参考文献

We report for the first time a microdevice that enables the selective enrichment,culture,and identification of tumor-initiating cells on native polydimethylsiloxane (PDMS). For nearly a decade,researchers have identified tumor-initiating breast cancer cells within heterogeneous populations of breast cancer cells by utilizing low-attachment serum-free culture conditions,which lead to the formation of spheroidal colonies (mammospheres) that are enriched for tumor-initiating cells. However,the utility of this assay has been limited by difficulties in combining this culture-plate-based technique with other cellular and molecular analyses. Integrating the mammosphere technique into a microsystem can enable it to be combined directly with a number of functions,such as cell sorting,drug screens,and molecular assays. In this work,we demonstrate mammosphere culture within a PDMS microdevice. We first prove that a native hydrophobic PDMS surface is as effective as commercial low-attachment plates at selectively promoting the formation of mammospheres. We then experimentally assess the PDMS microdevice. Time-lapse images of mammosphere formation within the microdevice show that mammospheres form from single cells or small clusters of cells. Following formation of the mammospheres,it is desirable to evaluate the cells within the spheroids for enrichment of tumor initiating cells. To perform assays such as this (which require the loading and rinsing of reagents) without flushing the cells (which are in suspension) from the device,the culture chamber is separated from a reagent reservoir by a commercially available microporous membrane,and thus reagents are exchanged between the reservoir and the culture chamber by diffusion only. Using this capability,we verify that the mammospheres are enriched for tumor initiating cells by staining aldehyde dehydrogenase activity,a cancer stem cell marker. To the best of our knowledge,this is the first assay that enables the direct observation of tumor-initiating cells within a suspended mammosphere. View Publication

In this study,we sought to establish a novel method to prospectively and dynamically identify live human oligodendrocyte precursor cells (OPCs) and oligodendrocyte lineage cells from brain dissociates and pluripotent stem cell culture. We selected a highly conserved enhancer element of the Sox10 gene,known as MCS5,which directs reporter expression to oligodendrocyte lineage cells in mouse and zebrafish. We demonstrate that lentiviral Sox10-MCS5 induced expression of GFP at high levels in a subpopulation of human CD140a/PDGF??R-sorted OPCs as well as their immature oligodendrocyte progeny. Furthermore,we show that almost all Sox10-MCS5:GFPhigh cells expressed OPC antigen CD140a and human OPCs expressing SOX10,OLIG2,and PDGFRA mRNAs could be prospectively identified using GFP based fluorescence activated cells sorting alone. Additionally,we established a human induced pluripotent cell (iPSC) line transduced with the Sox10-MCS5:GFP reporter using a Rex-Neo cassette. Similar to human primary cells,GFP expression was restricted to embryoid bodies containing both oligodendrocyte progenitor and oligodendrocyte cells and co-localized with NG2 and O4-positive cells respectively. As such,we have developed a novel reporter system that can track oligodendrocyte commitment in human cells,establishing a valuable tool to improve our understanding and efficiency of human oligodendrocyte derivation. ?? 2013 Elsevier Inc. View Publication

We report that a single growth factor,NM23-H1,enables serial passaging of both human ES and iPS cells in the absence of feeder cells,their conditioned media or bFGF in a fully defined xeno-free media on a novel defined,xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1� View Publication

Diabetes is the most common chronic disease in the world and causes complications with many diseases,such as heart disease and osteoporosis. Osteoporosis is a systemic bone disease characterized by imbalance in bone resorption and bone formation. Osteoclast is type of bone cell that functions in bone resorption and plays a critical role in bone remodeling. Rosiglitazone and pioglitazone,which belong to Thiazolidinediones(TZDs),are commonly used antidiabetic drugs. As PPARγ full agonists,they can activate PPARγ in a ligand-dependent way. Recent studies indicate that these PPARγ full agonists have some side effects,such as weight gain and bone loss,which may increase the risk of osteoporosis. In contrast,selective PPARγ Modulators (SPPARγMs) are novel PPARγ ligands that can activate PPARγ in different ways and lead to distinct downstream genes. Mice bone marrow cells were stimulated with recombinant mouse RANKL and M-CSF to generate osteoclasts. To determine the effect on osteoclasts formation,PPARγ ligands (Rosiglitazone,Fmoc-L-Leu,and Telmisartan) were added at the beginning of the culture. Rosiglitazone significantly increased the differentiation of multinucleated osteoclasts,while osteoclasts formation triggered by SPPARγMs was much less than that displayed by rosiglitazone. We found that the enhancement of PPARγ ligands may be associated with TRAF6 and downstream ERK signal pathway. We also demonstrated osteoclasts show characteristic M2 phenotype and can be further promoted by PPARγ ligands,especially rosiglitazone. In conclusion,reduced osteoclasts differentiation characteristic of SPPARγMs highlights SPPARγMs potential as therapeutic targets in diabetes,versus traditional antidiabetic drugs. View Publication

Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development,but the soundness of this model has been challenged by others,who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7+ within 48 h,with minimal expression of mesoderm markers,including T (Brachyury). Instead,they begin to express a series of trophoblast markers,including HLA-G,demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium,and,over time,produce extensive amounts of human chorionic gonadotropin,progesterone,placental growth factor,and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling,which at day 2 provide colonies that are entirely KRT7+ and in which the majority of cells are transiently CDX2+. Colonies grown on two chemically defined media,including the one in which BMP4 was reported to drive mesoderm formation,also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts. View Publication

Transforming growth factor beta (TGF-$$) signaling has been implicated in driving tumor progression and metastasis by inducing stem cell-like features in some human cancer cell lines. In this study,we have utilized a novel murine cell line NMuMG-ST,which acquired cancer stem cell (CSC) phenotypes during spontaneous transformation of the untransformed murine mammary cell line NMuMG,to investigate the role of autocrine TGF-$$ signaling in regulating their survival,metastatic ability,and the maintenance of cancer stem cell characteristics. We have retrovirally transduced a dominant-negative TGF-$$ type II receptor (DNRII) into the NMuMG-ST cell to abrogate autocrine TGF-$$ signaling. The expression of DNRII reduced TGF-$$ sensitivity of the NMuMG-ST cells in various cell-based assays. The blockade of autocrine TGF-$$ signaling reduced the ability of the cell to grow anchorage-independently and to resist serum deprivation-induced apoptosis. These phenotypes were associated with reduced levels of active and phosphorylated AKT and ERK,and Gli1 expression suggesting that these pathways contribute to the growth and survival of this model system. More interestingly,the abrogation of autocrine TGF-$$ signaling also led to the attenuation of several features associated with mammary stem cells including epithelial-mesenchymal transition,mammosphere formation,and expression of stem cell markers. When xenografted in athymic nude mice,the DNRII cells were also found to undergo apoptosis and induced significantly lower lung metastasis burden than the control cells even though they formed similar size of xenograft tumors. Thus,our results indicate that autocrine TGF-$$ signaling is involved in the maintenance and survival of stem-like cell population resulting in the enhanced metastatic ability of the murine breast cancer cells. View Publication

A fundamental impediment to functional recovery from spinal cord injury (SCI) and traumatic brain injury is the lack of sufficient axonal regeneration in the adult central nervous system. There is thus a need to develop agents that can stimulate axon growth to re-establish severed connections. Given the critical role played by protein kinases in regulating axon growth and the potential for pharmacological intervention,small molecule protein kinase inhibitors present a promising therapeutic strategy. Here,we report a robust cell-based phenotypic assay,utilizing primary rat hippocampal neurons,for identifying small molecule kinase inhibitors that promote neurite growth. The assay is highly reliable and suitable for medium-throughput screening,as indicated by its Z'-factor of 0.73. A focused structurally diverse library of protein kinase inhibitors was screened,revealing several compound groups with the ability to strongly and consistently promote neurite growth. The best performing bioassay hit robustly and consistently promoted axon growth in a postnatal cortical slice culture assay. This study can serve as a jumping-off point for structure activity relationship (SAR) and other drug discovery approaches toward the development of drugs for treating SCI and related neurological pathologies. View Publication

Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible,stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover,the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures,thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction,protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format,a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally,we demonstrate the utility of the system through the large-scale production (140-750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents. View Publication

