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There is ample evidence that the ubiquitin-proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover,proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells,acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein,we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4,and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog,but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion,our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however,efficient colony formation requires proteasome activity. Therefore,discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells. View Publication

Helper-dependent adenoviral vectors (HDAd) that express certain transgene products are impossible to produce because the transgene product is toxic to the producer cells,especially when made in large amounts during vector production. Downregulating transgene expression from the HDAd during vector production is a way to solve this problem. In this report,we show that this can be accomplished by inserting the target sequence for the adenoviral VA RNAI into the 3' untranslated region of the expression cassette in the HDAd. Thus during vector production,when the producer cells are coinfected with both the helper virus (HV) and the HDAd,the VA RNAI produced by the HV will target the transgene mRNA from the HDAd via the endogenous cellular RNAi pathway. Once the HDAd is produced and purified,transduction of the target cells results in unimpeded transgene expression because of the absence of HV. This simple and universal strategy permits for the robust production of otherwise recalcitrant HDAds. View Publication

ALS is a rapidly progressive,devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression,and molecular insights into pathogenesis and progression are sorely needed. In that context,we used high-depth,next generation RNA sequencing (RNAseq,Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned textgreater50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2,DEseq2,EdgeR) for identification of differentially expressed genes (DEG's). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples,with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNF$$-induced protein 2 (TNFAIP2) as a major network hub" gene (WGCNA). Using the oPOSSUM algorithm� View Publication

Utilizing a fast 31P magnetic resonance spectroscopy (MRS) 2-dimensional chemical shift imaging (2D-CSI) method,this study examined the heterogeneity of creatine kinase (CK) forward flux rate of hearts with postinfarction left ventricular (LV) remodeling. Immunosuppressed Yorkshire pigs were assigned to 4 groups: 1) A sham-operated normal group (SHAM,n = 6); 2) A 60 minutes distal left anterior descending coronary artery ligation and reperfusion (MI,n = 6); 3) Open patch group; ligation injury plus open fibrin patch over the site of injury (Patch,n = 6); and 4) Cell group,hiPSCs-cardiomyocytes,-endothelial cells,and -smooth muscle cells (2 million,each) were injected into the injured myocardium pass through a fibrin patch (Cell+Patch,n = 5). At 4 weeks,the creatine phosphate (PCr)/ATP ratio,CK forward flux rate (Flux PCr→ATP),and k constant of CK forward flux rate (kPCr→ATP) were severely decreased at border zone myocardium (BZ) adjacent to MI. Cell treatment results in significantly increase of PCr/ATP ratio and improve the value of kPCr→ATP and Flux PCr→ATP in BZ myocardium. Moreover,the BZ myocardial CK total activity and protein expression of CK mitochondria isozyme and CK myocardial isozyme were significantly reduced,but recovered in response to cell treatment. Thus,cell therapy results in improvement of BZ bioenergetic abnormality in hearts with postinfarction LV remodeling,which is accompanied by significantly improvements in BZ CK activity and CK isozyme expression. The fast 2D 31P MR CSI mapping can reliably measure the heterogeneity of bioenergetics in hearts with post infarction LV remodeling. View Publication

In the past decade,tissue microarray (TMA) technology has evolved as an innovative tool for high-throughput proteomics analysis and mainly for biomarker validation. Similarly,enormous amount of data can be obtained from the cell line macroarray (CLMA) technology,which developed from the TMA using formalin-fixed,paraffin-embedded cell pellets. Here,we applied CLMA technology in stem cell research and in particular to identify bona fide neogenerated human induced pluripotent stem cell (hiPSC) clones suitable for down the line differentiation. All hiPSC protocols generate tens of clones,which need to be tested to determine genetically stable cell lines suitable for differentiation. Screening methods generally rely on fluorescence-activated cell sorting isolation and coverslip cell growth followed by immunofluorescence; these techniques could be cumbersome. Here,we show the application of CLMA to identify neogenerated pluripotent cell colonies and neuronal differentiated cell products. We also propose the use of the automated image analyzer,TissueQuest,as a reliable tool to quickly select the best clones,based upon the level of expression of multiple pluripotent biomarkers. View Publication

