技术资料

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion,differentiation,migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin,which both localize to stem cell niches in vivo. This matrix allows clonal derivation,clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes. View Publication

Tunneling nanotubes (TnTs) are long,non-adherent,actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study,we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48. h; and this effect was most prominent in media conditions (low-serum,hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs,in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs,which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation,and also lipid raft formation as a potential biomarker for TnT-forming cells. textcopyright 2014 Elsevier Inc. View Publication

The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases,including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells,direct cell reprogramming driven by lineagedetermining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter,we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover,they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination,but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling,drug screening,and cell therapy of retinal degeneration. View Publication

Human pluripotent stem cells (PSCs) are critical in vitro tools forbackslashnunderstanding mechanisms that regulate lineage differentiation inbackslashnthe human embryo as well as a potentially unlimited supply of stembackslashncells for regenerative medicine. Pluripotent human and mouse embryonicbackslashnstem cells (ESCs) derived from the inner cell mass of blastocystsbackslashnshare a similar transcription factor network to maintain pluripotencybackslashnand self-renewal,yet there are considerable molecular differencesbackslashnreflecting the diverse environments in which mouse and human ESCsbackslashnare derived. In the current study we evaluated the role of Proteinbackslashnarginine methyltransferase 5 (PRMT5) in human ESC (hESC) self-renewalbackslashnand pluripotency given its critical role in safeguarding mouse ESCbackslashnpluripotency. Unlike the mouse,we discovered that PRMT5 has no rolebackslashnin hESC pluripotency. Using microarray analysis we discovered thatbackslashna significant depletion in PRMT5 RNA and protein from hESCs changedbackslashnthe expression of only 78 genes,with the majority being repressed.backslashnFunctionally,we discovered that depletion of PRMT5 had no effectbackslashnon expression of OCT4,NANOG or SOX2,and did not prevent teratomabackslashnformation. Instead,we show that PRMT5 functions in hESCs to regulatebackslashnproliferation in the self-renewing state by regulating the fractionbackslashnof cells in Gap 1 (G1) of the cell cycle and increasing expressionbackslashnof the G1 cell cycle inhibitor P57. Taken together our data unveilsbackslashna distinct role for PRMT5 in hESCs and identifies P57 as new target. View Publication

Charge-coupled device detectors are vulnerable to cosmic rays that can contaminate Raman spectra with positive going spikes. Because spikes can adversely affect spectral processing and data analyses,they must be removed. Although both hardware-based and software-based spike removal methods exist,they typically require parameter and threshold specification dependent on well-considered user input. Here,we present a fully automated spike removal algorithm that proceeds without requiring user input. It is minimally dependent on sample attributes,and those that are required (e.g.,standard deviation of spectral noise) can be determined with other fully automated procedures. At the core of the method is the identification and location of spikes with coincident second derivatives along both the spectral and spatiotemporal dimensions of two-dimensional datasets. The method can be applied to spectra that are relatively inhomogeneous because it provides fairly effective and selective targeting of spikes resulting in minimal distortion of spectra. Relatively effective spike removal obtained with full automation could provide substantial benefits to users where large numbers of spectra must be processed. View Publication

Molecular markers defining self-renewing pluripotent embryonic stem cells (ESCs) have been identified by relative comparisons between undifferentiated and differentiated cells. Most of analysis has been done under a specific differentiation condition that may present significantly different molecular changes over others. Therefore,it is currently unclear if there are true consensus markers defining undifferentiated hESCs. To identify a set of key genes consistently altered during differentiation of hESCs regardless of differentiation conditions we have performed microarray analysis on undifferentiated hESCs (H1 and H9) and differentiated EB's and validated our results using publicly available expression array data sets. We constructed consensus modules by Weighted Gene Correlation Analysis (WGCNA) and discovered novel markers that are consistently present in undifferentiated hESCs under various differentiation conditions. We have validated top markers (downregulated: LCK,KLKB1 and SLC7A3; upregulated: RhoJ,Zeb2 and Adam12) upon differentiation. Functional validation analysis of LCK in self-renewal of hESCs by using LCK inhibitor or gene silencing with siLCK resulted in a loss of undifferentiation characteristics- morphological change,reduced alkaline phosphatase activity and pluripotency gene expression,demonstrating a potential functional role of LCK in self-renewal of hESCs. We have designated hESC markers to interactive networks in the genome,identifying possible interacting partners and showing how new markers relate to each other. Furthermore,comparison of these data sets with available datasets from iPSCs revealed that the level of these newly identified markers were correlated to the establishment of iPSCs,which may imply a potential role of these markers in gaining of cellular potency. Stem Cells 2014. View Publication

