参考文献

Epigenomic regulation is likely to be important in the maintenance of genomic integrity of human pluripotent stem cells,however,the mechanisms are unknown. We explored the epigenomes and transcriptomes of human pluripotent stem cells before and after spontaneous transformation to abnormal karyotypes and in correlation to cancer cells. Our results reveal epigenetic silencing of Catalase,a key regulator of oxidative stress and DNA damage control in abnormal cells. Our findings provide novel insight into the mechanisms associated with spontaneous transformation of human pluripotent stem cells towards malignant fate. The same mechanisms may control the genomic stability of cells in somatic tissues. View Publication

Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells,only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study,we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1,a critical transcription factor for pancreatic development,leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore,in the presence of biochemical factors,200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin,glucagon,or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ,suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells. View Publication

Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans,but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells,we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently,precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo,including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy. View Publication

Although hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) are considered a promising model for predicting hepatotoxicity,their application has been restricted because of the low activity of drug metabolizing enzymes (DMEs). Here we found that the low expression of xenobiotic receptors (constitutive androstane receptor,CAR; and pregnane X receptor,PXR) contributes to the low activity of DMEs in hPSC-HLCs. Most CAR- and PXR-regulated DMEs and transporters were transcriptionally down-regulated in hPSC-HLC. Transcriptional expression of CAR and PXR was highly repressed in hPSC-HLCs,whereas mRNA levels of aryl hydrocarbon receptor (AHR) were comparable to those of adult liver. Furthermore,ligand-induced transcriptional activation was observed only at AHR in hPSC-HLCs. Bisulfite sequencing analysis demonstrated that promoter hypermethylation of CAR and PXR was associated with diminished transcriptional activity in hPSC-HLCs. Treatment with AHR-selective ligands increased the transcription of AHR-dependent target genes by direct AHR-DNA binding at the xenobiotic response element. In addition,an antagonist of AHR significantly inhibited AHR-dependent target gene expression. Thus,AHR may function intrinsically as a xenosensor as well as a ligand-dependent transcription factor in hPSC-HLCs. Our results indicate that hPSC-HLCs can be used to screen toxic substances related to AHR signaling and to identify potential AHR-targeted therapeutics. View Publication

Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently,the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed,respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last,screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together,our results highlight the potential of hiPSCs for studying human tumourigenesis. View Publication

The use of induced pluripotent stem cells (iPSCs) as a source of cells for cell-based therapy in regenerative medicine is hampered by the limited efficiency and safety of the reprogramming procedure and the low efficiency of iPSC differentiation to specialized cell types. Evidence suggests that iPSCs retain an epigenetic memory of their parental cells with a possible influence on their differentiation capacity in vitro. We reprogramme three cell types,namely human umbilical cord vein endothelial cells (HUVECs),endothelial progenitor cells (EPCs) and human dermal fibroblasts (HDFs),to iPSCs and compare their hematoendothelial differentiation capacity. HUVECs and EPCs were at least two-fold more efficient in iPSC reprogramming than HDFs. Both HUVEC- and EPC-derived iPSCs exhibited high potentiality toward endothelial cell differentiation compared with HDF-derived iPSCs. However,only HUVEC-derived iPSCs showed efficient differentiation to hematopoietic stem/progenitor cells. Examination of DNA methylation at promoters of hematopoietic and endothelial genes revealed evidence for the existence of epigenetic memory at the endothelial genes but not the hematopoietic genes in iPSCs derived from HUVECs and EPCs indicating that epigenetic memory involves an endothelial differentiation bias. Our findings suggest that endothelial cells and EPCs are better sources for iPSC derivation regarding their reprogramming efficiency and that the somatic cell type used for iPSC generation toward specific cell lineage differentiation is of importance. View Publication

The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation. View Publication

textlessptextgreater Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases,thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics,but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC) and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover,the protooncogene textlessitalictextgreatermyctextless/italictextgreater showed the strongest expression in HEP-iPSC,compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such as textlessitalictextgreatermyctextless/italictextgreater might be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stem cell-based approaches for liver metabolic diseases. textless/ptextgreater View Publication

RNA functions at enhancers remain mysterious. Here we show that the 7SK small nuclear RNA (snRNA) inhibits enhancer transcription by modulating nucleosome position. 7SK occupies enhancers and super enhancers genome wide in mouse and human cells,and it is required to limit enhancer-RNA initiation and synthesis in a manner distinct from promoter pausing. Clustered elements at super enhancers uniquely require 7SK to prevent convergent transcription and DNA-damage signaling. 7SK physically interacts with the BAF chromatin-remodeling complex,recruits BAF to enhancers and inhibits enhancer transcription by modulating chromatin structure. In turn,7SK occupancy at enhancers coincides with that of Brd4 and is exquisitely sensitive to the bromodomain inhibitor JQ1. Thus,7SK uses distinct mechanisms to counteract the diverse consequences of pervasive transcription that distinguish super enhancers,enhancers and promoters. View Publication

Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However,insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs),which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore,we investigated the effects of sodium fluoride (NaF) on the proliferation,differentiation and viability of H9 hESCs. For the first time,we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology,mitochondrial membrane potential (MMP),caspase activities,cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway,coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a pJNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs. View Publication