技术资料

BACKGROUND: Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo,where activity of inputs can be differentially regulated,but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here,we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. METHODOLOGY/PRINCIPAL FINDINGS: GluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons,both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures,synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures. CONCLUSIONS/SIGNIFICANCE: The enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde 'reward' signal generated by WT neurons,although in this paradigm there was no 'punishment' signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous system. View Publication

Monocytes have long been considered a heterogeneous group of cells both in terms of morphology and function. In humans,three distinct subsets have been described based on their differential expression of the cell surface markers CD14 and CD16. However,the relationship between these subsets and the production of cytokines has for the most part been based on ELISA measurements,making it difficult to draw conclusions as to their functional profile on the cellular level. In the present study,we have investigated lipoteichoic acid (LTA) and lipopolysaccharide (LPS) induced cytokine secretion by monocytes using the FluoroSpot technique. This method measures the number of cytokine secreting cells on the single cell level and uses fluorescent detection,allowing for the simultaneous analysis of two cytokines from the same population of isolated cells. By this approach,human monocytes from healthy volunteers could be divided into several subgroups as IL-1β,IL-6,TNF-α and MIP-1β were secreted by larger populations of responding cells (25.9-39.2%) compared to the smaller populations of GM-CSF (9.1%),IL-10 (1.3%) and IL-12p40 (1.2%). Furthermore,when studying co-secretion in FluoroSpot,an intricate relationship between the monocytes secreting IL-1β and/or IL-6 and those secreting TNF-α,MIP-1β,GM-CSF,IL-10 and IL-12p40 was revealed. In this way,dissecting the secretion pattern of the monocytes in response to TLR-2 or TLR-4 stimulation,several subpopulations with distinct cytokine secreting profiles could be identified. View Publication

Avian species are important model animals for developmental biology and disease research. However,unlike in mice,where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function,clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs),the first nonmammalian iPSCs,which were clonally isolated and propagated,important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes,indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes,oligodendrocytes,and neurons. Ultimately,qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers,extraembryonic tissues,and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals. View Publication

Metabolic conversion of vitamin A (retinol) into retinoic acid (RA) controls numerous physiological processes. 9-cis-retinoic acid (9cRA),an active metabolite of vitamin A,is a high affinity ligand for retinoid X receptor (RXR) and also activates retinoic acid receptor (RAR). Despite the identification of candidate enzymes that produce 9cRA and the importance of RXRs as established by knockout experiments,in vivo detection of 9cRA in tissue was elusive until recently when 9cRA was identified as an endogenous pancreas retinoid by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. This review will discuss the current status of the analysis,occurrence,and function of 9cRA. Understanding both the nuclear receptor-mediated and non-genomic mechanisms of 9cRA will aid in the elucidation of disease physiology and possibly lead to the development of new retinoid-based therapeutics. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism. View Publication

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expressing four transcription factors: Oct4,Sox2,Klf4,and c-Myc. Here we report that enhancing RA signaling by expressing RA receptors (RARs) or by RA agonists profoundly promoted reprogramming,but inhibiting it using a RAR-α dominant-negative form completely blocked it. Coexpressing Rarg (RAR-γ) and Lrh-1 (liver receptor homologue 1; Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells to ground-state iPSCs requires only 4 d induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous factor-independent iPSCs,which resembled ground-state mouse ES cells in growth properties,gene expression,and signaling dependency. Our findings demonstrate that signaling through RARs has critical roles in molecular reprogramming and that the synergistic interaction between Rarg and Lrh1 directs reprogramming toward ground-state pluripotency. The human iPSCs described here should facilitate functional analysis of the human genome. View Publication

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation,purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages. View Publication