The incidence of human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) has rapidly increased over the past 30 years,prompting the suggestion that an epidemic maybe on the horizon. Therefore,there is a clinical need to develop alternate therapeutic strategies to manage the growing number of HPV-positive HNSCC patients. High-risk HPV E6 inactivates p53 through two distinct mechanisms; association with E6AP to degrade p53 and association with p300 to block p300-mediated p53 acetylation and activation. In this study,we determined if targeting the E6-p300 interaction is an effective approach to reactivate p53 in HPV-positive HNSCC. Ectopic expression of the CH1 domain of p300 in HPV-positive HNSCC blocks the association between E6 and p300,increases total and acetylated p53 levels and enhances p53 transcriptional activity. Moreover,expression of p21,miR-34a and miR-200c are increased,demonstrating functional p53 reactivation. CH1 overexpression in HPV-positive HNSCC has a global anticancer effect resulting in a decrease in cell proliferation and clonogenic survival and an increase in apoptosis. The in vivo tumor-initiating ability of HPV-positive HNSCC is severely compromised with CH1 overexpression,in part through a reduction in the cancer-initiating cell population. A novel small-molecule CH1 inhibitor,CH1iB,reactivates p53 and potentiates the anticancer activity of cis-platinum in HPV-positive HNSCC cells. Our work shows that CH1-domain inhibitors represent a novel class of p53-reactivation therapeutics for managing HPV-positive HNSCC patients. View Publication

Tumor-initiating cells (TICs) are a sub-population of cells that exhibit a robust ability to self-renew and contribute to the formation of primary tumors,the relapse of previously treated tumors and the development of metastases. TICs have been identified in various tumors including those of the breast,and are particularly enriched in the basal-like and claudin-low subtypes of breast cancer. The signaling pathways that contribute to the function and maintenance of TICs are under intense study. We explored the potential involvement of the nuclear factor-$$B (NF-$$B) family of transcription factors in TICs in cell lines that are representative of basal-like and claudin-low breast cancer. NF-$$B was found to be activated in breast cancer cells that form tumorspheres efficiently. Moreover,both canonical and non-canonical NF-$$B signaling is required for these cells to self-renew in vitro and to form xenograft tumors efficiently in vivo using limiting dilutions of cells. Consistent with this fact,canonical and non-canonical NF-$$B signaling is activated in TICs isolated from breast cancer cell lines. Experimental results indicate that NF-$$B promotes the function of TICs by stimulating epithelial-to-mesenchymal transition and by upregulating the expression of the inflammatory cytokines interleukin-1$$ and interleukin-6. The results suggest the use of NF-$$B inhibitors for clinical therapy of certain breast cancers. View Publication

Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however,their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC),human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC),and compared their in-vitro tube formation,migration and cytokine expression profiles,and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless,BM-EC,hESC-EC and hiPSC-EC exhibited typical cobblestone morphology,had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein,and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (Ptextgreater0.05). While increased expression of major angiogenic factors including epidermal growth factor,hepatocyte growth factor,vascular endothelial growth factor,placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (Ptextless0.05),the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (Ptextless0.05). Compared with medium,transplanting BM-EC (n = 6),HUVEC (n = 6),hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion,functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases,and hESC-EC or iPSC-EC are readily available as off-the-shelf" format for the treatment of tissue ischemia." View Publication

Faithful execution of developmental gene expression programs occurs at multiple levels and involves many different components such as transcription factors,histone-modification enzymes,and mRNA processing proteins. Recent evidence suggests that nucleoporins,well known components that control nucleo-cytoplasmic trafficking,have wide-ranging functions in developmental gene regulation that potentially extend beyond their role in nuclear transport. Whether the unexpected role of nuclear pore proteins in transcription regulation,which initially has been described in fungi and flies,also applies to human cells is unknown. Here we show at a genome-wide level that the nuclear pore protein NUP98 associates with developmentally regulated genes active during human embryonic stem cell differentiation. Overexpression of a dominant negative fragment of NUP98 levels decreases expression levels of NUP98-bound genes. In addition,we identify two modes of developmental gene regulation by NUP98 that are differentiated by the spatial localization of NUP98 target genes. Genes in the initial stage of developmental induction can associate with NUP98 that is embedded in the nuclear pores at the nuclear periphery. Alternatively,genes that are highly induced can interact with NUP98 in the nuclear interior,away from the nuclear pores. This work demonstrates for the first time that NUP98 dynamically associates with the human genome during differentiation,revealing a role of a nuclear pore protein in regulating developmental gene expression programs. View Publication