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Human embryonic stem (hES) cells show an atypical cell-cycle regulation characterized by a high proliferation rate and a short G1 phase. In fact,a shortened G1 phase might protect ES cells from external signals inducing differentiation,as shown for certain stem cells. It has been suggested that self-renewal and pluripotency are intimately linked to cell-cycle regulation in ES cells,although little is known about the overall importance of the cell-cycle machinery in maintaining ES cell identity. An appealing model to address whether the acquisition of stem cell properties is linked to cell-cycle regulation emerged with the ability to generate induced pluripotent stem (iPS) cells by expression of defined transcription factors. Here,we show that the characteristic cell-cycle signature of hES cells is acquired as an early event in cell reprogramming. We demonstrate that induction of cell proliferation increases reprogramming efficiency,whereas cell-cycle arrest inhibits successful reprogramming. Furthermore,we show that cell-cycle arrest is sufficient to drive hES cells toward irreversible differentiation. Our results establish a link that intertwines the mechanisms of cell-cycle control with the mechanisms underlying the acquisition and maintenance of ES cell identity. View Publication

Meticulous characterization of human embryonic stem cells (hESC) is critical to their eventual use in cell-based therapies,particularly in view of the diverse methods for derivation and maintenance of these cell lines. However,characterization methods are generally not standardized and many currently used assays are subjective,making dependable and direct comparison of cell lines difficult. In order to address this problem,we selected 10 molecular-based high-resolution assays as components of a panel for characterization of hESC. The selection of the assays was primarily based on their quantitative or objective (rather than subjective) nature. We demonstrate the efficacy of this panel by characterizing 4 hESC lines,derived in two different laboratories using different derivation techniques,as pathogen free,genetically stable,and able to differentiate into derivatives of all three germ layers. Our panel expands and refines a characterization panel previously proposed by the International Stem Cell Initiative and is another step toward standardized hESC characterization and quality control,a crucial element of successful hESC research and clinical translation. View Publication

There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals,thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide,the use of human cell-teratogenicity assays has just started to be explored. Herein,a human pluripotent stem cell test (hPST) for identifying teratogens is described,benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically,71 drug-like compounds with known in vivo effects,including thalidomide,were examined in the hPST. A threshold of 5μM demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore,15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally,to assess the suitability of the hPST for high-throughput screens,a small library of 300 kinase inhibitors was tested,demonstrating the hPST platform's utility for interrogating teratogenic mechanisms and drug safety prediction. Thus,the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models. View Publication

The Sox6 transcription factor plays critical roles in various cell types,including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells,we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar,evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo,and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied,in erythroid cells,by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation,mediated by the double Sox6 binding site within its own promoter,may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation. View Publication

Chemical sensing on the nanoscale has been breaking new ground since the discovery of surface enhanced Raman scattering (SERS). For nanoparticles,controlled particle aggregation is necessary to achieve the largest SERS enhancements. Therefore,aggregating agents such as salts or linker molecules are used in conjunction with chemically sensitive reporters in order to develop robust environmentally sensitive SERS probes. While salt-induced colloidal nanosphere aggregates have produced robust SERS signals,their variability in aggregate size contributes significantly to poor SERS signal reproducibility,which can complicate their use in in vitro cellular studies. Such systems often also lack reproducibility in spectral measurements between different nanoparticle clusters. Preaggregation of colloids via linkers followed by surface functionalization with reporter molecules results in the linker occupying valuable SERS hotspot volume which could otherwise be utilized by additional reporter molecules. Ideally,both functionalities should be obtained from a single molecule. Here,we report the use of 3,5-dimercaptobenzoic acid,a single multifunctional molecule that creates SERS hotspots via the controlled aggregation of nanoparticles,and also reports pH values. We show that 3,5-dimercaptobenzoic acid bound to Au nanospheres results in an excellent pH nanoprobe,producing very robust,and highly reproducible SERS signals that can report pH across the entire physiological range with excellent pH resolution. To demonstrate the efficacy of our novel pH reporters,these probes were also used to image both the particle and pH distribution in the cytoplasm of human induced pluripotent stem cells (hiPSCs). View Publication

