技术资料

The fluorescence ubiquitination cell cycle indicator (FUCCI) system provides a powerful method to evaluate cell cycle mechanisms associated with stem cell self-renewal and cell fate specification. By integrating the FUCCI system into human pluripotent stem cells (hPSCs) it is possible to isolate homogeneous fractions of viable cells representative of all cell cycle phases. This method avoids problems associated with traditional tools used for cell cycle analysis such as synchronizing drugs,elutriation and temperature sensitive mutants. Importantly,FUCCI reporters allow cell cycle events in dynamic systems,such as differentiation,to be evaluated. Initial reports on the FUCCI system focused on its strengths in reporting spatio-temporal aspects of cell cycle events in living cells and developmental models. In this report,we describe approaches that broaden the application of FUCCI reporters in PSCs through incorporation of FACS. This approach allows molecular analysis of the cell cycle in stem cell systems that were not previously possible. View Publication

The bioorthogonal keto group has attracted interest for the site-specific chemical conjugation of recombinant proteins under mild conditions,e.g. with aminooxy-functionalised fluorescent probes,radiometal chelates,toxins or polymers. However,the cotranslational incorporation of the corresponding non-canonical amino acid p-acetyl-L-phenylalanine (Apa) into proteins expressed in Escherichia coli by means of amber suppression using a previously described system with a mutated tRNA and an engineered tyrosyl-tRNA synthetase from Methanococcus jannaschii shows limited efficiency and considerable promiscuity towards endogenous amino acids. Employing a one-plasmid system that encodes all three components required for selection,i.e. the modified aminoacyl-tRNA synthetase (aaRS),the cognate amber suppressor tRNA and the enhanced green fluorescent protein equipped with an amber stop codon and serving as reporter,we have generated an Apa-specific aaRS&tRNA pair with considerably improved efficiency (17-fold increased expression) and also fidelity (6-fold). To this end,both the aaRS and the tRNA were subjected to doped random mutagenesis and selection in altogether four evolutionary cycles using fluorescence-activated bacterial cell sorting as well as automated screening of microcultures. The resulting aaRS&tRNA pair was applied to the functionalisation of an Anticalin with specificity towards oncofetal fibronectin by introducing a keto group at a permissible site for subsequent conjugation with a fluorescent dye,thus allowing visualisation of this tumour target under the microscope. View Publication

DPF2,also named ubi-d4/requiem (REQU),interacts with a protein complex containing OCT4. This paper provides data in support of the research article entitled DPF2 regulates OCT4 protein level and nuclear distribution". The highlights include: (1) Denature-immunoprecipitation assay revealed ubiquitination of OCT4 in pluripotent H9 cells� View Publication

Activation of ErbB4 receptor signaling is instrumental in heart development,lack of which results in embryonic lethality. However,mechanism governing its intracellular signaling remains elusive. Using human pluripotent stem cells,we show that ErbB4 is critical for cardiogenesis whereby its genetic knockdown results in loss of cardiomyocytes. Phospho-proteome profiling and Western blot studies attribute this loss to inactivation of p38$\$ isoform which physically interacts with NKx2.5 and GATA4 transcription factors. Post-cardiomyocyte formation p38$\$/NKx2.5 downregulation is followed by p38$\$/MEF2c upregulation suggesting stage-specific developmental roles of p38 MAPK isoforms. Knockdown of p38$\$ similarly disrupts cardiomyocyte formation in spite of the presence of NKx2.5. Cell fractionation and NKx2.5 phosphorylation studies suggest inhibition of ErbB4-p38$\$ hinders NKx2.5 nuclear translocation during early cardiogenesis. This study reveals a novel pathway that directly links ErbB4 and p38$\$ the transcriptional machinery of NKx2.5-GATA4 complex which is critical for cardiomyocyte formation during mammalian heart development. View Publication

The in vitro toxicological evaluation of e-liquid aerosol is an important aspect of consumer protection,but the cell model is of great significance. Due to its water solubility,e-liquid aerosol is deposited in the conducting zone of the respiratory tract. Therefore,primary normal human bronchial epithelial (NHBE) cells are more suitable for e-liquid aerosol testing than the widely used alveolar cell line A549. Due to their prolonged lifespan,immortalized cell lines derived from primary NHBE cells,exhibiting a comparable in vitro differentiation,might be an alternative for acute toxicity testing. In our study,A549 cells freshly isolated NHBE cells and the immortalized cell line CL-1548 were exposed at the air-liquid interface to e-liquid aerosol and cigarette mainstream smoke in a CULTEX(®) RFS compact module. The cell viability was analyzed 24 h post-exposure. In comparison with primary NHBE cells,the CL-1548 cell line showed lower sensitivity to e-liquid aerosol but significantly higher sensitivity compared to A549 cells. Therefore,the immortalized cell line CL-1548 is recommended as a tool for the routine testing of e-liquid aerosol and is preferable to A549 cells. View Publication

