技术资料

Cohesin is implicated in establishing and maintaining pluripotency. Whether this is because of essential cohesin functions in the cell cycle or in gene regulation is unknown. Here we tested cohesin's contribution to reprogramming in systems that reactivate the expression of pluripotency genes in the absence of proliferation (embryonic stem [ES] cell heterokaryons) or DNA replication (nuclear transfer). Contrary to expectations,cohesin depletion enhanced the ability of ES cells to initiate somatic cell reprogramming in heterokaryons. This was explained by increased c-Myc (Myc) expression in cohesin-depleted ES cells,which promoted DNA replication-dependent reprogramming of somatic fusion partners. In contrast,cohesin-depleted somatic cells were poorly reprogrammed in heterokaryons,due in part to defective DNA replication. Pluripotency gene induction was rescued by Myc,which restored DNA replication,and by nuclear transfer,where reprogramming does not require DNA replication. These results redefine cohesin's role in pluripotency and reveal a novel function for Myc in promoting the replication-dependent reprogramming of somatic nuclei. View Publication

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate to cardiomyocytes in vitro,offering unique opportunities to investigate cardiac development and disease as well as providing a platform to perform drug and toxicity tests. Initial cardiac differentiation methods were based on either inductive co-culture or aggregation as embryoid bodies,often in the presence of fetal calf serum. More recently,monolayer differentiation protocols have evolved as feasible alternatives and are often performed in completely defined culture medium and substrates. Thus,our ability to efficiently and reproducibly generate cardiomyocytes from multiple different hESC and hiPSC lines has improved significantly.We have developed a directed differentiation monolayer protocol that can be used to generate cultures comprising ˜50% cardiomyocytes,in which both the culture of the undifferentiated human pluripotent stem cells (hPSCs) and the differentiation procedure itself are defined and serum-free. The differentiation method is also effective for hPSCs maintained in other culture systems. In this chapter,we outline the differentiation protocol and describe methods to assess cardiac differentiation efficiency as well as to identify and quantify the yield of cardiomyocytes. View Publication

Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14+CD16+ monocytes,a mature subpopulation of peripheral blood monocytes,are key mediators of HIV neuropathogenesis. Infected CD14+CD16+ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy,continued influx of CD14+CD16+ monocytes,both infected and uninfected,contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain,CD14+CD16+ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14+CD16+ monocytes and their contribution to HIV neuropathogenesis are not known. In this study,we showed that CD14+CD16+ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R,D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist,SKF38393,increased CD14+CD16+ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion,live cell imaging was used to monitor the accumulation of CD14+CD16+ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14+CD16+ monocytes in suspension settled to the dish surface. In a spreading assay,dopamine increased the area of CD14+CD16+ monocytes during the early stages of cell adhesion. In addition,adhesion assays showed that the overall total number of adherent CD14+CD16+ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14+CD16+ monocytes in dopamine rich brain regions. View Publication

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

The integration of up- and downstream unit operations can result in the elimination of hold steps,thus decreasing the footprint,and ultimately can create robust closed system operations. This type of design is desirable for the bioprocess of human mesenchymal stem cells (hMSC),where high numbers of pure cells,at low volumes,need to be delivered for therapy applications. This study reports a proof of concept of the integration of a continuous perfusion culture in bioreactors with a tangential flow filtration (TFF) system for the concentration and washing of hMSC. Moreover,we have also explored a continuous alternative for concentrating hMSC. Results show that expanding cells in a continuous perfusion operation mode provided a higher expansion ratio,and led to a shift in cells' metabolism. TFF operated either in continuous or discontinuous allowed to concentrate cells,with high cell recovery (>80%) and viability (>95%); furthermore,continuous TFF permitted to operate longer with higher cell concentrations. Continuous diafiltration led to higher protein clearance (98%) with lower cell death,when comparing to discontinuous diafiltration. Overall,an integrated process allowed for a shorter process time,recovering 70% of viable hMSC (>95%),with no changes in terms of morphology,immunophenotype,proliferation capacity and multipotent differentiation potential. 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

A large-conductance calcium channel in chicken dorsal root ganglion neurons was studied with patch-clamp recordings of unitary currents. In addition to the conventional pattern of Ca-channel gating previously described in neurons (mode 1")� 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