Myocardial ischemia-reperfusion (I/R) injury is unavoidable during cardioplegic arrest and open-heart surgery. Danshen is one of the most popular traditional herbal medicines in China,which has entered the Food and Drug Administration-approved phase III clinical trial. This study was aimed to develop a human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) model to mimic I/R injury and evaluate the cardioprotective effect of regular cardioplegic solution with Danshen. hiPSC-CMs were cultured with the crystalloid cardioplegic solution (Thomas group) and Thomas solution with 2 or 10 µg/mL Danshen (Thomas plus Danshen groups). The cells under normoxic culture condition served as baseline group. Then,the cells were placed in a modular incubator chamber. After 45 min hypoxia and 3 h reoxygenation,hiPSC-CMs subjected to hypoxia/reoxygenation resulted in a sharp increase of reactive oxygen species (ROS) content in Thomas group versus baseline group. Compared with the Thomas group,ROS accumulation was significant suppressed in Thomas plus Danshen groups,which might result from elevating the content of glutathione and enhanced activities of superoxide dismutase and glutathione peroxidase. The enhanced L-type Ca(2+) current in hiPSC-CMs after I/R injury was also significantly decreased by Danshen,and meanwhile intracellular Ca(2+) level was reduced and calcium overload was suppressed. Thomas plus Danshen groups also presented less irregular transients and lower apoptosis rates. As a result,Danshen could improve antioxidant and calcium handling in cardiomyocytes during I/R and lead to reduced arrhythmia events and apoptosis rates. hiPSC-CMs model offered a platform for the future translational study of the cardioplegia. View Publication

A patient specific point mutation (c.1288GtextgreaterT) of Men1 gene was introduced into wide type iPSC line with CRISPR/Cas9 and single-stranded donor oligonucleotides carrying the mutation. The mutated iPSC line has a heterozygous c.1288GtextgreaterT mutation on exon-9 of Men1 that was confirmed by sequencing analysis. The karyotype of this line was normal and the pluripotency was demonstrated by its ability to differentiate into three germ layers. These artificially created Men1 mutation in wild type iPSC line will help to dissect out the molecular basis of two patients carried the same mutation from one family who were differentially represented hypoglycemia. View Publication

Approximately 70% of KRAS-positive colorectal cancers (CRCs) have a CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes. The factors involved in,and the mechanistic basis of,CIMP is not understood. Among the CIMP genes are the tumor suppressors p14(ARF),p15(INK4B),and p16(INK4A),encoded by the INK4-ARF locus. In this study,we perform an RNA interference screen and identify ZNF304,a zinc-finger DNA-binding protein,as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS. In KRAS-positive human CRC cell lines and tumors,ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes. Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1,resulting in DNA hypermethylation and transcriptional silencing. KRAS promotes silencing through upregulation of ZNF304,which drives DNA binding. Finally,we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells. DOI: http://dx.doi.org/10.7554/eLife.02313.001. View Publication

Understanding the mechanisms that sustain pluripotency in human embryonic stem cells (hESCs) is an active area of research that may prove useful in regenerative medicine and will provide fundamental information relevant to development and cancer. hESCs and cancer cells share the unique ability to proliferate indefinitely and rapidly. Because the protein survivin is uniquely overexpressed in virtually all human cancers and in hESCs,we sought to investigate its role in supporting the distinctive capabilities of these cell types. Results presented here suggest that survivin contributes to the maintenance of pluripotency and that post-transcriptional control of survivin isoform expression is selectively regulated by microRNAs. miR-203 has been extensively studied in human tumors,but has not been characterized in hESCs. We show that miR-203 expression and activity is consistent with the expression and subcellular localization of survivin isoforms that in turn modulate expression of the Oct4 and Nanog transcription factors to sustain pluripotency. This study contributes to understanding of the complex regulatory mechanisms that govern whether hESCs proliferate or commit to lineages. textcopyright 2014 Wiley Periodicals,Inc. 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 histo-blood group i and I antigens have been characterized as straight and branched repeats of N-acetyllactosamine,respectively,and the conversion of the straight-chain i to the branched-chain I structure on red cells is regulated to occur after birth. It has been demonstrated that the human I locus expresses 3 IGnT transcripts,IGnTA,IGnTB,and IGnTC,and that the last of these is responsible for the I branching formation on red cells. In the present investigation,the K-562 cell line was used as a model to show that the i-to-I transition in erythroid differentiation is determined by the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha),which enhances transcription of the IGnTC gene,consequently leading to formation of the I antigen. Further investigation suggested that C/EBPalpha IGnTC-activation activity is modulated at a posttranslational level,and that the phosphorylation status of C/EBPalpha may have a crucial effect. Results from studies using adult and cord erythropoietic cells agreed with those derived using the K-562 cell model,with lentiviral expression of C/EBPalpha in CD34(+) hemopoietic cells demonstrating the determining role of C/EBPalpha in the induction of the IGnTC gene as well as in I antigen expression. View Publication