Tools for rapid and efficient transgenesis in safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE� View Publication

The generation of liquefied poly-ɛ-caprolactone (PCL) droplets by means of a microfluidic device results in uniform-sized microspheres,which are validated as microcarriers for human embryonic stem cell culture. Formed droplet size and size distribution,as well as the resulting PCL microsphere size,are correlated with the viscosity and flow rate ratio of the dispersed (Q d) and continuous (Q c) phases. PCL in dichloromethane increases its viscosity with concentration and molecular weight. Higher viscosity and Q d/Q c lead to the formation of larger droplets,within two observed formation modes: dripping and jetting. At low viscosity of dispersed phase and Q d/Q c,the microfluidic device is operated in dripping mode,which generates droplets and microspheres with greater size uniformity. Solutions with lower molecular weight PCL have lower viscosity,resulting in a wider concentration range for the dripping mode. When coated with extracellular matrix (ECM) proteins,the fabricated PCL microspheres are demonstrated capable of supporting the expansion of human embryonic stem cells. View Publication

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety,the ability to activate appropriate innate immune mediators upon vaccination,and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV),an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species,are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2,NS1,and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2,NS1,and VP7 BTV-4 genes in a transfer plasmid,the construction of recombinant MVAs,the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification. View Publication

Short bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving,such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes,new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs),called human intestinal organoids (HIOs),have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However,given that HIOs are small three-dimensional structures grown in vitro,methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds,and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro,the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast,HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine,which need to be explored further to develop them into fully functional tissue. View Publication

The present study describes and compares functional properties of Nuli-1 cells and primary human nasal epithelial cells (HNEC) including TLR expression and function. Differences in gene expression were identified for non-TLR genes that play a role in TLR response pathways. However,experiments comparing TLR gene expression for both Nuli-1 cells and HNECs indicated conserved expression in both cell types. Stimulation of the two cell types resulted in a conserved response to TLR3 agonists,but in differences in response to agonists for TLR5 and TLR6/2. HNECs were much more susceptible to infection with Staphylococcus aureus than NuLi-1 cells. Furthermore,when cultured at air-liquid interface (ALI),NuLi-1 cells possessed much lower trans-epithelial resistance than primary HNEC and did not exhibit maintenance of cell morphology or mucous production which was observed in HNECs. Nor did they produce the characteristic interconnecting pattern of tight junction complexes at the apicolateral margin of adjacent cells. Caution should therefore be exercised when selecting cell lines for immunological studies and a thorough screen of properties relevant to the study should always be carried out prior to commencement. View Publication

An 85- to 95 kDa class of lymphocyte surface molecules,defined in man by antibodies of the Hermes series,is involved in lymphocyte binding to high endothelial venules and is likely of central importance in the process of lymphocyte homing. In this report,we have examined the relationship between these Hermes-defined homing-receptors" and two other 80 to 95 kDa lymphocyte surface molecules that have been extensively studied--CD44 [In(Lu)-related p80] defined by mAb A1G3 and A3D8� View Publication

Primary dilated cardiomyopathy (DCM) is a non-ischemic heart disease with impaired pumping function of the heart. In this study,we used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a healthy volunteer and a primary DCM patient to investigate the impact of DCM on iPSC-CMs' responses to different types of anisotropic strain. A bioreactor system was established that generates cardiac-mimetic forces of 150 kPa at 5% anisotropic cyclic strain and 1. Hz frequency. After confirming cardiac induction of the iPSCs,it was determined that fibronectin was favorable to other extracellular matrix protein coatings (gelatin,laminin,vitronectin) in terms of viable cell area and density,and was therefore selected as the coating for further study. When iPSC-CMs were exposed to three strain conditions (no strain,5% static strain,and 5% cyclic strain),the static strain elicited significant induction of sarcomere components in comparison to other strain conditions. However,this induction occurred only in iPSC-CMs from a healthy volunteer (control iPSC-CMs")� View Publication