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Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions,women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures,and this action was attenuated by estrogen receptor beta (ER-$$) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line,NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4,-5,-6) mRNA expression. Together,these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium. View Publication

The impact of shipping temperatures and preservation media used during transport of either peripheral blood mononuclear cells (PBMCs) or Jurkat cells was assessed,in view of implementing of a proficiency testing scheme on mononuclear cell viability. Samples were analyzed before and after shipment at different temperatures (ambient temperature,dry ice,and liquid nitrogen) and in different preservation media (serum with cryoprotectant,commercial cryopreservation solution,and room temperature transport medium). Sample quality was assessed by viability assays (Trypan Blue dye exclusion,flow cytometry,Cell Analysis System cell counting (CASY)),and by ELISpot functional assay. The liquid nitrogen storage and shipment were found to be the most stable conditions to preserve cell viability and functionality. However,we show that alternative high quality shipment conditions for viable cells are dry ice shipment and commercial cryopreservation solution. These were also cost-efficient shipment conditions,satisfying the requirements of a proficiency testing scheme for viable mononuclear cells. Room temperature transport medium dramatically and adversely affected the integrity of mononuclear cells. View Publication

The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However,human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during induced pluripotent stem cell (iPSC) reprogramming. Here,we show that inhibition of Notch greatly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus,restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs. View Publication

Mobile elements are important evolutionary forces that challenge genomic integrity. Long interspersed element-1 (L1,also known as LINE-1) is the only autonomous transposon still active in the human genome. It displays an unusual pattern of evolution,with,at any given time,a single active L1 lineage amplifying to thousands of copies before getting replaced by a new lineage,likely under pressure of host restriction factors,which act notably by silencing L1 expression during early embryogenesis. Here,we demonstrate that in human embryonic stem (hES) cells,KAP1 (KRAB [Kruppel-associated box domain]-associated protein 1),the master cofactor of KRAB-containing zinc finger proteins (KRAB-ZFPs) previously implicated in the restriction of endogenous retroviruses,represses a discrete subset of L1 lineages predicted to have entered the ancestral genome between 26.8 million and 7.6 million years ago. In mice,we documented a similar chronologically conditioned pattern,albeit with a much contracted time scale. We could further identify an L1-binding KRAB-ZFP,suggesting that this rapidly evolving protein family is more globally responsible for L1 recognition. KAP1 knockdown in hES cells induced the expression of KAP1-bound L1 elements,but their younger,human-specific counterparts (L1Hs) were unaffected. Instead,they were stimulated by depleting DNA methyltransferases,consistent with recent evidence demonstrating that the PIWI-piRNA (PIWI-interacting RNA) pathway regulates L1Hs in hES cells. Altogether,these data indicate that the early embryonic control of L1 is an evolutionarily dynamic process and support a model in which newly emerged lineages are first suppressed by DNA methylation-inducing small RNA-based mechanisms before KAP1-recruiting protein repressors are selected. View Publication

IMPORTANCE: Uveal melanoma is characterized by mutations in GNAQ and GNA11,resulting in mitogen-activated protein kinase pathway activation. OBJECTIVE: To assess the efficacy of selumetinib,a selective,non-adenosine triphosphate competitive inhibitor of MEK1 and MEK2,in uveal melanoma. DESIGN,SETTING,AND PARTICIPANTS: Randomized,open-label,phase 2 clinical trial comparing selumetinib vs chemotherapy conducted from August 2010 through December 2013 among 120 patients with metastatic uveal melanoma at 15 academic oncology centers in the United States and Canada. INTERVENTIONS: One hundred one patients were randomized in a 1:1 ratio to receive selumetinib,75 mg orally twice daily on a continual basis (n = 50),or chemotherapy (temozolomide,150 mg/m2 orally daily for 5 of every 28 days,or dacarbazine,1000 mg/m2 intravenously every 21 days [investigator choice]; n = 51) until disease progression,death,intolerable adverse effects,or withdrawal of consent. After primary outcome analysis,19 patients were registered and 18 treated with selumetinib without randomization to complete the planned 120-patient enrollment. Patients in the chemotherapy group could receive selumetinib at the time of radiographic progression. MAIN OUTCOMES AND MEASURES: Progression-free survival,the primary end point,was assessed as of April 22,2013. Additional end points,including overall survival,response rate,and safety/toxicity,were assessed as of December 31,2013. RESULTS: Median progression-free survival among patients randomized to chemotherapy was 7 weeks (95% CI,4.3-8.4 weeks; median treatment duration,8 weeks; interquartile range [IQR],4.3-16 weeks) and among those randomized to selumetinib was 15.9 weeks (95% CI,8.4-21.1 weeks; median treatment duration,16.1 weeks; IQR,8.1-25.3 weeks) (hazard ratio,0.46; 95% CI,0.30-0.71; P textless .001). Median overall survival time was 9.1 months (95% CI,6.1-11.1 months) with chemotherapy and 11.8 months (95% CI,9.8-15.7 months) with selumetinib (hazard ratio,0.66; 95% CI,0.41-1.06; P = .09). No objective responses were observed with chemotherapy. Forty-nine percent of patients treated with selumetinib achieved tumor regression,with 14% achieving an objective radiographic response to therapy. Treatment-related adverse events were observed in 97% of patients treated with selumetinib,with 37% requiring at least 1 dose reduction. CONCLUSIONS AND RELEVANCE: In this hypothesis-generating study of patients with advanced uveal melanoma,selumetinib compared with chemotherapy resulted in a modestly improved progression-free survival and response rate; however,no improvement in overall survival was observed. Improvement in clinical outcomes was accompanied by a high rate of adverse events. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01143402. View Publication

