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Nature Neuroscience 17,1180 (2014). doi:10.1038/nn.3787 View Publication

BACKGROUND Acute respiratory distress syndrome (ARDS) is characterized by overwhelming inflammatory responses and lung remodeling. We hypothesized that leukocyte infiltration during the inflammatory response modulates epithelial remodeling through a mechanism of epithelial-mesenchymal transition (EMT). METHODS Human lung epithelial cells were treated for 30 min with hydrochloric acid (HCl). Human monocytes were then cocultured with the epithelial cells for up to 48 h,in the presence or absence of blocking peptides against lymphocyte function-associated antigen-1 (LFA-1),or tyrphostin A9,a specific inhibitor for platelet-derived growth factor (PDGF) receptor tyrosine kinase. RESULTS Exposure of lung epithelial cells to HCl resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and production of interleukin (IL)-8 at 24 h. The expression of the epithelial markers E-cadherin decreased while the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) increased at 24 h and remained high at 48 h. The addition of monocytes augmented the profiles of lower expression of epithelial markers and higher mesenchymal markers accompanied by increased collagen deposition. This EMT profile was associated with an enhanced production of IL-8 and PDGF. Treatment of the lung epithelial cells with the LAF-1 blocking peptides CD11a237-246 or/and CD18112-122 suppressed monocyte adhesion,production of IL-8,PDGF and hydroxyproline as well as EMT markers. Treatment with tyrphostin A9 prevented the EMT profile shift induced by HCl stimulation. CONCLUSIONS The interaction between epithelial cells and monocytes enhanced epithelial remodelling after initial injury through EMT signalling that is associated with the release of soluble mediators,including IL-8 and PDGF. View Publication

Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency,and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3,H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers,such as OCT4 (POU5F1),TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors. View Publication

Mesenchymal stem cells (MSCs) may be used as powerful tools for the repair and regeneration of damaged tissues. However,isolating tissue specific-derived MSCs may cause pain and increased infection rates in patients,and repetitive isolations may be required. To overcome these difficulties,we have examined alternative methods for MSC production. Here,we show that induced pluripotent stem cells (iPSCs) may be differentiated into mesenchymal stem cells (iMSCs) following exposure to SB431542. Purified iMSCs were administered to mdx mice to study skeletal muscle regeneration in a murine model of muscular dystrophy. Purified iMSCs displayed fibroblast-like morphology,formed three-dimensional spheroid structures,and expressed characteristic mesenchymal stem cell surface markers such as CD29,CD33,CD73,CD90,and CD105. Moreover,iMSCs were capable of differentiating into adipogenic,osteogenic,and chondrogenic lineages. Transplanting iMSC cells to tibialis anterior skeletal muscle tissue in mdx mice lowered oxidative damage as evidenced by a reduction in nitrotyrosine levels,and normal dystrophin expression levels were restored. This study demonstrates the therapeutic potential of purified iMSCs in skeletal muscle regeneration in mdx mice,and suggests that iPSCs are a viable alternate source for deriving MSCs as needed. textcopyright 2014 Elsevier Inc. View Publication

AbstractBack to TopbackslashnNeurons produced from stem cells have emerged as a tool to identify new therapeutic targets for neurological diseases such as amyotrophic lateral sclerosis (ALS). However,it remains unclear to what extent these new mechanistic insights will translate to animal models,an important step in the validation of new targets. Previously,we found that glia from mice carrying the SOD1G93A mutation,a model of ALS,were toxic to stem cell–derived human motor neurons. We use pharmacological and genetic approaches to demonstrate that the prostanoid receptor DP1 mediates this glial toxicity. Furthermore,we validate the importance of this mechanism for neural degeneration in vivo. Genetic ablation of DP1 in SOD1G93A mice extended life span,decreased microglial activation,and reduced motor neuron loss. Our findings suggest that blocking DP1 may be a therapeutic strategy in ALS and demonstrate that discoveries from stem cell models of disease can be corroborated in vivo. View Publication

Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets,a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors,32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3),insulin receptor substrate 2 (IRS2),docking protein 1 (DOK1),Src homology 2 domain containing transforming protein 1 (SHC1),and sprouty homologue 1 (SPRY1) as validating targets,and SPRY2,SH2 domain containing 2A (SH2D2A),and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse,rat,and lower vertebrate genomes. Among tissues and lineages,RHEX was elevated in EPCs,occurred as a plasma membrane protein,was rapidly PY-phosphorylated textgreater20-fold upon EPO exposure,and coimmunoprecipitated with the EPOR. In UT7epo cells,knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation,and RHEX coupling to GRB2. In primary human EPCs,shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development. View Publication

THAP1 encodes a transcription factor but its regulation is largely elusive. TOR1A was shown to be repressed by THAP1 in vitro. Notably,mutations in both of these genes lead to dystonia (DYT6 or DYT1). Surprisingly,expressional changes of TOR1A in THAP1 mutation carriers have not been detected indicating additional levels of regulation. Here,we investigated whether THAP1 is able to autoregulate its own expression. Using in-silico prediction,luciferase reporter gene assays,and (quantitative) chromatin immunoprecipitation (ChIP),we defined the THAP1 minimal promoter to a 480. bp-fragment and demonstrated specific binding of THAP1 to this region which resulted in repression of the THAP1 promoter. This autoregulation was disturbed by different DYT6-causing mutations. Two mutants (Ser6Phe,Arg13His) were shown to be less stable than wildtype THAP1 adding to the effect of reduced binding to the THAP1 promoter. Overexpressed THAP1 is preferably degraded through the proteasome. Notably,endogenous THAP1 expression was significantly reduced in cells overexpressing wildtype THAP1 as demonstrated by quantitative PCR. In contrast,higher THAP1 levels were detected in induced pluripotent stem cell (iPS)-derived neurons from THAP1 mutation carriers. Thus,we identified a feedback-loop in the regulation of THAP1 expression and demonstrated that mutant THAP1 leads to higher THAP1 expression levels. This compensatory autoregulation may contribute to the mean age at onset in the late teen years or even reduced penetrance in some THAP1 mutation carriers. View Publication