Human embryonic stem cells (hESCs) can be differentiated into multiple cell types with great therapeutic potential. However,optimizing the often multi-week cultures to obtain sufficient differentiated cell yields has been in part limited by the high variability of even parallel hESC differentiation cultures. We describe the isolation and features of a subline of CA1 hESCs (CA1S) that display a very high 25% cloning efficiency while retaining many properties of the parental hESCs,including being karyotypically normal and their ability to generate teratomas containing all three germ layers. Although more detailed analysis revealed that CA1S cells have a 3.8 Mb genomic duplication on chromosome 20,they remain highly useful. In particular,CA1S cells are readily expanded at high yields in culture and possess greatly reduced well-to-well variation even when seeded at 100 cells/well. Thus,108 CA1S cells can be generated within one week from 106 cells to seed 106 wells. We determined that CA1S cells have the capacity to follow established in vitro differentiation protocols to pancreatic progenitors and subsequent hormone-positive cell types and used CA1S cells to explore definitive endoderm induction in a high performance screen (Z-factor = 0.97). This system revealed that CA1S cells do not require WNT3A to efficiently form definitive endoderm,a finding that was confirmed with H1 hESCs,although H1 cells did show modest benefits of high WNT3A doses. Proliferative index measurements of CA1S cells were shown to rapidly reflect their differentiation status in a high throughput system. Though results obtained with CA1S cells will need to be confirmed using conventional hESC lines,these cells should ease the development of optimized hESC growth and differentiation protocols. In particular,they should limit the more arduous secondary screens using hESCs to a smaller number of variables and doses. Biotechnol. Bioeng. 2013;110: 2706–2716. textcopyright 2013 Wiley Periodicals,Inc. View Publication

Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes,with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations,leading to behavioural pathologies in humans,remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome,we narrowed this cellular phenotype to a single gene candidate,frizzled 9 (FZD9). At the neuronal stage,layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites,increased numbers of spines and synapses,aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain. View Publication

Genome-wide association studies (GWASs) have reported many single nucleotide polymorphisms (SNPs) associated with psychiatric disorders,but knowledge is lacking regarding molecular mechanisms. Here we show that risk alleles spanning multiple genes across the 10q24.32 schizophrenia-related locus are associated in the human brain selectively with an increase in the expression of both BLOC-1 related complex subunit 7 (BORCS7) and a previously uncharacterized,human-specific arsenite methyltransferase (AS3MT) isoform (AS3MT(d2d3)),which lacks arsenite methyltransferase activity and is more abundant in individuals with schizophrenia than in controls. Conditional-expression analysis suggests that BORCS7 and AS3MT(d2d3) signals are largely independent. GWAS risk SNPs across this region are linked with a variable number tandem repeat (VNTR) polymorphism in the first exon of AS3MT that is associated with the expression of AS3MT(d2d3) in samples from both Caucasians and African Americans. The VNTR genotype predicts promoter activity in luciferase assays,as well as DNA methylation within the AS3MT gene. Both AS3MT(d2d3) and BORCS7 are expressed in adult human neurons and astrocytes,and they are upregulated during human stem cell differentiation toward neuronal fates. Our results provide a molecular explanation for the prominent 10q24.32 locus association,including a novel and evolutionarily recent protein that is involved in early brain development and confers risk for psychiatric illness. View Publication

Generation of midbrain dopaminergic (mDA) neurons from human pluripotent stem cells provides a platform for inquiry into basic and translational studies of Parkinson's disease (PD). However,heterogeneity in differentiation in vitro makes it difficult to identify mDA neurons in culture or in vivo following transplantation. Here,we report the generation of a human embryonic stem cell (hESC) line with a tyrosine hydroxylase (TH)-RFP (red fluorescent protein) reporter. We validated that RFP faithfully mimicked TH expression during differentiation. Use of this TH-RFP reporter cell line enabled purification of mDA-like neurons from heterogeneous cultures with subsequent characterization of neuron transcriptional and epigenetic programs (global binding profiles of H3K27ac,H3K4me1,and 5-hydroxymethylcytosine [5hmC]) at four different stages of development. We anticipate that the tools and data described here will contribute to the development of mDA neurons for applications in disease modeling and/or drug screening and cell replacement therapies for PD. 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