技术资料

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

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

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

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 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

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

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

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

Retinal pigment epithelium-specific 65 kDa (RPE65)-associated Leber congenital amaurosis is an autosomal recessive disease that results in reduced visual acuity and night blindness beginning at birth. It is one of the few retinal degenerative disorders for which promising clinical gene transfer trials are currently underway. However,the ability to enroll patients in a gene augmentation trial is dependent on the identification of 2 bona fide disease-causing mutations,and there are some patients with the phenotype of RPE65-associated disease who might benefit from gene transfer but are ineligible because 2 disease-causing genetic variations have not yet been identified. Some such patients have novel mutations in RPE65 for which pathogenicity is difficult to confirm. The goal of this study was to determine if an intronic mutation identified in a 2-year-old patient with presumed RPE65-associated disease was truly pathogenic and grounds for inclusion in a clinical gene augmentation trial. Sequencing of the RPE65 gene revealed 2 mutations: (1) a previously identified disease-causing exonic leucine-to-proline mutation (L408P) and (2) a novel single point mutation in intron 3 (IVS3-11) resulting in an AtextgreaterG change. RT-PCR analysis using RNA extracted from control human donor eye-derived primary RPE,control iPSC-RPE cells,and proband iPSC-RPE cells revealed that the identified IVS3-11 variation caused a splicing defect that resulted in a frameshift and insertion of a premature stop codon. In this study,we demonstrate how patient-specific iPSCs can be used to confirm pathogenicity of unknown mutations,which can enable positive clinical outcomes. View Publication

Summary The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14,when expressed in tumors,causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor,rather than host,is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia,thereby extending lifespan and improving quality of life for cancer patients. View Publication

This paper proposes a bio-driven algorithm that detects cell regions automatically in the human embryonic stem cell (hESC) images obtained using a phase contrast microscope. The algorithm uses both statistical intensity distributions of foreground/hESCs and background/substrate as well as cell property for cell region detection. The intensity distributions of foreground/hESCs and background/substrate are modeled as a mixture of two Gaussians. The cell property is translated into local spatial information. The algorithm is optimized by parameters of the modeled distributions and cell regions evolve with the local cell property. The paper validates the method with various videos acquired using different microscope objectives. In comparison with the state-of-the-art methods,the proposed method is able to detect the entire cell region instead of fragmented cell regions. It also yields high marks on measures such as Jacard similarity,Dice coefficient,sensitivity and specificity. Automated detection by the proposed method has the potential to enable fast quantifiable analysis of hESCs using large data sets which are needed to understand dynamic cell behaviors. View Publication

Interactions between stem cells and their microenvironment are critical for regulation and maintenance of stem cell function. To elucidate the molecular interactions within the human limbal epithelial stem/progenitor cell (LEPC) niche,which is essential for maintaining corneal transparency and vision,we performed a comprehensive expression analysis of cell adhesion molecules (CAMs) using custom-made quantitative real-time polymerase chain reaction (qRT-PCR) arrays and laser capture-microdissected LEPC clusters,comprising LEPCs,melanocytes,mesenchymal cells,and transmigrating immune cells. We show that LEPCs are anchored to their supporting basement membrane by the laminin receptors $\$3$\$1 and $\$6$\$4 integrin and the dystroglycan complex,while intercellular contacts between LEPCs and melanocytes are mediated by N-,P-,and E-cadherin together with L1-CAM,a member of the immunoglobulin superfamily (Ig)CAMs. In addition to the LEPC-associated heparan sulfate proteoglycans syndecan-2,glypican-3,and glypican-4,the IgCAM members ICAM-1 and VCAM-1 were found to be variably expressed on LEPCs and associated niche cells and to be dynamically regulated in response to chemokines such as interferon-$\$ enhance interactions with immune cells. Moreover,junctional adhesion molecule JAM-C accumulating in the subepithelial limbal matrix,appeared to be involved in recruitment of immune cells,while mesenchymal stromal cells appeared to use the nephronectin receptor integrin $\$8 for approaching the limbal basement membrane. In summary,we identified a novel combination of cell surface receptors that may regulate both stable and dynamic cell-matrix and cell-cell interactions within the limbal niche. The findings provide a solid foundation for further functional studies and for advancement of our current therapeutic strategies for ocular surface reconstruction. View Publication