技术资料

Despite the recent identification of the transcriptional regulatory circuitry involving SOX2,NANOG,and OCT-4,the intracellular signaling networks that control pluripotency of human embryonic stem cells (hESCs) remain largely undefined. Here,we demonstrate an essential role for the serine/threonine protein kinase mammalian target of rapamycin (mTOR) in regulating hESC long-term undifferentiated growth. Inhibition of mTOR impairs pluripotency,prevents cell proliferation,and enhances mesoderm and endoderm activities in hESCs. At the molecular level,mTOR integrates signals from extrinsic pluripotency-supporting factors and represses the transcriptional activities of a subset of developmental and growth-inhibitory genes,as revealed by genome-wide microarray analyses. Repression of the developmental genes by mTOR is necessary for the maintenance of hESC pluripotency. These results uncover a novel signaling mechanism by which mTOR controls fate decisions in hESCs. Our findings may contribute to effective strategies for tissue repair and regeneration. View Publication

Cripto is a developmental oncoprotein that signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK),phosphatidylinositol 3-kinase (PI3K)/Akt and Smad2/3 pathways. However,the molecular basis for Cripto coupling to these pathways during embryogenesis and tumorigenesis is not fully understood. In this regard,we recently demonstrated that Cripto forms a cell surface complex with the HSP70 family member glucose-regulated protein-78 (GRP78). Here,we provide novel functional evidence demonstrating that cell surface GRP78 is a necessary mediator of Cripto signaling in human tumor,mammary epithelial and embryonic stem cells. We show that targeted disruption of the cell surface Cripto/GRP78 complex using shRNAs or GRP78 immunoneutralization precludes Cripto activation of MAPK/PI3K pathways and modulation of activin-A,activin-B,Nodal and transforming growth factor-beta1 signaling. We further demonstrate that blockade of Cripto binding to cell surface GRP78 prevents Cripto from increasing cellular proliferation,downregulating E-Cadherin,decreasing cell adhesion and promoting pro-proliferative responses to activin-A and Nodal. Thus,disrupting the Cripto/GRP78 binding interface blocks oncogenic Cripto signaling and may have important therapeutic value in the treatment of cancer. View Publication

OBJECTIVE The aim of this study was to evaluate the distribution,concentration and toxicity of cinnamaldehyde in electronic cigarette (e-cigarette) refill fluids and aerosols. METHODS The distribution and concentration of cinnamaldehyde were determined in 39 e-cigarette refill fluids plus 6 duplicates using gas chromatography and mass spectrometry (GC/MS). A cinnamaldehyde toxicity profile was established for embryonic and adult cells using a live cell imaging assay,immunocytochemistry,the comet assay and a recovery assay. RESULTS Twenty of the 39 refill fluids contained cinnamaldehyde at concentrations that are cytotoxic to human embryonic and lung cells in the MTT assay. Cinnamon Ceylon aerosol produced in a cartomizer-style e-cigarette was cytotoxic. Cinnamon Ceylon aerosols and refill fluid aerosols (80% propylene glycol or cinnamaldehyde/propylene glycol) made using a tank/boxmod e-cigarette were more cytotoxic at 5 V than 3 V. Using GC/MS,aerosols produced at 5 V contained 10 additional peaks not present in aerosol generated at 3 V. One of these,2,3-butandione (diacetyl),was confirmed with an authentic standard. Cinnamaldehyde depolymerised microtubules in human pulmonary fibroblasts. At concentrations that produced no effect in the MTT assay,cinnamaldehyde decreased growth,attachment and spreading; altered cell morphology and motility; increased DNA strand breaks; and increased cell death. At the MTT IC50 concentration,lung cells were unable to recover from cinnamaldehyde after 2 hours of treatment,whereas embryonic cells recovered after 8 hours. CONCLUSIONS Cinnamaldehyde-containing refill fluids and aerosols are cytotoxic,genotoxic and low concentrations adversely affect cell processes and survival. These data indicate that cinnamaldehyde in e-cigarette refill fluids/aerosols may impair homeostasis in the respiratory system. View Publication

