技术资料

Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background,yet the landscapes of in vivo tissues remain largely uncharted. Here,we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages,lineages,and cellular environments. They also reveal global features of the epigenome,related to nuclear architecture,that also vary across cellular phenotypes. Specifically,developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture,and discuss their implications for lineage fidelity,cellular senescence,and reprogramming. ?? 2013 Elsevier Inc. View Publication

Induced pluripotent stem cells (iPSCs) hold great clinical potential for regenerative medicine. Much work has been done to investigate the mechanisms of their generation,focusing on the cell nucleus. However,the roles of specific organelles and in particular mitochondria in the potential mechanisms of nuclear reprogramming remain unclear. In this study,we sought to determine the role of mitochondrial metabolism transition in nuclear reprogramming. We found that the mitochondrial cristae had remodeled in iPSCs. The efficiency of iPSC generation was significantly reduced by down-regulation of mitochondrial inner membrane protein (IMMT),which regulates the morphology of mitochondrial cristae. Moreover,cells with the oxidative phosphorylation (OXPHOS) advantage had higher reprogramming efficiency than normal cells and the glycolysis intermediate lactic acid enhanced the efficiency of iPSCs generation. Our results show that the remodeling of mitochondrial cristae couples with the generation of iPSCs,suggesting mitochondrial metabolism transition plays an important role in nuclear reprogramming. View Publication

In this study,we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor $$-positive (ER$$(+)) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ER$$,HER-2/Neu overexpression and loss of CD24 surface receptor (CD24(-/low)). Importantly,expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover,aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5,indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT,stemness and ultimately tumor progression. Importantly,pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24(+) epithelial phenotype that was coupled to ER$$ expression,downregulation of HER-2/Neu,inhibition of EMT and impaired self-renewal ability,resulting in the suppression of distant metastases. Taken together,our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ER$$(+) breast cancer cells. Moreover,this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ER$$(+) breast cancer patients resistant to conventional endocrine therapy. View Publication

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations,including their reliance on exogenous materials or electrical fields,which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material,such as carbon nanotubes,proteins,and siRNA,to 11 cell types,including embryonic stem cells and immune cells. When used for the delivery of transcription factors,the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed,its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications. View Publication

Mesenchymal progenitors or stromal cells have shown promise as a therapeutic strategy for a range of diseases including heart failure. In this context,we explored the growth and differentiation potential of mesenchymal progenitors (MPs) derived in vitro from human embryonic stem cells (hESCs). Similar to MPs isolated from bone marrow,hESC derived MPs (hESC-MPs) efficiently differentiated into archetypical mesenchymal derivatives such as chondrocytes and adipocytes. Upon treatment with 5-Azacytidine or TGF-β1,hESC-MPs modified their morphology and up-regulated expression of key cardiac transcription factors such as NKX2-5,MEF2C,HAND2 and MYOCD. Nevertheless,NKX2-5+ hESC-MP derivatives did not form contractile cardiomyocytes,raising questions concerning the suitability of these cells as a platform for cardiomyocyte replacement therapy. Gene profiling experiments revealed that,although hESC-MP derived cells expressed a suite of cardiac related genes,they lacked the complete repertoire of genes associated with bona fide cardiomyocytes. Our results suggest that whilst agents such as TGF-β1 and 5-Azacytidine can induce expression of cardiac related genes,but treated cells retain a mesenchymal like phenotype. View Publication

Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors,but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue,we blocked Notch activity in MCF-7 cells by expressing a dominant-negative MAML-GFP (dnMAML) construct,which inhibits signaling through all Notch receptors,and quantified the effect on tumor-initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor-initiating cell numbers in vivo showed a significant decrease,but not a complete abrogation,of these cells in dnMAML-expressing cells. Interestingly,when assessed in secondary assays in vitro or in vivo,there was no difference in tumor-initiating activity between the dnMAML-expressing cells and control cells. The fact that a subpopulation of dnMAML-expressing cells was capable of forming primary and secondary tumors indicates that there are Notch-independent tumor-initiating cells in the breast cancer cell line MCF-7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool,which will require combination therapies against multiple oncogenic pathways to eliminate the tumor-initiating cell population. View Publication

BACKGROUND: The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them,fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However,downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study,we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2,an inhibitor of protein kinase C (PKC),GF109203X (GFX),increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β),suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator,phorbol 12-myristate 13-acetate whereas Gö6976,a selective inhibitor of PKCα,β,and γ isoforms could not counteract the effect of PMA. Intriguingly,functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ,PKCε,and ζ,the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ,and the phosphorylation of AKT was reduced by PKCε in hPS cells. CONCLUSIONS/SIGNIFICANCE: Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT,mitogen-activated protein kinase/ERK-1/2 kinase (MEK),PKC/ERK-1/2 kinase,and PKC/GSK-3β. Addition of GFX with a MEK inhibitor,U0126,in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells. View Publication

