技术资料

About half of the human genome consists of highly repetitive elements,most of which are considered dispensable for human life. Here,we report that repetitive elements originating from endogenous retroviruses (ERVs) are systematically transcribed during human early embryogenesis in a stage-specific manner. Our analysis highlights that the long terminal repeats (LTRs) of ERVs provide the template for stage-specific transcription initiation,thereby generating hundreds of co-expressed,ERV-derived RNAs. Conversion of human embryonic stem cells (hESCs) to an epiblast-like state activates blastocyst-specific ERV elements,indicating that their activity dynamically reacts to changes in regulatory networks. In addition to initiating stage-specific transcription,many ERV families contain preserved splice sites that join the ERV segment with non-ERV exons in their genomic vicinity. In summary,we find that ERV expression is a hallmark of cellular identity and cell potency that characterizes the cell populations in early human embryos. View Publication

The effort and cost of obtaining neurons for large-scale screens has limited drug discovery in neuroscience. To overcome these obstacles,we fabricated arrays of releasable polystyrene micro-rafts to generate thousands of uniform,mobile neuron mini-cultures. These mini-cultures sustain synaptically-active neurons which can be easily transferred,thus increasing screening throughput by textgreater30-fold. Compared to conventional methods,micro-raft cultures exhibited significantly improved neuronal viability and sample-to-sample consistency. We validated the screening utility of these mini-cultures for both mouse neurons and human induced pluripotent stem cell-derived neurons by successfully detecting disease-related defects in synaptic transmission and identifying candidate small molecule therapeutics. This affordable high-throughput approach has the potential to transform drug discovery in neuroscience. View Publication

Pluripotent human embryonic stem cells (hESCs) have the capability of differentiating into different lineages based on specific environmental cues. We had previously shown that hESCs can be primed to differentiate into either neurons or glial cells,depending on the arrangement,geometry and size of their substrate topography. In particular,anisotropically patterned substrates like gratings were found to favour the differentiation of hESCs into neurons rather than glial cells. In this study,our aim is to elucidate the underlying mechanisms of topography-induced differentiation of hESCs towards neuronal lineages. We show that high actomyosin contractility induced by a nano-grating topography is crucial for neuronal maturation. Treatment of cells with the myosin II inhibitor (blebbistatin) and myosin light chain kinase inhibitor (ML-7) greatly reduces the expression level of microtubule-associated protein 2 (MAP2). On the other hand,our qPCR array results showed that PAX5,BRN3A and NEUROD1 were highly expressed in hESCs grown on nano-grating substrates as compared to unpatterned substrates,suggesting the possible involvement of these genes in topography-mediated neuronal differentiation of hESCs. Interestingly,YAP was localized to the cytoplasm of differentiating hESCs. Taken together,our study has provided new insights in understanding the mechanotransduction of topographical cues during neuronal differentiation of hESCs. View Publication

Three-dimensional (3D) printing is advantageous over conventional technologies for the fabrication of sophisticated structures such as 3D micro-channels for future applications in tissue engineering and drug screening. We aimed to apply this technology to cell-based assays using polydimethylsiloxane (PDMS),the most commonly used material for fabrication of micro-channels used for cell culture experiments. Useful properties of PDMS include biocompatibility,gas permeability and transparency. We developed a simple and robust protocol to generate PDMS-based devices using a soft lithography mold produced by 3D printing. 3D chemical gradients were then generated to stimulate cells confined to a micro-channel. We demonstrate that concentration gradients of growth factors,important regulators of cell/tissue functions in vivo,influence the survival and growth of human embryonic stem cells. Thus,this approach for generation of 3D concentration gradients could have strong implications for tissue engineering and drug screening. View Publication

Current protocols for human pluripotent stem cell (hPSC) expansion require feeder cells or matrices from animal sources that have been the major obstacle to obtain clinical grade hPSCs due to safety issues,difficulty in quality control,and high expense. Thus,feeder-free,chemically defined synthetic platforms have been developed,but are mostly confined to typical polystyrene culture plates. Here,we report a chemically defined,material-independent,bio-inspired surface coating allowing for feeder-free expansion and maintenance of self-renewal and pluripotency of hPSCs on various polymer substrates and devices. Polydopamine (pDA)-mediated immobilization of vitronectin (VN) peptides results in surface functionalization of VN-dimer/pDA conjugates. The engineered surfaces facilitate adhesion,proliferation,and colony formation of hPSCs via enhanced focal adhesion,cell-cell interaction,and biophysical signals,providing a chemically defined,xeno-free culture system for clonal expansion and long-term maintenance of hPSCs. This surface engineering enables the application of clinically-relevant hPSCs to a variety of biomedical systems such as tissue-engineering scaffolds and medical devices. View Publication

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript,we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore,we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages,further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays. View Publication

The liver is the main organ responsible for the modification,clearance,and transformational toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes. However,the scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes; however,current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly,fetal hepatocytes acquire mature CYP450 expression only postpartum,suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid,a by-product of intestinal flora,activate pregnane X receptor (PXR) and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes,while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear factor 4 alpha (HNF4α) positive,permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R(2) correlation of 0.94 between TC50 values obtained in stem cell-derived hepatocytes and primary cells,compared to 0.62 for HepG2 cells. Finally,stem cell-derived hepatocytes demonstrate all toxicological endpoints examined,including steatosis,apoptosis,and cholestasis,when exposed to nine known hepatotoxins. CONCLUSION: Our work provides fresh insights into liver development,suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues results in the first functional,inducible,hPSC-derived hepatocyte for predictive toxicology. (Hepatology 2015). View Publication