mAbs constitute an important biological tool for influenza virus haemagglutinin (HA) epitope mapping through the generation of escape mutants,which could provide insights into immune evasion mechanisms and may benefit the future development of vaccines. Several influenza A (H1N1) pandemic 2009 (pdm09) HA escape mutants have been recently described. However,the HA antigenic sites of the previous seasonal A/Brisbane/59/2007 (H1N1) (Bris07) virus remain poorly documented. Here,we produced mAbs against pdm09 and Bris07 HA proteins expressed in human HEK293 cells. Escape mutants were generated using mAbs that exhibited HA inhibition and neutralizing activities. The resulting epitope mapping of the pdm09 HA protein revealed 11 escape mutations including three that were previously described (G172E,N173D and K256E) and eight novel ones (T89R,F128L,G157E,K180E,A212E,R269K,N311T and G478E). Among the six HA mutations that were part of predicted antigenic sites (Ca1,Ca2,Cb,Sa or Sb),three (G172E,N173D and K180E) were within the Sa site. Eight escape mutations (H54N,N55D,N55K,L60H,N203D,A231T,V314I and K464E) were obtained for Bris07 HA,and all but one (N203D,Sb site) were outside the predicted antigenic sites. Our results suggest that the Sa antigenic site is immunodominant in pdm09 HA,whereas the N203D mutation (Sb site),present in three different Bris07 escape mutants,appears as the immunodominant epitope in that strain. The fact that some mutations were not part of predicted antigenic sites reinforces the necessity of further characterizing the HA of additional H1N1 strains. View Publication

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently,optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally,hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However,these methods have several major limitations,including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods,we have recently developed a new method,which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here,we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor),phenylbenzodioxane (ROCK II inhibitor),and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover,based on NCM,we have demonstrated efficient transfection or transduction of plasmid DNAs,lentiviral particles,and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture,and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies,stem cell research,and drug discovery. View Publication

Adaptive immunity to self-antigens causes autoimmune disorders,such as multiple sclerosis,psoriasis and type 1 diabetes; paradoxically,T- and B-cell responses to amyloid-$\$(A$\$) reduce Alzheimer's disease (AD)-associated pathology and cognitive impairment in mouse models of the disease. The manipulation of adaptive immunity has been a promising therapeutic approach for the treatment of AD,although vaccine and anti-A$\$ approaches have proven difficult in patients,thus far. CD4(+) T cells have a central role in regulating adaptive immune responses to antigens,and A$\$-specific CD4(+) T cells have been shown to reduce AD pathology in mouse models. As these cells may facilitate endogenous mechanisms that counter AD,an evaluation of their abundance before and during AD could provide important insights. A$\$-CD4see is a new assay developed to quantify A$\$-specific CD4(+) T cells in human blood,using dendritic cells derived from human pluripotent stem cells. In tests of textgreater50 human subjects A$\$-CD4see showed an age-dependent decline of A$\$-specific CD4(+) T cells,which occurs earlier in women than men. In aggregate,men showed a 50% decline in these cells by the age of 70 years,but women reached the same level before the age of 60 years. Notably,women who carried the AD risk marker apolipoproteinE-ɛ4 (ApoE4) showed the earliest decline,with a precipitous drop between 45 and 52 years,when menopause typically begins. A$\$-CD4see requires a standard blood draw and provides a minimally invasive approach for assessing changes in A$\$ that may reveal AD-related changes in physiology by a decade. Furthermore,CD4see probes can be modified to target any peptide,providing a powerful new tool to isolate antigen-specific CD4(+) T cells from human subjects. View Publication

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function,little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet,understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore,we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs,from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties,including resting membrane potential,action potential,sodium and potassium channel currents,somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons,the resting membrane potential became more negative,the expression of voltage-gated sodium channels increased,the membrane became capable of generating action potentials following adequate depolarization and,at day 48-55,50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step,of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology,as electrophysiological properties of iPSC-derived neurons mature over time. View Publication

The anisotropic alignment of cardiomyocytes in native myocardium tissue is a functional feature that is absent in traditional in vitro cardiac cell culture. Microenvironmental factors cue structural organization of the myocardium,which promotes the mechanical contractile properties and electrophysiological patterns seen in mature cardiomyocytes. Current nano- and microfabrication techniques,such as photolithography,generate simplified cell culture topographies that are not truly representative of the multifaceted and multi-scale fibrils of the cardiac extracellular matrix. In addition,such technologies are costly and require a clean room for fabrication. This chapter offers an easy,fast,robust,and inexpensive fabrication of biomimetic multi-scale wrinkled surfaces through the process of plasma treating and shrinking prestressed thermoplastic. Additionally,this chapter includes techniques for culturing stem cells and their cardiac derivatives on these substrates. Importantly,this wrinkled cell culture platform is compatible with both fluorescence and bright-field imaging; real-time physiological monitoring of CM action potential propagation and contraction properties can elucidate cardiotoxicity drug effects. View Publication