技术资料

Human embryonic stem cells (hESCs) are emerging as an attractive alternative source for cell replacement therapy since the cells can be expanded in culture indefinitely and differentiated into any cell types in the body. In order to optimize cell-to-cell interaction,cell proliferation and differentiation into specific lineages as well as tissue organization,it is important to provide a microenvironment for the hESCs which mimics the stem cell niche. One approach is to provide a three-dimensional (3D) environment such as encapsulation. We present an approach to culture and differentiate hESCs into midbrain dopamine (mdDA) neurons in a 3D microenvironment using alginate microcapsules for the first time. A detailed gene and protein expression analysis during neuronal differentiation showed an increased gene and protein expression of various specific DA neuronal markers,particularly tyrosine hydroxylase (TH) by textgreater100 folds after 2weeks and at least 50% higher expression after 4weeks respectively,compared to cells differentiated under conventional two-dimensional (2D) platform. The encapsulated TH+ cells co-expressed mdDA neuronal markers,forkhead box protein A-2 (FOXA2) and pituitary homeobox-3 (PITX3) after 4weeks and secreted approximately 60pg/ml/106 cells higher DA level when induced. We propose that the 3D platform facilitated an early onset of DA neuronal generation compared to that with conventional 2D system which also secretes more DA under potassium-induction. It is a very useful model to study the proliferation and directed differentiation of hESCs to various lineages,particularly to mdDA neurons. This 3D system also allows the separation of feeder cells from hESCs during the process of differentiation and also has potential for immune-isolation during transplantation studies. ?? 2013 Elsevier B.V. View Publication

Transposon gene delivery systems offer an alternative,non-viral-based approach to generate induced pluripotent stem cells (iPSCs). Here we used the Sleeping Beauty (SB) transposon to generate four human iPSC lines from foetal fibroblasts. In contrast to other gene delivery systems,the SB transposon does not exhibit an integration bias towards particular genetic elements,thereby reducing the risk of insertional mutagenesis. Furthermore,unlike the alternative transposon piggyBac,SB has no SB-like elements within the human genome,minimising the possibility of mobilising endogenous transposon elements. All iPSC lines exhibited the expected characteristics of pluripotent human cells,including the ability to differentiate to derivatives of all three germ layers in vitro. Re-expression of the SB transposase in the iPSCs after reprogramming resulted in the mobilisation of some of the transposons. These results indicate that the SB transposon system is a useful addition to methods for generating human iPSCs,both for basic and applied biomedical research,and in the context of future therapeutic application. textcopyright 2013 International Society of Differentiation. View Publication

Patients with triple-negative breast cancer display the highest rates of early relapse of all patients with breast cancer. The basal-like subtype,a subgroup of triple-negative breast cancer,exhibits high levels of constitutively active NF-κB signalling. Here we show that NF-κB activation,induced by inflammatory cytokines or by epigenetically dysregulated NIK expression,cell-autonomously upregulates JAG1 expression in non-cancer stem cells. This upregulation stimulates NOTCH signalling in cancer stem cells in trans,leading to an expansion of cancer stem cell populations. Among breast cancers,the NF-κB-dependent induction of JAG1 and the NOTCH-dependent expansion of the cancer stem cell population occur only in the basal-like subtype. Collectively,our results indicate that NF-κB has a non-cell-autonomous role in regulating cancer stem cell populations by forming intratumoural microenvironments composed of JAG1-expressing non-cancer stem cells with a basal-like subtype. View Publication

The neural differentiation of human embryonic stem cells (ESCs) is a potential tool for elucidating the key mechanisms involved in human neurogenesis. Nestin and ??-III-tubulin,which are cytoskeleton proteins,are marker proteins of neural stem cells (NSCs) and neurons,respectively. However,the expression patterns of nestin and ??-III-tubulin in neural derivatives from human ESCs remain unclear. In this study,we found that neural progenitor cells (NPCs) derived from H9 cells express high levels of nestin and musashi-1. In contrast,??-III-tubulin was weakly expressed in a few NPCs. Moreover,in these cells,nestin formed filament networks,whereas ??-III-tubulin was distributed randomly as small particles. As the differentiation proceeded,the nestin filament networks and the ??-III-tubulin particles were found in both the cell soma and the cellular processes. Moreover,the colocalization of nestin and ??-III-tubulin was found mainly in the cell processes and neurite-like structures and not in the cell soma. These results may aid our understanding of the expression patterns of nestin and ??-III-tubulin during the neural differentiation of H9 cells. ?? 2013 Elsevier Inc. View Publication