Breast cancer exhibits a propensity to metastasize to bone,resulting in debilitating skeletal complications associated with significant morbidity and poor prognosis. The cross-talk between metastatic cancer cells and bone is critical to the development and progression of bone metastases. We have shown the involvement of the HGF/c-MET system in tumor-bone interaction contributing to human breast cancer metastasis. Therefore,disruption of HGF/c-MET signaling is a potential targeted approach to treating metastatic bone disease. In this study,we evaluated the effects of c-MET inhibition by both an oral,selective,small-molecule c-MET inhibitor,tivantinib,and a specific short hairpin RNA (shRNA) against c-MET in a mouse model of human breast cancer. Tivantinib exhibited dose-dependent antimetastatic activity in vivo,and the 120 mg/kg dose,proven to be suboptimal in reducing subcutaneous tumor growth,induced significant inhibition of metastatic growth of breast cancer cells in bone and a noteworthy reduction of tumor-induced osteolysis. shRNA-mediated c-MET silencing did not affect in vitro proliferation of bone metastatic cells,but significantly reduced their migration,and this effect was further enhanced by tivantinib. Both observations were confirmed in vivo. Indeed,more pronounced tumor growth suppression with concomitant marked decreases of lytic lesions and prolongation of survival were achieved by dual c-MET inhibition using both tivantinib and RNA interference strategies. Overall,our findings highlighted the effectiveness of c-MET inhibition in delaying the onset and progression of bone metastases and strongly suggest that targeting c-MET may have promising therapeutic value in the treatment of bone metastases from breast cancer. View Publication

The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here,we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells,as predicted from a numerical model of cell migration,and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further,reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications. View Publication

Partial cDNA and genomic clones of rat stem cell factor (SCF) have been isolated. Using probes based on the rat sequence,partial and full-length cDNA and genomic clones of human SCF have been isolated. Based on the primary structure of the 164 amino acid protein purified from BRL-3A cells,truncated forms of the rat and human proteins have been expressed in E. coli and mammalian cells and have been shown to possess biological activity. SCF is able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures. SCF exhibits potent synergistic activities in conjunction with colony-stimulating factors,resulting in increased colony numbers and colony size. View Publication

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids,pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction,the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step,3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors,and they proliferate and expand over 1-3 months to give rise to intestinal tissue,complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date,this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro. View Publication

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) are a potentially unlimited source of cardiomyocytes (CMs) for cardiac transplantation therapies. The establishment of pure PSC-CM populations is important for this application,but is hampered by a lack of CM-specific surface markers suitable for their identification and sorting. Contemporary purification techniques are either non-specific or require genetic modification. We report a second harmonic generation (SHG) signal detectable in PSC-CMs that is attributable to sarcomeric myosin,dependent on PSC-CM maturity,and retained while PSC-CMs are in suspension. Our study demonstrates the feasibility of developing a SHG-activated flow cytometer for the non-invasive purification of PSC-CMs. View Publication

INTRODUCTION: The induced pluripotent stem cell (iPSC) technology allows generation of patient-specific pluripotent stem cells,thereby providing a novel cell-therapy platform for severe degenerative diseases. One of the key issues for clinical-grade iPSC derivation is the accessibility of donor cells used for reprogramming. METHODS: We examined the feasibility of reprogramming mobilized GMP-grade hematopoietic progenitor cells (HPCs) and peripheral blood mononuclear cells (PBMCs) and tested the pluripotency of derived iPS clones. RESULTS: Ectopic expression of OCT4,SOX2,KLF4,and c-MYC in HPCs and PBMCs resulted in rapid iPSC derivation. Long-term time-lapse imaging revealed efficient iPSC growth under serum- and feeder-free conditions with frequent mitotic events. HPC- and PBMC-derived iPS cells expressed pluripotency-associated markers,including SSEA-4,TRA-1-60,and NANOG. The global gene-expression profiles demonstrated the induction of endogenous pluripotent genes,such as LIN28,TERT,DPPA4,and PODXL,in derived iPSCs. iPSC clones from blood and other cell sources showed similar ultrastructural morphologies and genome-wide gene-expression profiles. On spontaneous and guided differentiation,HPC- and PBMC-derived iPSCs were differentiated into cells of three germ layers,including insulin-producing cells through endodermal lineage,verifying the pluripotency of the blood-derived iPSC clones. CONCLUSIONS: Because the use of blood cells allows minimally invasive tissue procurement under GMP conditions and rapid cellular reprogramming,mobilized HPCs and unmobilized PBMCs would be ideal somatic cell sources for clinical-grade iPSC derivation,especially from diabetes patients complicated by slow-healing wounds. View Publication