Normalization of human RNA-seq experiments employing chimpanzee RNA as a spike-in standard is reported. Human and chimpanzee RNAs exhibit single nucleotide variations (SNVs) in average 210-bp intervals. Spike-in chimpanzee RNA would behave the same as the human counterparts during the whole NGS procedures owing to the high sequence similarity. After discrimination of species origins of the NGS reads based on SNVs,the chimpanzee reads were used to read-by-read normalize biases and variations of human reads. By this approach,as many as 10,119 transcripts were simultaneously normalized for the entire NGS procedures leading to accurate and reproducible quantification of differential gene expression. In addition,incomparable data sets from different in-process degradations or from different library preparation methods were made well comparable by the normalization. Based on these results,we expect that the normalization approaches using near neighbor genomes as internal standards could be employed as a standard protocol,which will improve both accuracy and comparability of NGS results across different sample batches,laboratories and NGS platforms. View Publication

We performed gene expression microarray analysis coupled with spherical self-organizing map (sSOM) for artificially developed cancer stem cells (CSCs). The CSCs were developed from human induced pluripotent stem cells (hiPSCs) with the conditioned media of cancer cell lines,whereas the CSCs were induced from primary cell culture of human cancer tissues with defined factors (OCT3/4,SOX2,and KLF4). These cells commonly expressed human embryonic stem cell (hESC)/hiPSC-specific genes (POU5F1,SOX2,NANOG,LIN28,and SALL4) at a level equivalent to those of control hiPSC 201B7. The sSOM with unsupervised method demonstrated that the CSCs could be divided into three groups based on their culture conditions and original cancer tissues. Furthermore,with supervised method,sSOM nominated TMED9,RNASE1,NGFR,ST3GAL1,TNS4,BTG2,SLC16A3,CD177,CES1,GDF15,STMN2,FAM20A,NPPB,CD99,MYL7,PRSS23,AHNAK,and LOC152573 genes commonly upregulating among the CSCs compared to hiPSC,suggesting the gene signature of the CSCs. View Publication

Cell type-specific surface markers offer a powerful tool for purifying defined cell types for restorative therapies and drug screenings. Midbrain dopaminergic neurons (mesDA) are the nerve cells preferentially lost in the brains of Parkinson's disease patients. Clinical trials of transplantation of fetal neural precursors suggest that cell therapy may offer a cure for this devastating neurological disease. Many lines of preclinical studies demonstrate that neural progenitors committed to dopaminergic fate survive and integrate better than postmitotic DA neurons. We show that the folate-receptor 1 (FolR1),a GPI-anchored cell surface molecule,specifically marks mesDA neural progenitors and immature mesDA neurons. FolR1 expression superimposes with Lmx1a,a bona-fide mesDA lineage marker,during the active phase of mesDA neurogenesis from E9.5 to E14.5 during mouse development,as well as in ESC-derived mesDA lineage. FolR1(+) neural progenitors can be isolated by FACS or magnetic sorting (MAC) which give rise to dopamine neurons expressing TH and Pitx3,whilst FolR1 negative cells generate non-dopaminergic neurons and glia cells. This study identifies FolR1 as a new cell surface marker selectively expressed in mesDA progenitors in vivo and in vitro and that can be used to enrich in vitro differentiated TH neurons. View Publication

BACKGROUND Aside from its importance in reproduction,estrogen (E2) is known to regulate the proliferation and differentiation of hematopoietic stem cells in rodents. However,the regulatory role of E2 in human hematopoietic system has not been investigated. The purpose of this study is to investigate the effect of E2 on hematopoietic differentiation using human pluripotent stem cells (hPSCs). RESULTS E2 improved hematopoietic differentiation of hPSCs via estrogen receptor alpha (ER-$$)-dependent pathway. During hematopoietic differentiation of hPSCs,ER-$$ is persistently maintained and hematopoietic phenotypes (CD34 and CD45) were exclusively detected in ER-$$ positive cells. Interestingly,continuous E2 signaling is required to promote hematopoietic output from hPSCs. Supplementation of E2 or an ER-$$ selective agonist significantly increased the number of hemangioblasts and hematopoietic progenitors,and subsequent erythropoiesis,whereas ER-$$ selective agonist did not. Furthermore,ICI 182,780 (ER antagonist) completely abrogated the E2-induced hematopoietic augmentation. Not only from hPSCs but also from human umbilical cord bloods,does E2 signaling potentiate hematopoietic development,suggesting universal function of E2 on hematopoiesis. CONCLUSIONS Our study identifies E2 as positive regulator of human hematopoiesis and suggests that endocrine factors such as E2 influence the behavior of hematopoietic stem cells in various physiological conditions. View Publication

