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Crosstalk between intracellular signaling pathways has been extensively studied to understand the pluripotency of human pluripotent stem cells (hPSCs),including human embryonic stem cells and human induced pluripotent stem cells (hiPSCs); however,the contribution of NAD(+) -dependent pathways remains largely unknown. Here,we show that NAD(+) depletion by FK866 (a potent inhibitor of NAD(+) biosynthesis) was fatal in hPSCs,particularly when deriving pluripotent cells from somatic cells and maintaining pluripotency. NAD and its precursors (nicotinamide [NAM] and nicotinic acid) fully replenished the NAD(+) depletion by FK866 in hPSCs. However,only NAM effectively enhanced the reprogramming efficiency and kinetics of hiPSC generation and was also significantly advantageous for the maintenance of undifferentiated hPSCs. Our molecular and functional studies reveal that NAM lowers the barriers to reprogramming by accelerating cell proliferation and protecting cells from apoptosis and senescence by alleviating oxidative stress,reactive oxygen species accumulation,and subsequent mitochondrial membrane potential collapse. We provide evidence that the positive effects of NAM (occurring at concentrations well above the physiological range) on pluripotency control are molecularly associated with the repression of p53,p21,and p16. Our findings establish that adequate intracellular NAD(+) content is crucial for pluripotency; the distinct effects of NAM on pluripotency may be dependent not only on its metabolic advantage as a NAD(+) precursor but also on the ability of NAM to enhance resistance to cellular stress. View Publication

Background: The ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP). Method: Embryoid bodies were made from a hiPS cell line reprogrammed with Oct4,Nanog,Lin28 and Sox2. Sharp microelectrodes were used to record APs from beating-clusters (BC) and patch-clamp techniques were used to record Ito in single hiPSC-CM. mRNA levels of Kv1.4,KChIP2 and Kv4.3 were quantified from BCs. Results: BCs exhibited spontaneous beating (60.5??2.6bpm) and maximum-diastolic-potential (MDP) of 67.8??0.8mV (n=155). A small 4-aminopyridine-sensitive phase-1-repolarization was observed in only 6/155 BCs. A robust Ito was recorded in the majority of cells (13.7??1.9 pA/pF at +40mV; n=14). Recovery of Ito from inactivation (at -80mV) showed slow kinetics (??1=200??110ms (12%) and ??2=2380??240ms (80%)) accounting for its minimal contribution to the AP. Transcript data revealed relatively high expression of Kv1.4 and low expression of KChIP2 compared to human native ventricular tissues. Mathematical modeling predicted that restoration of IK1 to normal levels would result in a more negative MDP and a prominent phase-1-repolarization. Conclusion: The slow recovery kinetics of Ito coupled with a depolarized MDP account for the lack of an AP notch in the majority of hiPSC-CM. These characteristics reveal a deficiency for the development of in vitro models of inherited cardiac arrhythmia syndromes in which Ito-induced AP notch is central to the disease phenotype. ?? 2013 Elsevier Ltd. View Publication

Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study,we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4,hSox2,hKlf4,and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci,a hallmark of DM1,were detected in DM1 iPSCs,neural stem cells (NSCs),and terminally differentiated neurons and astrocytes. In conclusion,we have successfully established disease-specific human DM1 iPSC lines,NSCs,and neuronal lineages with pathognomonic intranuclear RNA foci,which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development. View Publication

We demonstrate substantial differences in 'adhesive signature' between human pluripotent stem cells (hPSCs),partially reprogrammed cells,somatic cells and hPSC-derived differentiated progeny. We exploited these differential adhesion strengths to rapidly (over approximately 10 min) and efficiently isolate fully reprogrammed induced hPSCs (hiPSCs) as intact colonies from heterogeneous reprogramming cultures and from differentiated progeny using microfluidics. hiPSCs were isolated label free,enriched to 95%-99% purity with textgreater80% survival,and had normal transcriptional profiles,differentiation potential and karyotypes. We also applied this strategy to isolate hPSCs (hiPSCs and human embryonic stem cells) during routine culture and show that it may be extended to isolate hPSC-derived lineage-specific stem cells or differentiated cells. View Publication

