参考文献

Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study,we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures,single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells,corresponding to an increased expression of pluripotency markers OCT4 and NANOG,and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed,aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols,with induction of primitive streak cells using bone morphogenetic protein 4 and activin A,followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5,thus indicating the production of large numbers of immature cardiomyocytes (˜65,000/cm(2) or ˜1.5 per input hESC). This protocol was shown to be effective in HES3,H9,and,to a lesser,extent,MEL1 hESC lines. In addition,we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression,whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation,and potentially for the future treatment of heart failure. View Publication

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation,a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation,as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data,possibly due to their crosstalk with Notch Signaling. Overall,this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs. View Publication

Activation of P2X(7) receptor (P2X(7)R) and pannexin have been implicated in membrane permeabilization associated with ischemic cell death and many other inflammatory processes. P2X(7)R has a unique property of forming large pore upon repeated or prolonged application of agonist like ATP or 2',3'-(4-benzoyl) benzoyl ATP. It has been proposed that pannexin 1 (panx1) hemichannel associates with P2X(7)R to form large pore,though the actual mechanism is not yet understood. Calcium concentration in extracellular milieu drops in many patho-physiological conditions,e.g. ischemia,when P2X(7)R/pannexin is also known to be activated. Therefore,we hypothesize that extracellular calcium ([Ca(2+)](o)) plays an important role in the coupling of P2X(7)R-panx1 and subsequent membrane permeabilization. In this study we show that membrane permeability of the P2X(7)R and panx1 expressing N2A cell increases in ([Ca(2+)](o))-free solution. In [Ca(2+)](o)-free solution,fluorescent dye calcein trapped cells exhibited time-dependent dye leakage resulting in about 50% decrease of fluorescence intensity in 30 min. Control cells in 2 mM [Ca(2+)](o) did not show such leakage. Like N2A cells,mixed culture of neuron and glia,derived from hippocampal progenitor cells showed similar dye leakage. Dye leakage was blocked either by pannexin-specific blocker,carbenoxolone or P2X(7)R antagonists,Brilliant Blue G,and oxidized ATP. Furthermore P2X(7)R and panx1 were co-immunoprecipitated. The amount of P2X(7)R protein pulled-down with panx1,increased by twofold when cells were incubated 30 min in [Ca(2+)](o)-free buffer. Taken together,the results of this study demonstrate the activation and association of P2X(7)R-panx1,triggered by the removal of [Ca(2+)](o). View Publication

Histone deacetylases (HDAC) are key enzymes in the epigenetic control of gene expression. Recently,inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis,colitis,airway inflammation and asthma. So far,little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins,the class III HDAC. In this study,we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC),a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol,a specific sirtuin inhibitor,in HDMEC response to pro-inflammatory cytokines. We found that,even though sirtinol treatment alone affected only long-term cell proliferation,it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNF)α and interleukin (IL)-1β. In fact,sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1β-stimulated cells,as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably,sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2,we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally,we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether,these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement. View Publication

BACKGROUND: Human cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However,methods suitable for clinical practice have yet to be fully established. METHODOLOGY/PRINCIPAL FINDINGS: In this study,we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo,and identified Garcinol,a plant-derived histone acetyltransferase (HAT) inhibitor,as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34(+)CD38(-) HSCs supplemented with stem cell factor and thrombopoietin,Garcinol increased numbers of CD34(+)CD38(-) HSCs/PCs more than 4.5-fold and Isogarcinol,a derivative of Garcinol,7.4-fold. Furthermore,during a 7-day culture of CD34(+) HSCs/PCs,Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones,while inactive derivatives did not. CONCLUSIONS/SIGNIFICANCE: Our findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs. View Publication