Reduced expression 1 (REX1) is a widely used pluripotency marker,but little is known about its roles in pluripotency. Here,we show that REX1 is functionally important in the reacquisition and maintenance of pluripotency. REX1-depleted human pluripotent stem cells (hPSCs) lose their self-renewal capacity and full differentiation potential,especially their mesoderm lineage potential. Cyclin B1/B2 expression was found to parallel that of REX1. REX1 positively regulates the transcriptional activity of cyclin B1/B2 through binding to their promoters. REX1 induces the phosphorylation of DRP1 at Ser616 by cyclin B/CDK1,which leads to mitochondrial fission and appears to be important for meeting the high-energy demands of highly glycolytic hPSCs. During reprogramming to pluripotency by defined factors (OCT4,SOX2,KLF4,and c-MYC),the reprogramming kinetics and efficiency are markedly improved by adding REX1 or replacing KLF4 with REX1. These improvements are achieved by lowering reprogramming barriers (growth arrest and apoptosis),by enhancing mitochondrial fission,and by conversion to glycolytic metabolism,dependent on the cyclin B1/B2-DRP1 pathway. Our results show that a novel pluripotency regulator,REX1,is essential for pluripotency and reprogramming. View Publication

Human macrophages are specialised hosts for HIV-1,dengue virus,Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens,because available myeloid cell lines are,by definition,not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined,feeder- and serum-free culture conditions and produces very consistent,pure,high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively,up to ∼3×10(7) monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14(+),CD16(low),CD163(+)). Differentiation with M-CSF produces macrophages that are highly phagocytic,HIV-1-infectable,and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate,as they recognise foreign nucleic acids; the lentivector system described here overcomes this,as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells,surmounting issues of transgene silencing. Overall,the method we describe here is an efficient,effective,scalable system for the reproducible production and genetic modification of human macrophages,facilitating the interrogation of human macrophage biology. View Publication

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm,which is manifested by rosette formation,with consecutive differentiation into neural progenitors and early glial-like cells. In this study,we examined the involvement of early neural markers - OTX2,PAX6,Sox1,Nestin,NR2F1,NR2F2,and IRX2 - in the onset of rosette formation,during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation,which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation,when rosettes comprise no more than 3-5 cells,and that its expression precedes that of established markers of early neuronal differentiation. Importantly,the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly,we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro,and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice. ?? 2013 Elsevier B.V. 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

Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro,but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal,we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray,and tested their ability to perform mature hepatocyte functions (albumin and urea secretion,cytochrome activity). By these measures,ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. View Publication

Remodelling of the human embryo at implantation is indispensable for successful pregnancy. Yet it has remained mysterious because of the experimental hurdles that beset the study of this developmental phase. Here,we establish an in vitro system to culture human embryos through implantation stages in the absence of maternal tissues and reveal the key events of early human morphogenesis. These include segregation of the pluripotent embryonic and extra-embryonic lineages,and morphogenetic rearrangements leading to generation of a bilaminar disc,formation of a pro-amniotic cavity within the embryonic lineage,appearance of the prospective yolk sac,and trophoblast differentiation. Using human embryos and human pluripotent stem cells,we show that the reorganization of the embryonic lineage is mediated by cellular polarization leading to cavity formation. Together,our results indicate that the critical remodelling events at this stage of human development are embryo-autonomous,highlighting the remarkable and unanticipated self-organizing properties of human embryos. View Publication

Human embryonic stem cells (hESCs) are promising in regenerative medicine (RM) due to their differentiation plasticity and proliferation potential. However,a major challenge in RM is the generation of a vascular system to support nutrient flow to newly synthesized tissues. Here we refined an existing method to generate tight vessels by differentiating hESCs in CD34(+) vascular progenitor cells using chemically defined media and growth conditions. We selectively purified these cells from CD34(-) outgrowth populations also formed. To analyze these differentiation processes,we compared the proteomes of the hESCs with those of the CD34(+) and CD34(-) populations using high resolution mass spectrometry,label-free quantification,and multivariate analysis. Eighteen protein markers validate the differentiated phenotypes in immunological assays; nine of these were also detected by proteomics and show statistically significant differential abundance. Another 225 proteins show differential abundance between the three cell types. Sixty-three of these have known functions in CD34(+) and CD34(-) cells. CD34(+) cells synthesize proteins implicated in endothelial cell differentiation and smooth muscle formation,which support the bipotent phenotype of these progenitor cells. CD34(-) cells are more heterogeneous synthesizing muscular/osteogenic/chondrogenic/adipogenic lineage markers. The remaining textgreater150 differentially abundant proteins in CD34(+) or CD34(-) cells raise testable hypotheses for future studies to probe vascular morphogenesis. View Publication

Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs,especially for long-term studies. If 3D culture systems were to be used for such purposes,it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time,and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality,as demonstrated by improved urea production and hepatic marker expression. In addition,hPSC-HLCs in the scaffolds exhibit a more mature phenotype,as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays,making this system highly attractive for drug testing applications. View Publication