Raman spectra often contain undesirable,randomly positioned,intense,narrow-bandwidth,positive,unidirectional spectral features generated when cosmic rays strike charge-coupled device cameras. These must be removed prior to analysis,but doing so manually is not feasible for large data sets. We developed a quick,simple,effective,semi-automated procedure to remove cosmic ray spikes from spectral data sets that contain large numbers of relatively homogenous spectra. Although some inhomogeneous spectral data sets can be accommodated—it requires replacing excessively modified spectra with the originals and removing their spikes with a median filter instead—caution is advised when processing such data sets. In addition,the technique is suitable for interpolating missing spectra or replacing aberrant spectra with good spectral estimates. The method is applied to baseline-flattened spectra and relies on fitting a third-order (or higher) polynomial through all the spectra at every wavenumber. Pixel intensities in excess of a threshold of 3× the noise standard deviation above the fit are reduced to the threshold level. Because only two parameters (with readily specified default values) might require further adjustment,the method is easily implemented for semi-automated processing of large spectral sets. View Publication

BackgroundmicroRNAs (miRNAs) have been implicated in the control of many biological processes and their deregulation has been associated with many cancers. In recent years,the cancer stem cell (CSC) concept has been applied to many cancers including pediatric. We hypothesized that a common signature of deregulated miRNAs in the CSCs fraction may explain the disrupted signaling pathways in CSCs.Methodology/ResultsUsing a high throughput qPCR approach we identified 26 CSC associated differentially expressed miRNAs (DEmiRs). Using BCmicrO algorithm 865 potential CSC associated DEmiR targets were obtained. These potential targets were subjected to KEGG,Biocarta and Gene Ontology pathway and biological processes analysis. Four annotated pathways were enriched: cell cycle,cell proliferation,p53 and TGF-beta/BMP. Knocking down hsa-miR-21-5p,hsa-miR-181c-5p and hsa-miR-135b-5p using antisense oligonucleotides and small interfering RNA in cell lines led to the depletion of the CSC fraction and impairment of sphere formation (CSC surrogate assays).ConclusionOur findings indicated that CSC associated DEmiRs and the putative pathways they regulate may have potential therapeutic applications in pediatric cancers. View Publication

Reprogramming of somatic cells into patient-specific pluripotent analogues of human embryonic stem cells (ESCs) emerges as a prospective therapeutic angle in molecular medicine and a tool for basic stem cell biology. However,the combination of relative inefficiency and high variability of non-defined culture conditions precluded the use of this technique in a clinical setting and impeded comparability between laboratories. To overcome these obstacles,we sequentially devised a reprogramming protocol using one lentiviral-based polycistronic reprogramming construct,optimized for high co-expression of OCT4,SOX2,KLF4 and MYC in conjunction with small molecule inhibitors of non-permissive signaling cascades,such as transforming growth factor $\$(SB431542),MEK/ERK (PD0325901) and Rho-kinase signaling (Thiazovivin),in a defined extracellular environment. Based on human fetal liver fibroblasts we could efficiently derive induced pluripotent stem cells (iPSCs) within 14 days. We attained efficiencies of up to 10.97±1.71% resulting in 79.5- fold increase compared to non-defined reprogramming using four singular vectors. We show that the overall increase of efficiency and temporal kinetics is a combinatorial effect of improved lentiviral vector design,signaling inhibition and definition of extracellular matrix (Matrigel®) and culture medium (mTESR®1). Using this protocol,we could derive iPSCs from patient fibroblasts,which were impermissive to classical reprogramming efforts,and from a patient suffering from familial platelet disorder. Thus,our defined protocol for highly efficient reprogramming to generate patient-specific iPSCs,reflects a big step towards therapeutic and broad scientific application of iPSCs,even in previously unfeasible settings. View Publication