Measuring spatial and temporal patterns of cytochemical variation in human embryonic stem cell (hESC) colonies is necessary for understanding the role of cellular communication in spontaneous differentiation,the mechanisms of biological niche creation,and structure-generating developmental processes. Such insights will ultimately facilitate directed differentiation and therewith promote advances in tissue engineering and regenerative medicine. However,the patterns of cytochemical inhomogeneities of hESC colonies are not well studied and their causes are not fully understood. We used Raman spectroscopic mapping to contrast supracellular variations in cytochemical composition across pluripotent and partly differentiated hESC colonies to gain a better understanding of the early-stage (i.e.,5 days) effects of the differentiation process on the nature and evolution of these patterns. Higher protein-to-nucleic acid ratios,a differentiation status indicator observed previously using Raman spectroscopy,confirmed reported results that spontaneous differentiation is more pronounced on the edges of a colony than elsewhere. In addition,pluripotent and partly differentiated colonies also showed higher lipid concentrations relative to nucleic acids at colony edges in contrast to relative glycogen concentrations,which were up to 400% more pronounced in the colony centers compared to their edges. Pluripotent and partly differentiated colonies differed,with the latter having higher average protein-to-nucleic acid and lipid-to-nucleic acid ratios but a lower glycogen-to-nucleic acid ratio. In both cases,cell density,pluripotency,and high glycogen appeared to vary in tandem. Spatial variations in glycogen- and protein-to-nucleic acid ratios have features on the order of 100 μm and larger. These dimensions are consistent with those reported for stem cell niches and suggest that cytochemical inhomogeneities may provide colony-level information about niches and niche formation. These results demonstrate Raman mapping to be a potentially useful technique for revealing the complexities in the spatial organization of hESC cultures and thus for monitoring the evolution of engineered hESC niches. View Publication

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism,pantothenate and CoA biosynthesis,glutathione metabolism,and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information,including Stemina Biomarker Discovery's predictive DevTox® model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity. View Publication

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper,we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells,we develop an image processing methodology with a recognition capability of multiple features,e.g.,cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern,in which single as well as multiple laser traps are employed for cell transportation,and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach. View Publication

Human pluripotent stem cells hold promising potential in many therapeutics applications including regenerative medicine and drug discovery. Over the past three decades,embryonic stem cell research has illustrated that embryonic stem cells possess two important and distinct properties: the ability to continuously self-renew and the ability to differentiate into all specialized cell types. In this article,we will discuss the continuing evolution of human pluripotent stem cell culture by examining requirements needed for the maintenance of self-renewal in vitro. We will also elaborate on the future direction of the field toward generating a robust and completely defined culture system,which has brought forth collaborations amongst biologists and engineers. As human pluripotent stem cell research progresses towards identifying solutions for debilitating diseases,it will be critical to establish a defined,reproducible and scalable culture system to meet the requirements of these clinical applications. View Publication

This unit describes the generation of tetracycline-inducible short hairpin RNA interference (shRNAi) human embryonic stem cell (hESC) lines. Using this vector-based approach enables stable and long-term expression of target hairpins under the control of doxycycline/tetracycline. Target degradation can be controlled in both a dose- and time-dependent manner that can even be switched off,depending upon the particular requirements of the study. View Publication

The midbody is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. Midbodies persist in cells long after division as midbody derivatives (MB(d)s),but their fate is unclear. Here we show that MB(d)s are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells,induced pluripotent stem cells and potential cancer 'stem cells' in vivo and in vitro. MB(d) loss accompanies stem-cell differentiation,and involves autophagic degradation mediated by binding of the autophagic receptor NBR1 to the midbody protein CEP55. Differentiating cells and normal dividing cells do not accumulate MB(d)s and possess high autophagic activity. Stem cells and cancer cells accumulate MB(d)s by evading autophagosome encapsulation and exhibit low autophagic activity. MB(d) enrichment enhances reprogramming to induced pluripotent stem cells and increases the in vitro tumorigenicity of cancer cells. These results indicate unexpected roles for MB(d)s in stem cells and cancer 'stem cells'. View Publication

ETV6-RUNX1 gene fusion is usually an early,prenatal event in childhood acute lymphoblastic leukemia (ALL). Transformation results in the generation of a persistent (> 14 years) preleukemic clone,which postnatally converts to ALL after the acquisition of necessary secondary genetic alterations. Many cancer cells show some expression of the erythropoietin receptor (EPOR) gene,although the functionality" of any EPOR complexes and their relevant signaling pathways in nonerythroid cells has not been validated. EPOR mRNA is selectively and ectopically expressed in ETV6-RUNX1(+) ALL but the presence of a functional EPOR on the cell surface and its role in leukemogenesis driven by ETV6-RUNX1 remains to be identified. Here we show that ETV6-RUNX1 directly binds the EPOR promoter and that expression of ETV6-RUNX1 alone in normal pre-B cells is sufficient to activate EPOR transcription. We further reveal that murine and human ETV6-RUNX1(+) cells expressing EPOR mRNA have EPO ligand binding activity that correlates with an increased cell survival through activation of the JAK2-STAT5 pathway and up-regulation of antiapoptotic BCL-XL. These data support the contention that ETV6-RUNX1 directly activates ectopic expression of a functional EPOR and provides cell survival signals that may contribute critically to persistence of covert premalignant clones in children. View Publication