SAG treatment also produced physiological effects and partially rescued both N-methyl-d-aspartate (NMDA) receptor-dependent synaptic plasticity and NMDA/AMPA receptor ratio,physiological measures associated with memory. These outcomes confirm an important role for the hedgehog pathway in cerebellar development and raise the possibility for its direct influence in hippocampal function. The positive results from this approach suggest a possible direction for therapeutic intervention to improve cognitive function for this population. View Publication

Diblock copolymers consisting of poly(ethylene glycol)-block-poly(γ-4-(((2-(piperidin-1-yl)ethyl)amino)methyl)benzyl-l-glutamate) (PEG-b-PVBLG-8) were synthesized and evaluated for their ability to mediate gene delivery in hard-to-transfect cells like IMR-90 human fetal lung fibroblasts and human embryonic s View Publication

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence,which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here,we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity,simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity. textcopyright 2013 Elsevier Inc. View Publication

Wnt/β-catenin signalling has a variety of roles in regulating stem cell fates. Its specific role in mouse epiblast stem cell self-renewal,however,remains poorly understood. Here we show that Wnt/β-catenin functions in both self-renewal and differentiation in mouse epiblast stem cells. Stabilization and nuclear translocation of β-catenin and its subsequent binding to T-cell factors induces differentiation. Conversely,retention of stabilized β-catenin in the cytoplasm maintains self-renewal. Cytoplasmic retention of β-catenin is effected by stabilization of Axin2,a downstream target of β-catenin,or by genetic modifications to β-catenin that prevent its nuclear translocation. We also find that human embryonic stem cell and mouse epiblast stem cell fates are regulated by β-catenin through similar mechanisms. Our results elucidate a new role for β-catenin in stem cell self-renewal that is independent of its transcriptional activity and will have broad implications in understanding the molecular regulation of stem cell fate. View Publication

Mast cells are unique hematopoietic cells that are richly distributed in the skin and mucosal surfaces of the respiratory and gastrointestinal tract. They play a key role in allergic inflammation by releasing a cocktail of granular constituents,including histamine,serine proteases,and various eicosanoids and cytokines. As such,a number of drugs target either inhibition of mast cell degranulation or the products of degranulation. To identify potential novel drugs and mechanisms in mast cell biology,assays were developed to identify inhibitors of mast cell degranulation and activation in a phenotypic screen. Due to the challenges associated with obtaining primary mast cells,cord blood-derived mononuclear cells were reproducibly differentiated to mast cells and assays developed to monitor tryptase release and prostaglandin D2 generation. The tryptase assay was particularly sensitive,requiring only 500 cells per data point,which permitted a set of approximately 12,000 compounds to be screened robustly and cost-effectively. Active compounds were tested for concomitant inhibition of prostaglandin D2 generation. This study demonstrates the robustness and effectiveness of this approach in the identification of potential novel compounds and mechanisms targeting mast cell-driven inflammation,to enable innovative drug discovery efforts to be prosecuted. View Publication

Recently developed genomics-based tools are allowing repositioning of Food and Drug Administration (FDA)-approved drugs as cancer treatments,which were employed to identify drugs that target cancer stem cells (CSCs) of breast cancer. Gene expression datasets of CSCs from six studies were subjected to connectivity map to identify drugs that may ameliorate gene expression patterns unique to CSCs. All-trans retinoic acid (ATRA) was negatively connected with gene expression in CSCs. ATRA reduced mammosphere-forming ability of a subset of breast cancer cells,which correlated with induction of apoptosis,reduced expression of SOX2 but elevated expression of its antagonist CDX2. SOX2/CDX2 ratio had prognostic relevance in CSC-enriched breast cancers. K-ras mutant breast cancer cell line enriched for CSCs was resistant to ATRA,which was reversed by MAP kinase inhibitors. Thus,ATRA alone or in combination can be tested for efficacy using SOX2,CDX2,and K-ras mutation/MAPK activation status as biomarkers of response. View Publication

Cancer stem cells (CSCs) are responsible for tumor progression,metastases,resistance to therapy,and tumor recurrence. Therefore,the identification of molecules involved in CSC self-renewal is a necessary step toward more effective therapies. To this aim,through the transcription profiling of the murine ErbB2(+) tumor cell line TUBO vs. derived CSC-enriched mammospheres,Toll-like receptor 2 (TLR2) was identified as 2-fold overexpressed in CSCs,as confirmed by qPCR and cytofluorimetric analysis. TLR2 signaling inhibition impaired in vitro mammosphere generation in murine TUBO (60%) and 4T1 (30%) and human MDA-MB-231 (50%),HCC1806 (60%),and MCF7 (50%) cells. In CSC,TLR2 was activated by endogenous high-mobility-group box 1 (HMGB1),inducing I$$B$$ phosphorylation,IL-6 and TGF$$ secretion,and,consequently,STAT3 and Smad3 activation. In vivo TLR2 inhibition blocked TUBO tumor takes in 9/14 mice and induced a 2-fold reduction in lung metastases development by decreasing cell proliferation and vascularization and increasing apoptosis. Collectively,these results demonstrate that murine and human mammary CSCs express TLR2 and its ligand HMGB1; this autocrine loop plays a pivotal role in CSC self-renewal,tumorigenesis,and metastatic ability. These findings,while providing evidence against the controversial use of TLR2 agonists in antitumor therapy,lay out new paths toward the design of anticancer treatments. View Publication

