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Huntington's disease (HD) is a fatal neurodegenerative disease,caused by expansion of polyglutamine repeats in the Huntingtin gene,with longer expansions leading to earlier ages of onset. The HD iPSC Consortium has recently reported a new in vitro model of HD based on the generation of induced pluripotent stem cells (iPSCs) from HD patients and controls. The current study has furthered the disease in a dish model of HD by generating new non-integrating HD and control iPSC lines. Both HD and control iPSC lines can be efficiently differentiated into neurons/glia; however,the HD-derived cells maintained a significantly greater number of nestin-expressing neural progenitor cells compared with control cells. This cell population showed enhanced vulnerability to brain-derived neurotrophic factor (BDNF) withdrawal in the juvenile-onset HD (JHD) lines,which appeared to be CAG repeat-dependent and mediated by the loss of signaling from the TrkB receptor. It was postulated that this increased death following BDNF withdrawal may be due to glutamate toxicity,as the N-methyl-d-aspartate (NMDA) receptor subunit NR2B was up-regulated in the cultures. Indeed,blocking glutamate signaling,not just through the NMDA but also mGlu and AMPA/Kainate receptors,completely reversed the cell death phenotype. This study suggests that the pathogenesis of JHD may involve in part a population of 'persistent' neural progenitors that are selectively vulnerable to BDNF withdrawal. Similar results were seen in adult hippocampal-derived neural progenitors isolated from the BACHD model mouse. Together,these results provide important insight into HD mechanisms at early developmental time points,which may suggest novel approaches to HD therapeutics. View Publication

BACKGROUND Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes (VCMs),but hESC-VCMs are developmentally immature. Previous attempts to profile hESC-VCMs primarily relied on transcriptomic approaches,but the global proteome has not been examined. Furthermore,most hESC-CM studies focus on pathways important for cardiac differentiation,rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs,hESC-derived VCMs,human fetal and human adult ventricular and atrial CMs. METHODS AND RESULTS Using two-dimensional-differential-in-gel electrophoresis,121 differentially expressed (textgreater1.5-fold; Ptextless0.05) proteins were detected. The data set implicated a role of the peroxisome proliferator-activated receptor $\$ in cardiac maturation. Consistently,WY-14643,a peroxisome proliferator-activated receptor $\$,increased fatty oxidative enzyme level,hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line,treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in engineered human ventricular cardiac microtissue by 3-fold,signifying their maturation. CONCLUSIONS We conclude that the peroxisome proliferator-activated receptor $\$ thyroid hormone pathways modulate the metabolism and maturation of hESC-VCMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs. View Publication

Recombinant lentiviral vectors are powerful tools to stably manipulate human pluripotent stem cells. They can be used to deliver ectopic genes,shRNAs,miRNAs,or any possible genetic DNA sequence into diving and nondividing cells. Here we describe a general protocol for the production of self-inactivating lentiviral vector particles and their purification to high titers by either ultracentrifugation or ultrafiltration. Next we provide a basic procedure to transduce human pluripotent stem cells and propagate clonal cell lines. View Publication

Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders,some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here,we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program,TALENSeek,(2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol,and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify,construct,and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity. View Publication

Emerging evidence shows that glioblastoma multiforme (GBM) originates from cancer stem cells (CSCs). Characterization of CSC-specific signalling pathways would help identify new therapeutic targets and perhaps lead to the development of more efficient therapies selectively targeting CSCs. Here; we successfully dedifferentiated two patient-derived GBM cell lines into CSC-like cells (induced glioma stem cells,iGSCs) through expression of Oct4,Sox2 and Nanog transcription factors. Transformed cells exhibited significant suppression of epidermal growth factor receptor and its downstream pathways. Compared with parental GBM cells,iGSCs formed large neurospheres even in the absence of exogenous mitogens; they exhibited significant sensitivity to salinomycin and chemoresistance to temozolomide. Further characterization of iGSCs revealed induction of NOTCH1 and Wnt/β-catenin signalling and expression of CD133,CD44 and ALDH1A1. Our results indicate that iGSCs may help us understand CSC physiology and lead to development of potential therapeutic interventions aimed at differentiating tumour cells to render them more sensitive to chemotherapy or other standard agents. View Publication

Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling,drug development,screening,and the potential for patient-matched" cellular therapies in neurodegenerative diseases. In this study� View Publication

Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here,we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids,which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung,organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing,we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles,suggesting that HLOs are an excellent model to study human lung development,maturation and disease. View Publication