OBJECTIVE The KLF4 gene has been shown to be inactivated in cervical carcinogenesis as a tumor suppressor. However,the mechanism of KLF4 silencing in cervical carcinomas has not yet been identified. DNA methylation plays a key role in stable suppression of gene expression. METHODS The methylation status of the KLF4 promoter CpG islands was analyzed by bisulfite sequencing (BSQ) in tissues of normal cervix and cervical cancer. KLF4 gene expression was detected by RT-PCR,immunohistochemistry and western blot. KLF4 promoter methylation in cervical cancer cell line was determined by BSQ and methylation-specific polymerase chain reaction (MS-PCR). Cell proliferation ability was detected by cell growth curve and MTT assay. RESULTS The methylated allele was found in 41.90% of 24 cervical cancer tissues but only in 11.11% of 11 normal cervix tissues (Ptextless0.005). KLF4 mRNA levels were significantly reduced in cervical cancer tissues compared with normal cervix tissues (Ptextless0.01) and KLF4 mRNA expression showed a significant negative correlation with the promoter hypermethylation (r = -0.486,P = 0.003). Cervical cancer cell lines also showed a significant negative correlation between KLF4 expression and hypermethylation. After treatment with the demethylating agent 5-Azacytidine (5-Aza),the expression of KLF4 in the cervical cancer cell lines at both mRNA and protein levels was drastically increased,the cell proliferation ability was inhibited and the chemosensitivity for cisplatin was significantly increased. CONCLUSION KLF4 gene is inactivated by methylation-induced silencing mechanisms in a large subset of cervical carcinomas and KLF4 promoter hypermethylation inactivates the gene's function as a tumor suppressor in cervical carcinogenesis. View Publication

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture,they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands,a process which we call vasculogenesis,was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum,fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response,which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis,the development of blood vessels from in situ differentiating endothelial cells,and angiogenesis,the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated. View Publication

AIM To enumerate and characterize mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) for clinical application. MATERIALS & METHODS hESC were differentiated into hESC-MSC and characterized by the expression of surface markers using flow cytometry. hESC-MSC were evaluated with respect to growth kinetics,colony-forming potential,as well as osteogenic and adipogenic differentiation capacity. Immunosuppressive effects were assessed using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS hESC-MSC showed similar morphology,and cell surface markers as adipose (AMSC) and bone marrow-derived MSC (BMSC). hESC-MSC exhibited a higher growth rate during early in vitro expansion and equivalent adipogenic and osteogenic differentiation and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive effects as AMSC and BMSC. CONCLUSION hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications. View Publication

The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription,thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs,which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ˜15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together,our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species. View Publication

Potential teratogenic effects of alcohol on fetal development have been documented. Especially studies have demonstrated deleterious effect of ethanol exposure on neuronal development in animal models and on the maintenance and differentiation of neuronal precursor cells derived from stem cells. To better understand the molecular effect of alcohol on the process of neural differentiation,we have performed gene expression microarray analysis on human embryonic stem cells being directed to neural rosettes and neural precursor cells in the presence of ethanol treatment. Here we provide detailed experimental methods,analysis and information associated with our data deposited into Gene Expression Omnibus (GEO) under GSE56906. Our data provide scientific insight on potential molecular effects of fetal alcohol exposure on neural differentiation of early embryo development. View Publication

Human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM)-based assays are emerging as a promising tool for the in vitro preclinical screening of QT interval-prolonging side effects of drugs in development. A major impediment to the widespread use of human iPSC-CM assays is the low throughput of the currently available electrophysiological tools. To test the precision and applicability of the near-infrared fluorescent voltage-sensitive dye 1-(4-sulfanatobutyl)-4-β[2-(di-n-butylamino)-6-naphthyl]butadienylquinolinium betaine (di-4-ANBDQBS) for moderate-throughput electrophysiological analyses,we compared simultaneous transmembrane voltage and optical action potential (AP) recordings in human iPSC-CM loaded with di-4-ANBDQBS. Optical AP recordings tracked transmembrane voltage with high precision,generating nearly identical values for AP duration (AP durations at 10%,50%,and 90% repolarization). Human iPSC-CMs tolerated repeated laser exposure,with stable optical AP parameters recorded over a 30-min study period. Optical AP recordings appropriately tracked changes in repolarization induced by pharmacological manipulation. Finally,di-4-ANBDQBS allowed for moderate-throughput analyses,increasing throughput textgreater10-fold over the traditional patch-clamp technique. We conclude that the voltage-sensitive dye di-4-ANBDQBS allows for high-precision optical AP measurements that markedly increase the throughput for electrophysiological characterization of human iPSC-CMs. View Publication