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BACKGROUND Accumulating evidence suggests that some long noncoding RNAs (lncRNAs) are involved in certain diseases,such as cancer. The lncRNA,CCDC26,is related to childhood acute myeloid leukemia (AML) because its copy number is altered in AML patients. RESULTS We found that CCDC26 transcripts were abundant in the nuclear fraction of K562 human myeloid leukemia cells. To examine the function of CCDC26,gene knockdown (KD) was performed using short hairpin RNAs (shRNAs),and four KD clones,in which CCDC26 expression was suppressed to 1% of its normal level,were isolated. This down-regulation included suppression of CCDC26 intron-containing transcripts (the CCDC26 precursor mRNA),indicating that transcriptional gene suppression (TGS),not post-transcriptional suppression,was occurring. The shRNA targeting one of the two CCDC26 splice variants also suppressed the other splice variant,which is further evidence for TGS. Growth rates of KD clones were reduced compared with non-KD control cells in media containing normal or high serum concentrations. In contrast,enhanced growth rates in media containing much lower serum concentrations and increased survival periods after serum withdrawal were observed for KD clones. DNA microarray and quantitative polymerase chain reaction screening for differentially expressed genes between KD clones and non-KD control cells revealed significant up-regulation of the tyrosine kinase receptor,KIT,hyperactive mutations of which are often found in AML. Treatment of KD clones with ISCK03,a KIT-specific inhibitor,eliminated the increased survival of KD clones in the absence of serum. CONCLUSIONS We suggest that CCDC26 controls growth of myeloid leukemia cells through regulation of KIT expression. A KIT inhibitor might be an effective treatment against the forms of AML in which CCDC26 is altered. View Publication

Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis,and in neurons,synaptic vesicle assembly,neurotransmission,and plasticity. Protein networks,or interactomes,downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. Here,we conducted a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. We identified components of the vesicle fusion machinery as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons,among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa,and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function,we examined the role for NSF in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described,we found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However,neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. Our results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity. View Publication

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes,cells from the corneal stroma,may have the potential for restoration of vision in cell therapy and biomedical engineering applications,but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells,maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype. View Publication

Human pluripotent stem cells (hPSCs),such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),provide a powerful model system for studies of cellular identity and early mammalian development,which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein,a simple and reliable biosensor for stem cell detection was established. In this biosensor system,stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment,and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells,showing that it is promising for specific and handy detection of human pluripotent stem cells. View Publication

PURPOSE We conducted an open-label,single-arm Phase I/II clinical trial in metastatic CRPC (mCRPC) patients eligible for docetaxel combined with treatment with autologous mature dendritic cells (DCs) pulsed with killed LNCaP prostate cancer cells (DCVAC/PCa). The primary and secondary endpoints were safety and immune responses,respectively. Overall survival (OS),followed as a part of the safety evaluation,was compared to the predicted OS according to the Halabi and MSKCC nomograms. EXPERIMENTAL DESIGN Twenty-five patients with progressive mCRPC were enrolled. Treatment comprised of initial 7 days administration of metronomic cyclophosphamide 50 mg p.o. DCVAC/PCa treatment consisted of a median twelve doses of 1 × 107 dendritic cells per dose injected s.c. (Aldara creme was applied at the site of injection) during a one-year period. The initial 2 doses of DCVAC/PCa were administered at a 2-week interval,followed by the administration of docetaxel (75 mg/m2) and prednisone (5 mg twice daily) given every 3 weeks until toxicity or intolerance was observed. The DCVAC/PCa was then injected every 6 weeks up to the maximum number of doses manufactured from one leukapheresis. RESULTS No serious DCVAC/PCa-related adverse events have been reported. The median OS was 19 months,whereas the predicted median OS was 11.8 months with the Halabi nomogram and 13 months with the MSKCC nomogram. Kaplan-Meier analyses showed that patients had a lower risk of death compared with both MSKCC (Hazard Ratio 0.26,95% CI: 0.13-0.51) and Halabi (Hazard Ratio 0.33,95% CI: 0.17-0.63) predictions. We observed a significant decrease in Tregs in the peripheral blood. The long-term administration of DCVAC/PCa led to the induction and maintenance of PSA specific T cells. We did not identify any immunological parameter that significantly correlated with better OS. CONCLUSIONS In patients with mCRPC,the combined chemoimmunotherapy with DCVAC/PCa and docetaxel was safe and resulted in longer than expected survival. Concomitant chemotherapy did not preclude the induction of specific anti-tumor cytotoxic T cells. View Publication