PURPOSE High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However,the existence of cells with high ALDH activity (ALDH(bright)) in primary prostate cancer specimens has not been shown so far. We investigated the presence,phenotype,and clinical significance of ALDH(bright) populations in clinical prostate cancer specimens. EXPERIMENTAL DESIGN We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDH(bright) populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate,benign prostatic hyperplasia (BPH),and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH,prostatic intraepithelial neoplasia (PIN),and multistage prostate cancer. RESULTS ALDH(bright) cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM((+)) and Trop2((+)) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer,only ALDH1A1 gene expression significantly correlated with ALDH activity (P textless 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly,ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably,ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients. CONCLUSIONS Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance. View Publication

The cell cycle is a series of integrated and coordinated physiological events that results in cell growth and replication. Besides observing the event of cell division it is not feasible to determine the cell cycle phase without fatal and/or perturbing invasive procedures such as cell staining,fixing,and/or dissociation. Raman microspectroscopy (RMS) is a chemical imaging technique that exploits molecular vibrations as a contrast mechanism; it can be applied to single living cells noninvasively to allow unperturbed analysis over time. We used RMS to determine the cell cycle phase based on integrating the composite 783 cm(-1) nucleic acid band intensities across individual cell nuclei. After correcting for RNA contributions using the RNA 811 cm(-1) band,the measured intensities essentially reflected DNA content. When quantifying Raman images from single cells in a population of methanol-fixed human embryonic stem cells,the histogram of corrected 783 cm(-1) band intensities exhibited a profile analogous to that obtained using flow-cytometry with nuclear stains. The two population peaks in the histogram occur at Raman intensities corresponding to a 1-fold and 2-fold diploid DNA complement per cell,consistent with a distribution of cells with a population peak due to cells at the end of G1 phase (1-fold) and a peak due to cells entering M phase (2-fold). When treated with EdU to label the replicating DNA and block cell division,cells with higher EdU-related fluorescence generally had higher integrated Raman intensities. This provides proof-of-principle of an analytical method for label-free RMS determination in situ of cell cycle phase in adherent monolayers or even single adherent cells. View Publication

Developing efficacious vaccines against enteric diseases is a global challenge that requires a better understanding of cellular recruitment dynamics at the mucosal surfaces. The current paradigm of T cell homing to the gastrointestinal (GI) tract involves the induction of $$4$$7 and CCR9 by Peyer's patch and mesenteric lymph node (MLN) dendritic cells (DCs) in a retinoic acid-dependent manner. This paradigm,however,cannot be reconciled with reports of GI T cell responses after intranasal (i.n.) delivery of antigens that do not directly target the GI lymphoid tissue. To explore alternative pathways of cellular migration,we have investigated the ability of DCs from mucosal and nonmucosal tissues to recruit lymphocytes to the GI tract. Unexpectedly,we found that lung DCs,like CD103(+) MLN DCs,up-regulate the gut-homing integrin $$4$$7 in vitro and in vivo,and induce T cell migration to the GI tract in vivo. Consistent with a role for this pathway in generating mucosal immune responses,lung DC targeting by i.n. immunization induced protective immunity against enteric challenge with a highly pathogenic strain of Salmonella. The present report demonstrates novel functional evidence of mucosal cross talk mediated by DCs,which has the potential to inform the design of novel vaccines against mucosal pathogens. View Publication

Although promising new radiation therapy techniques such as hadrontherapy are currently being evaluated in the treatment of head and neck malignancies,local control of head and neck squamous cell carcinoma (HNSCC) remains low. Here,we investigated the involvement of cancer stem-like cells (CSCs) in a radioresistant HNSCC cell line (SQ20B). Stem-like cells SQ20B/SidePopulation(SP)/CD44(+)/ALDH(high) were more resistant to both photon and carbon ion irradiation compared with non-CSCs. This was confirmed by a BrdU labeling experiment,which suggests that CSCs were able to proliferate and to induce tumorigenicity after irradiation. SQ20B/SP/CD44(+)/ALDH(high) were capable of an extended G2/M arrest phase in response to photon or carbon ion irradiation compared with non-CSCs. Moreover,our data strongly suggest that resistance of CSCs may result from an imbalance between exacerbated self-renewal and proliferative capacities and the decrease in apoptotic cell death triggering. In order to modulate these processes,two targeted pharmacological strategies were tested. Firstly,UCN-01,a checkpoint kinase (Chk1) inhibitor,induced the relapse of G2/M arrest and radiosensitization of SQ20B-CSCs. Secondly,all-trans retinoic acid (ATRA) resulted in an inhibition of ALDH activity,and induction of the differentiation and radiosensitization of SQ20B/SP/CD44(+)/ALDH(high) cells. The combination of ATRA and UCN-01 treatments with irradiation drastically decreased the surviving fraction at 2Gy of SQ20B-CSCs from 0.85 to 0.38 after photon irradiation,and from 0.45 to 0.21 in response to carbon ions. Taken together,our results suggest that the combination of UCN-01 and ATRA represent a promising pharmacological-targeted strategy that significantly sensitizes CSCs to photon or carbon ion radiation. View Publication