The hepatocyte growth factor (HGF)/MNNG HOS transforming gene (MET) pathway regulates cell growth,survival,and migration. MET is mutated or amplified in several malignancies. In myeloma,MET is not mutated,but patients have high plasma concentrations of HGF,high levels of MET expression,and gene copy number,which are associated with poor prognosis and advanced disease. Our previous studies demonstrated that MET is critical for myeloma cell survival and its knockdown induces apoptosis. In our current study,we tested tivantinib (ARQ 197),a small-molecule pharmacological MET inhibitor. At clinically achievable concentrations,tivantinib induced apoptosis by textgreater50% in all 12 human myeloma cell lines tested. This biologic response was associated with down-regulation of MET signaling and inhibition of the mitogen-activated protein kinase and phosphoinositide 3-kinase pathways,which are downstream of the HGF/MET axis. Tivantinib was equally effective in inducing apoptosis in myeloma cell lines resistant to standard chemotherapy (melphalan,dexamethasone,bortezomib,and lenalidomide) as well as in cells that were co-cultured with a protective bone marrow microenvironment or with exogenous cytokines. Tivantinib induced apoptosis in CD138+ plasma cells from patients and demonstrated efficacy in a myeloma xenograft mouse model. On the basis of these data,we initiated a clinical trial for relapsed/refractory multiple myeloma (MM). In conclusion,MET inhibitors may be an attractive target-based strategy for the treatment of MM. View Publication

BACKGROUND: Chronic rhinosinusitis (CRS) is a cluster of disorders that result in sinonasal mucosal inflammation. Staphylococcus aureus (S. aureus) is associated with severe and recalcitrant CRS. The purpose of our study was to investigate the effect of S. aureus on respiratory epithelial barrier structure and function. METHODS: Conditioned media from S. aureus reference strains (American Type Culture Collection [ATCC] 13565,14458,and 25923) was applied to air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs) and transepithelial electrical resistance (TEER) was measured to assess cell-to-cell integrity. Electron microscopy was used to gauge the ciliated area and tight junctions (TJs). Additionally,the expression of the TJ protein zona occludens-1 (ZO-1) was examined via immunofluorescence. Statistical analysis was performed using analysis of variance (ANOVA) with pairwise Bonferroni-adjusted t tests. RESULTS: Secreted products applied to ALI cultures from S. aureus strain 13565 caused a concentration-dependent decline in electrical impedance compared to controls and reference strains 14458 and 25923 (p textless 0.001). Electron microscopy showed a distinct separation between adjacent cells apically,in the region of TJs. The ciliated area was not affected; however,ZO-1 expression became discontinuous in HNECs exposed to the 13565 strain's conditioned media. CONCLUSION: Conditioned media of the S. aureus strain 13565 damages the airway epithelium by disrupting the TJs between primary HNECs grown at an ALI. These findings suggest that strain-specific S. aureus-secreted product(s) compromise epithelial barrier function,which may constitute 1 of the roles played by S. aureus in the pathophysiology of recalcitrant CRS. Further research is required to uncover the relevant molecular mechanisms. View Publication

Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) $$$$ and $$$$ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells. View Publication

Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability,with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia,coarsened facial features and intellectual disability,due to truncating mutations in the sorting nexin gene SNX14,encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate,a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma,accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction. View Publication

BACKGROUND Anagrelide is a cytoreductive agent used to lower platelet counts in essential thrombocythemia. Although the drug has been known to selectively inhibit megakaryopoiesis for many years,the molecular mechanism accounting for this activity is still unclear. OBJECTIVES AND METHODS To address this issue we have compared the global gene expression profiles of human hematopoietic cells treated ex-vivo with and without anagrelide while growing under megakaryocyte differentiation conditions,using high-density oligonucleotide microarrays. Gene expression data were validated by the quantitative polymerase chain reaction and mined to identify functional subsets and regulatory pathways. RESULTS We identified 328 annotated genes differentially regulated by anagrelide,including many genes associated with platelet functions and with the control of gene transcription. Prominent among the latter was TRIB3,whose expression increased in the presence of anagrelide. Pathway analysis revealed that anagrelide up-regulated genes that are under the control of the transcription factor ATF4,a known TRIB3 inducer. Notably,immunoblot analysis demonstrated that anagrelide induced the phosphorylation of eIF2α,which is an upstream regulator of ATF4,and increased ATF4 protein levels. Furthermore,salubrinal,an inhibitor of eIF2α dephosphorylation,increased the expression of ATF4-regulated genes and blocked megakaryocyte growth. CONCLUSIONS These findings link signaling through eIF2α/ATF4 to the anti-megakaryopoietic activity of anagrelide and identify new potential modulators of megakaryopoiesis. View Publication