Tissue morphogenesis and organ formation are the consequences of biochemical and biophysical cues that lead to cellular spatial patterning in development. To model such events in vitro,we use PEG-patterned substrates to geometrically confine human pluripotent stem cell colonies and spatially present mechanical stress. Modulation of the WNT/β-catenin pathway promotes spatial patterning via geometric confinement of the cell condensation process during epithelial-mesenchymal transition,forcing cells at the perimeter to express an OCT4+ annulus,which is coincident with a region of higher cell density and E-cadherin expression. The biochemical and biophysical cues synergistically induce self-organizing lineage specification and creation of a beating human cardiac microchamber confined by the pattern geometry. These highly defined human cardiac microchambers can be used to study aspects of embryonic spatial patterning,early cardiac development and drug-induced developmental toxicity. View Publication

Substrate composition significantly impacts human pluripotent stem cell (hPSC) self-renewal and differentiation,but relatively little is known about the role of endogenously produced extracellular matrix (ECM) components in regulating hPSC fates. Here we identify $\$-5 laminin as a signature ECM component endogenously synthesized by undifferentiated hPSCs cultured on defined substrates. Inducible shRNA knockdown and Cas9-mediated disruption of the LAMA5 gene dramatically reduced hPSC self-renewal and increased apoptosis without affecting the expression of pluripotency markers. Increased self-renewal and survival was restored to wild-type levels by culturing the LAMA5-deficient cells on exogenous laminin-521. Furthermore,treatment of LAMA5-deficient cells with blebbistatin or a ROCK inhibitor partially restored self-renewal and diminished apoptosis. These results demonstrate that endogenous $\$-5 laminin promotes hPSC self-renewal in an autocrine and paracrine manner. This finding has implications for understanding how stem cells dynamically regulate their microenvironment to promote self-renewal and provides guidance for efforts to design substrates for stem cell bioprocessing. View Publication

GTF2IRD1 is one of the three members of the GTF2I gene family,clustered on chromosome 7 within a 1.8 Mb region that is prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. These copy number variations disturb a variety of developmental systems and neurological functions. Human mapping data and analyses of knockout mice show that GTF2IRD1 and GTF2I underpin the craniofacial abnormalities,mental retardation,visuospatial deficits and hypersociability of WBS. However,the cellular role of the GTF2IRD1 protein is poorly understood due to its very low abundance and a paucity of reagents. Here,for the first time,we show that endogenous GTF2IRD1 has a punctate pattern in the nuclei of cultured human cell lines and neurons. To probe the functional relationships of GTF2IRD1 in an unbiased manner,yeast two-hybrid libraries were screened,isolating 38 novel interaction partners,which were validated in mammalian cell lines. These relationships illustrate GTF2IRD1 function,as the isolated partners are mostly involved in chromatin modification and transcriptional regulation,whilst others indicate an unexpected role in connection with the primary cilium. Mapping of the sites of protein interaction also indicates key features regarding the evolution of the GTF2IRD1 protein. These data provide a visual and molecular basis for GTF2IRD1 nuclear function that will lead to an understanding of its role in brain,behaviour and human disease. View Publication

Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins,the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of the Pneumovirinae F proteins,and as extension of previous work (Palomo et al.,2014),a general strategy was designed to obtain polyclonal and particularly monoclonal antibodies specific of the 6HB motif of the Pneumovirinae fusion protein. The antibodies reported here should assist in the characterization of the structural changes that the F protein of human metapneumovirus or respiratory syncytial virus experiences during the process of membrane fusion. View Publication

The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA,nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently,there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions,we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription� View Publication

Significant efforts have been invested into the differentiation of stem cells into functional hepatocyte-like cells that can be used for cell therapy,disease modeling and drug screening. Most of these efforts have been concentrated on the use of growth factors to recapitulate developmental signals under in vitro conditions. Using small molecules instead of growth factors would provide an attractive alternative since small molecules are cell-permeable and cheaper than growth factors. We have developed a protocol for the differentiation of human embryonic stem cells into hepatocyte-like cells using a predominantly small molecule-based approach (SM-Hep). This 3 step differentiation strategy involves the use of optimized concentrations of LY294002 and bromo-indirubin-3'-oxime (BIO) for the generation of definitive endoderm; sodium butyrate and dimethyl sulfoxide (DMSO) for the generation of hepatoblasts and SB431542 for differentiation into hepatocyte-like cells. Activin A is the only growth factor required in this protocol. Our results showed that SM-Hep were morphologically and functionally similar or better compared to the hepatocytes derived from the growth-factor induced differentiation (GF-Hep) in terms of expression of hepatic markers,urea and albumin production and cytochrome P450 (CYP1A2 and CYP3A4) activities. Cell viability assays following treatment with paradigm hepatotoxicants Acetaminophen,Chlorpromazine,Diclofenac,Digoxin,Quinidine and Troglitazone showed that their sensitivity to these drugs was similar to human primary hepatocytes (PHHs). Using SM-Hep would result in 67% and 81% cost reduction compared to GF-Hep and PHHs respectively. Therefore,SM-Hep can serve as a robust and cost effective replacement for PHHs for drug screening and development. 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