Pluripotent stem cells display significant heterogeneity in gene expression,but whether this diversity is an inherent feature of the pluripotent state remains unknown. Single-cell gene expression analysis in cell subsets defined by surface antigen expression revealed that human embryonic stem cell cultures exist as a continuum of cell states,even under defined conditions that drive self-renewal. The majority of the population expressed canonical pluripotency transcription factors and could differentiate into derivatives of all three germ layers. A minority subpopulation of cells displayed high self-renewal capacity,consistently high transcripts for all pluripotency-related genes studied,and no lineage priming. This subpopulation was characterized by its expression of a particular set of intercellular signaling molecules whose genes shared common regulatory features. Our data support a model of an inherently metastable self-renewing population that gives rise to a continuum of intermediate pluripotent states,which ultimately become primed for lineage specification. ?? 2014 The Authors. View Publication

Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However,scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard,suspension cultures are a viable alternative,because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However,the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here,we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production. ?? 2014 The Authors. View Publication

NK cells are innate immune cells known for their cytolytic activities toward tumors and infections. They are capable of expressing diverse killer Ig-like receptors (KIRs),and KIRs are implicated in susceptibility to Crohn's disease (CD),a chronic intestinal inflammatory disease. However,the cellular mechanism of this genetic contribution is unknown. In this study,we show that the licensing" of NK cells� View Publication

We evaluated a cocktail of HLA-A2-specific peptides including heteroclitic XBP1 US184-192 (YISPWILAV),heteroclitic XBP1 SP367-375 (YLFPQLISV),native CD138260-268 (GLVGLIFAV) and native CS1239-247 (SLFVLGLFL),for their ability to elicit multipeptide-specific cytotoxic T lymphocytes (MP-CTLs) using T cells from smoldering multiple myeloma (SMM) patients. Our results demonstrate that MP-CTLs generated from SMM patients' T cells show effective anti-MM responses including CD137 (4-1BB) upregulation,CTL proliferation,interferon-γ production and degranulation (CD107a) in an HLA-A2-restricted and peptide-specific manner. Phenotypically,we observed increased total CD3(+)CD8(+) T cells (textgreater80%) and cellular activation (CD69(+)) within the memory SMM MP-CTL (CD45RO(+)/CD3(+)CD8(+)) subset after repeated multipeptide stimulation. Importantly,SMM patients could be categorized into distinct groups by their level of MP-CTL expansion and antitumor activity. In high responders,the effector memory (CCR7(-)CD45RO(+)/CD3(+)CD8(+)) T-cell subset was enriched,whereas the remaining responders' CTL contained a higher frequency of the terminal effector (CCR7(-)CD45RO(-)/CD3(+)CD8(+)) subset. These results suggest that this multipeptide cocktail has the potential to induce effective and durable memory MP-CTL in SMM patients. Therefore,our findings provide the rationale for clinical evaluation of a therapeutic vaccine to prevent or delay progression of SMM to active disease. View Publication

Most patients with acute myelogenous leukemia (AML) relapse and die of their disease. Increasing evidence indicates that AML relapse is driven by the inability to eradicate leukemia stem cells (LSC). Thus,it is imperative to identify novel therapies that can ablate LSCs. Using an in silico gene expression-based screen for compounds evoking transcriptional effects similar to the previously described anti-LSC agent parthenolide,we identified AR-42 (OSU-HDAC42),a novel histone deacetylase inhibitor that is structurally similar to phenylbutyrate,but with improved activity at submicromolar concentrations. Here,we report that AR-42 induces NF-κB inhibition,disrupts the ability of Hsp90 to stabilize its oncogenic clients,and causes potent and specific cell death of LSCs but not normal hematopoietic stem and progenitor cells. Unlike parthenolide,the caspase-dependent apoptosis caused by AR-42 occurs without activation of Nrf-2-driven cytoprotective pathways. As AR-42 is already being tested in early clinical trials,we expect that our results can be extended to the clinic. View Publication

Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex,undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices,we systematically developed an optimized cardiac differentiation strategy,using a chemically defined medium consisting of just three components: the basal medium RPMI 1640,L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation,this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification. View Publication

Accumulating evidence suggests elements within tumors induce exhaustion of effector T cells and infiltration of immunosuppressive regulatory T cells (Tregs),thus preventing the development of durable antitumor immunity. Therefore,the discovery of agents that simultaneously block Treg suppressive function and reinvigorate effector function of lymphocytes is key to the development of effective cancer immunotherapy. Previous studies have shown that TLR ligands (TLRLs) could modulate the function of these T cell targets; however,those studies relied on cell-free or accessory cell-based assay systems that do not accurately reflect in vivo responses. In contrast,we used a human PBMC-based proliferation assay system to simultaneously monitor the effect of TLRLs on T cells (CD4(+),CD8(+),Tregs),B cells,and NK cells,which gave different and even conflicting results. We found that the TLR7/8L:CL097 could simultaneously activate CD8(+) T cells,B cells,and NK cells plus block Treg suppression of T cells and B cells. The TLRLs TLR1/2L:Pam3CSK4,TLR5L:flagellin,TLR4L:LPS,and TLR8/7L:CL075 also blocked Treg suppression of CD4(+) or CD8(+) T cell proliferation,but not B cell proliferation. Besides CL097,TLR2L:PGN,CL075,and TLR9L:CpG-A,CpG-B,and CpG-C) were strong activators of NK cells. Importantly,we found that Pam3CSK4 could: 1) activate CD4(+) T cell proliferation,2) inhibit the expansion of IL-10(+) naturally occurring FOXP3(+) Tregs and induction of IL-10(+) CD4(+) Tregs (IL-10-producing type 1 Treg),and 3) block naturally occurring FOXP3(+) Tregs suppressive function. Our results suggest these agents could serve as adjuvants to enhance the efficacy of current immunotherapeutic strategies in cancer patients. View Publication