Oncogenic transformation in Ewing sarcoma tumors is driven by the fusion oncogene EWS-FLI1. However,despite the well-established role of EWS-FLI1 in tumor initiation,the development of models of Ewing sarcoma in human cells with defined genetic elements has been challenging. Here,we report a novel approach to model the initiation of Ewing sarcoma tumorigenesis that exploits the developmental and pluripotent potential of human embryonic stem cells. The inducible expression of EWS-FLI1 in embryoid bodies,or collections of differentiating stem cells,generates cells with properties of Ewing sarcoma tumors,including characteristics of transformation. These cell lines exhibit anchorage-independent growth,a lack of contact inhibition and a strong Ewing sarcoma gene expression signature. Furthermore,these cells also demonstrate a requirement for the persistent expression of EWS-FLI1 for cell survival and growth,which is a hallmark of Ewing sarcoma tumors.Oncogene advance online publication,12 October 2015; doi:10.1038/onc.2015.368. View Publication

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g.,gene knock-in,knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs,microinjection of piPSCs into embryos,embryo transfer and production of chimeric animals based on successful protocols. View Publication

Fibroblasts from patients with Type I bipolar disorder (BPD) and their unaffected siblings were obtained from an Old Order Amish pedigree with a high incidence of BPD and reprogrammed to induced pluripotent stem cells (iPSCs). Established iPSCs were subsequently differentiated into neuroprogenitors (NPs) and then to neurons. Transcriptomic microarray analysis was conducted on RNA samples from iPSCs,NPs and neurons matured in culture for either 2 weeks (termed early neurons,E) or 4 weeks (termed late neurons,L). Global RNA profiling indicated that BPD and control iPSCs differentiated into NPs and neurons at a similar rate,enabling studies of differentially expressed genes in neurons from controls and BPD cases. Significant disease-associated differences in gene expression were observed only in L neurons. Specifically,328 genes were differentially expressed between BPD and control L neurons including GAD1,glutamate decarboxylase 1 (2.5 fold) and SCN4B,the voltage gated type IV sodium channel beta subunit (-14.6 fold). Quantitative RT-PCR confirmed the up-regulation of GAD1 in BPD compared to control L neurons. Gene Ontology,GeneGo and Ingenuity Pathway Analysis of differentially regulated genes in L neurons suggest that alterations in RNA biosynthesis and metabolism,protein trafficking as well as receptor signaling pathways may play an important role in the pathophysiology of BPD. View Publication

Replication stress causes DNA damage at fragile sites in the genome. DNA damage at telomeres can initiate breakage-fusion-bridge cycles and chromosome instability,which can result in replicative senescence or tumor formation. Little is known about the extent of replication stress or telomere dysfunction in human embryonic stem cells (hESCs). hESCs are grown in culture with the expectation of being used therapeutically in humans,making it important to minimize the levels of replication stress and telomere dysfunction. Here,the hESC line UCSF4 was cultured in a defined medium with growth factor Activin A,exogenous nucleosides,or DNA polymerase inhibitor aphidicolin. We used quantitative fluorescence in situ hybridization to analyze individual telomeres for dysfunction and observed that it can be increased by aphidicolin or Activin A. In contrast,adding exogenous nucleosides relieved dysfunction,suggesting that telomere dysfunction results from replication stress. Whether these findings can be applied to other hESC lines remains to be determined. However,because the loss of telomeres can lead to chromosome instability and cancer,we conclude that hESCs grown in culture for future therapeutic purposes should be routinely checked for replication stress and telomere dysfunction. View Publication

How BMP signaling integrates into and destabilizes the pluripotency circuitry of human pluripotent stem cells (hPSCs) to initiate differentiation into individual germ layers is a long-standing puzzle. Here we report muscle segment homeobox 2 (MSX2),a homeobox transcription factor of msh family,as a direct target gene of BMP signaling and a master mediator of hPSCs' differentiation to mesendoderm. Enforced expression of MSX2 suffices to abolish pluripotency and induce directed mesendoderm differentiation of hPSCs,while MSX2 depletion impairs mesendoderm induction. MSX2 is a direct target gene of the BMP pathway in hPSCs,and can be synergistically activated by Wnt signals via LEF1 during mesendoderm induction. Furthermore,MSX2 destabilizes the pluripotency circuitry through direct binding to the SOX2 promoter and repression of SOX2 transcription,while MSX2 controls mesendoderm lineage commitment by simultaneous suppression of SOX2 and induction of NODAL expression through direct binding and activation of the Nodal promoter. Interestingly,SOX2 can promote the degradation of MSX2 protein,suggesting a mutual antagonism between the two lineage-specifying factors in the control of stem cell fate. Together,our findings reveal crucial new mechanisms of destabilizing pluripotency and directing lineage commitment in hPSCs. View Publication