Human embryonic stem cells and mouse epiblast stem cells represent a primed pluripotent stem cell state that requires TGF-β/activin signaling. TGF-β and/or activin are commonly thought to regulate transcription through both Smad2 and Smad3. However,the different contributions of these two Smads to primed pluripotency and the downstream events that they may regulate remain poorly understood. We addressed the individual roles of Smad2 and Smad3 in the maintenance of primed pluripotency. We found that Smad2,but not Smad3,is required to maintain the undifferentiated pluripotent state. We defined a Smad2 regulatory circuit in human embryonic stem cells and mouse epiblast stem cells,in which Smad2 acts through binding to regulatory promoter sequences to activate Nanog expression while in parallel repressing autocrine bone morphogenetic protein signaling. Increased autocrine bone morphogenetic protein signaling caused by Smad2 down-regulation leads to cell differentiation toward the trophectoderm,mesoderm,and germ cell lineages. Additionally,induction of Cdx2 expression,as a result of decreased Smad2 expression,leads to repression of Oct4 expression,which,together with the decreased Nanog expression,accelerates the loss of pluripotency. These findings reveal that Smad2 is a unique integrator of transcription and signaling events and is essential for the maintenance of the mouse and human primed pluripotent stem cell state. View Publication

A satisfactory in vitro model of vocal fold mucosa does not exist,thus precluding a systematic,controlled study of vocal fold biology and biomechanics. We sought to create a valid,reproducible three-dimensional (3D) in vitro model of human origin of vocal fold mucosa of human origin. We hypothesized that coculture of human embryonic stem cell (hESC)-derived simple epithelial cells with primary vocal fold fibroblasts under appropriate conditions would elicit morphogenesis of progenitor cells into vocal fold epithelial-like cells and creation of a basement membrane. Using an in vitro prospective study design,hESCs were differentiated into cells that coexpressed the simple epithelial cell marker,keratin 18 (K18),and the transcription factor,p63. These simple epithelial cells were cocultured with primary vocal fold fibroblasts seeded in a collagen gel scaffold. The cells were cultured for 3 weeks in a keratinocyte medium at an air–liquid interface. After that time,the engineered mucosa demonstrated a stratified,squamous epithelium and a continuous basement membrane recapitulating the key morphologic and phenotypic characteristics of native vocal fold mucosa. hESC-derived epithelial cells exhibited positive staining for vocal fold stratified,squamous epithelial markers,keratin 13 (K13) and 14 (K14),as well as tight junctions,adherens junctions,gap junctions,and desmosomes. Despite the presence of components critical for epithelial structural integrity,the epithelium demonstrated greater permeability than native tissue indicating compromised functional integrity. While further work is warranted to improve functional barrier integrity,this study demonstrates that hESC-derived epithelial progenitor cells can be engineered to create a replicable 3D in vitro model of vocal fold mucosa featuring a multilayered,terminally differentiated epithelium. View Publication

Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration,conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ESCs (hESCs) contain 5-hydroxymethylcytosine (5hmC),which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly owing to TET1 activation,and this hydroxymethylation change is critical for optimal epigenetic reprogramming,but does not compromise primed pluripotency. Compared with hESCs,we find that iPSCs tend to form large-scale (100 kb–1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions,most of which exhibit incomplete hydroxymethylation on CG sites. Strikingly,these 5hmC aberrant hotspots largely coincide (∼ 80%) with aberrant iPSC–ESC non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute to the epigenetic variation of iPSCs and iPSC–hESC differences. View Publication

Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of,for example,ion selectivity,gating mechanism,composition,or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs) and their somatic cell source,keratinocytes from plucked human hair. This comparison revealed that 26&x25; of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6&x25; were downregulated. Additionally,iPSCs express a much higher number of ion channels compared to keratinocytes. Further,to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny,namely,neurons and cardiomyocytes derived from iPS cells. To conclude,hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms. View Publication

Definitive endoderm can be derived from human embryonic stem cells using low serum medium with cytokines involved in the epithelial-to-mesenchymal transition,including Activin A and Wnt3A. The purpose of this study was to develop an improved protocol that permits the induction of definitive endoderm while avoiding the high rate of cell death that often occurs with existing protocols. By including insulin and other nutrients,we demonstrate that cell viability can be preserved throughout differentiation. In addition,modifying a matrigel sandwich method previously reported to induce precardiac mesoderm allows for enhanced endodermal differentiation based on expression of endoderm-associated genes. The morphological and migratory characteristics of cells cultured by the technique,as well as gene expression patterns,indicate that the protocol can emulate key events in gastrulation towards the induction of definitive endoderm. View Publication