Robust control of human induced pluripotent stem cell (hIPSC) differentiation is essential to realize its patient-tailored therapeutic potential. Here,we demonstrate a novel application of Y-27632,a small molecule Rho-associated protein kinase (ROCK) inhibitor,to significantly influence the differentiation of hIPSCs in a lineage-specific manner. The application of Y-27632 to hIPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration and time-dependent manner. Such changes in cell and colony morphology were associated with decreased expression of E-cadherin,a cell-cell junctional protein,proportional to the increased exposure to Y-27632. Interestingly,gene and protein expression of pluripotency markers such as NANOG and OCT4 were not downregulated by an exposure to Y-27632 up to 36h. Simultaneously,epithelial-to-mesenchymal (EMT) transition markers were upregulated with an exposure to Y-27632. These EMT-like changes in the cells with longer exposure to Y-27632 resulted in a significant increase in the subsequent differentiation efficiency towards mesendodermal lineage. In contrast,an inhibitory effect was observed when cells were subjected to ectodermal differentiation after prolonged exposure to Y-27632. Collectively,these results present a novel method for priming hIPSCs to modulate their differentiation potential with a simple application of Y-27632. View Publication

Engineered heart tissues made from human pluripotent stem cell-derived cardiomyocytes have been used for modeling cardiac pathologies,screening new therapeutics,and providing replacement cardiac tissue. Current methods measure the functional performance of engineered heart tissue by their twitch force and beating frequency,typically obtained by optical measurements. In this article,we describe a novel method for assessing twitch force and beating frequency of engineered heart tissue using magnetic field sensing,which enables multiple tissues to be measured simultaneously. The tissues are formed as thin structures suspended between two silicone posts,where one post is rigid and another is flexible and contains an embedded magnet. When the tissue contracts it causes the flexible post to bend in proportion to its twitch force. We measured the bending of the post using giant magnetoresistive (GMR) sensors located underneath a 24-well plate containing the tissues. We validated the accuracy of the readings from the GMR sensors against optical measurements. We demonstrated the utility and sensitivity of our approach by testing the effects of three concentrations of isoproterenol and verapamil on twitch force and beating frequency in real-time,parallel experiments. This system should be scalable beyond the 24-well format,enabling greater automation in assessing the contractile function of cardiomyocytes in a tissue-engineered environment. View Publication

Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However,existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study,we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because of the capacity of Sendai virus (SeV) nonstructural C proteins to specifically inhibit viral RNA synthesis,overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression,target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore,the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications. View Publication

Cell division is characterized by a sequence of events by which a cell gives rise to two daughter cells. Quantitative measurements of cell-cycle dynamics in single cells showed that despite variability in G1-,S-,and G2 phases,duration of mitosis is short and remarkably constant. Surprisingly,there is no correlation between cell-cycle length and mitotic duration,suggesting that mitosis is temporally insulated from variability in earlier cell-cycle phases. By combining live cell imaging and computational modeling,we showed that positive feedback is the molecular mechanism underlying the temporal insulation of mitosis. Perturbing positive feedback gave rise to a sluggish,variable entry and progression through mitosis and uncoupled duration of mitosis from variability in cell cycle length. We show that positive feedback is important to keep mitosis short,constant,and temporally insulated and anticipate it might be a commonly used regulatory strategy to create modularity in other biological systems. View Publication

Fragile X syndrome (FXS) is a common cause of intellectual disability that is most often due to a CGG-repeat expansion mutation in the FMR1 gene that triggers epigenetic gene silencing. Epigenetic modifying drugs can only transiently and modestly induce FMR1 reactivation in the presence of the elongated CGG repeat. As a proof-of-principle,we excised the expanded CGG-repeat in both somatic cell hybrids containing the human fragile X chromosome and human FXS iPS cells using the CRISPR/Cas9 genome editing. We observed transcriptional reactivation in approximately 67% of the CRISPR cut hybrid colonies and in 20% of isolated human FXS iPSC colonies. The reactivated cells produced FMRP and exhibited a decline in DNA methylation at the FMR1 locus. These data demonstrate the excision of the expanded CGG-repeat from the fragile X chromosome can result in FMR1 reactivation. View Publication