The antitumor activity of LPS was first described by Dr. William Coley. However,its role in lung cancer remains unclear. The aim of our study was to elucidate the dose-dependent effects of LPS (0.1-10 μg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth increased at 3 and 7 d after the administration of low-dose LPS (0.1 μg/mouse) compared with control mice. This was associated with an influx of plasmacytoid dendritic cells (pDCs),regulatory T cells,myeloid-derived suppressor cells,and CD8(+) regulatory T cells. In contrast,high-dose LPS (10 μg/mouse) reduced lung tumor burden and was associated with a greater influx of pDCs,as well as a stronger Th1 and Th17 polarization. Depletion of pDCs during low-dose LPS administration resulted in a decreased lung tumor burden. Depletion of pDCs during high-dose LPS treatment resulted in an increased tumor burden. The dichotomy in LPS effects was due to the phenotype of pDCs,which were immunosuppressive after the low-dose LPS,and Th1- and T cytotoxic-polarizing cells after the high-dose LPS. Adoptive transfer of T cells into nude mice demonstrated that CD8(+) T cells were responsible for pDC recruitment following low-dose LPS administration,whereas CD4(+) T cells were required for pDC influx after the high-dose LPS. In conclusion,our data suggest differential effects of low-dose versus high-dose LPS on pDC phenotype and tumor progression or regression in the lungs of mice. View Publication

A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development,a basement membrane surrounds the neural plate that forms a tight,apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium,in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia. View Publication

The ST6Gal-I sialyltransferase adds an $$2-6-linked sialic acid to the N-glycans of certain receptors. ST6Gal-I mRNA has been reported to be upregulated in human cancer,but a prior lack of antibodies has limited immunochemical analysis of the ST6Gal-I protein. Here,we show upregulated ST6Gal-I protein in several epithelial cancers,including many colon carcinomas. In normal colon,ST6Gal-I localized selectively to the base of crypts,where stem/progenitor cells are found,and the tissue staining patterns were similar to the established stem cell marker ALDH1. Similarly,ST6Gal-I expression was restricted to basal epidermal layers in skin,another stem/progenitor cell compartment. ST6Gal-I was highly expressed in induced pluripotent stem (iPS) cells,with no detectable expression in the fibroblasts from which iPS cells were derived. On the basis of these observations,we investigated further an association of ST6Gal-I with cancer stem cells (CSC). Selection of irinotecan resistance in colon carcinoma cells led to a greater proportion of CSCs compared with parental cells,as measured by the CSC markers CD133 and ALDH1 activity (Aldefluor). These chemoresistant cells exhibited a corresponding upregulation of ST6Gal-I expression. Conversely,short hairpin RNA (shRNA)-mediated attenuation of ST6Gal-I in colon carcinoma cells with elevated endogenous expression decreased the number of CD133/ALDH1-positive cells present in the cell population. Collectively,our results suggest that ST6Gal-I promotes tumorigenesis and may serve as a regulator of the stem cell phenotype in both normal and cancer cell populations. View Publication

Magnesium alloys have attracted great interest for medical applications due to their unique biodegradable capability and desirable mechanical properties. When designed for medical applications,these alloys must have suitable degradation properties,i.e.,their degradation rate should not exceed the rate at which the degradation products can be excreted from the body. Cellular responses and tissue integration around the Mg-based implants are critical for clinical success. Four magnesium–zinc–strontium (ZSr41) alloys were developed in this study. The degradation properties of the ZSr41 alloys and their cytocompatibility were studied using an in vitro human embryonic stem cell (hESC) model due to the greater sensitivity of hESCs to known toxicants which allows to potentially detect toxicological effects of new biomaterials at an early stage. Four distinct ZSr41 alloys with 4 wt% zinc and a series of strontium compositions (0.15,0.5,1,and 1.5 wt% Sr) were produced through metallurgical processing. Their degradation was characterized by measuring total mass loss of samples and pH change in the cell culture media. The concentration of Mg ions released from ZSr41 alloy into the cell culture media was analyzed using inductively coupled plasma atomic emission spectroscopy. Surface microstructure and composition before and after culturing with hESCs were characterized using field emission scanning electron microscopy and energy dispersive X-ray spectroscopy. Pure Mg was used as a control during cell culture studies. Results indicated that the Mg–Zn–Sr alloy with 0.15 wt% Sr provided slower degradation and improved cytocompatibility as compared with pure Mg control. View Publication

All trans-retinoic acid (ATRA) is widely used to direct the differentiation of cultured stem cells. When exposed to the pluripotent human embryonal carcinoma (EC) stem cell line,TERA2.cl.SP12,ATRA induces ectoderm differentiation and the formation of neuronal cell types. We have previously generated synthetic analogues of retinoic acid (EC23 and EC19) which also induce the differentiation of EC cells. Even though EC23 and EC19 have similar chemical structures,they have differing biochemical effects in terms of EC cell differentiation. EC23 induces neuronal differentiation in a manner similar to ATRA,whereas EC19 directs the cells to form epithelial-like derivatives. Previous MALDI-TOF MS analysis examined the response of TERA2.cl.SP12 cells after exposure to ATRA,EC23 and EC19 and further demonstrated the similarly in the effect of ATRA and EC23 activity whilst responses to EC19 were very different. In this study,we show that Fourier Transform Infrared Micro-Spectroscopy (FT-IRMS) coupled with appropriate scatter correction and multivariate analysis can be used as an effective tool to further investigate the differentiation of human pluripotent stem cells and monitor the alternative affects different retinoid compounds have on the induction of differentiation. FT-IRMS detected differences between cell populations as early as 3 days of compound treatment. Populations of cells treated with different retinoid compounds could easily be distinguished from one another during the early stages of cell differentiation. These data demonstrate that FT-IRMS technology can be used as a sensitive screening technique to monitor the status of the stem cell phenotype and progression of differentiation along alternative pathways in response to different compounds. View Publication