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Teratogens,substances that may cause fetal abnormalities during development,are responsible for a significant number of birth defects. Animal models used to predict teratogenicity often do not faithfully correlate to human response. Here,we seek to develop a more predictive developmental toxicity model based on an in vitro method that utilizes both human embryonic stem (hES) cells and metabolomics to discover biomarkers of developmental toxicity. We developed a method where hES cells were dosed with several drugs of known teratogenicity then LC-MS analysis was performed to measure changes in abundance levels of small molecules in response to drug dosing. Statistical analysis was employed to select for specific mass features that can provide a prediction of the developmental toxicity of a substance. These molecules can serve as biomarkers of developmental toxicity,leading to better prediction of teratogenicity. In particular,our work shows a correlation between teratogenicity and changes of greater than 10% in the ratio of arginine to asymmetric dimethylarginine levels. In addition,this study resulted in the establishment of a predictive model based on the most informative mass features. This model was subsequently tested for its predictive accuracy in two blinded studies using eight drugs of known teratogenicity,where it correctly predicted the teratogenicity for seven of the eight drugs. Thus,our initial data shows that this platform is a robust alternative to animal and other in vitro models for the prediction of the developmental toxicity of chemicals that may also provide invaluable information about the underlying biochemical pathways. ?? 2010 Elsevier Inc. View Publication

OBJECTIVE: The molecular mechanisms that maintain human pluripotent stem (PS) cells are not completely understood. Here we sought to identify new candidate PS cell regulators to facilitate future improvements in their generation,expansion,and differentiation. MATERIALS AND METHODS: We used bioinformatic analyses of multiple serial-analysis-of-gene-expression libraries (generated from human PS cells and their differentiated derivatives),together with small interfering RNA (siRNA) screening to identify candidate pluripotency regulators. Validation of candidate regulators involved promoter analyses,Affymetrix profiling,real-time PCR,and immunoprecipitation. RESULTS: Promoter analysis of genes differentially expressed across multiple serial-analysis-of-gene-expression libraries identified E2F motifs in the promoters of many PS cell-specific genes (e.g.,POU5F1,NANOG,SOX2,FOXD3). siRNA analyses identified two retinoblastoma binding proteins (RBBP4,RBBP9) as required for maintenance of multiple human PS cell types. Both RBBPs were bound to RB in human PS cells,and E2F motifs were present in the promoters of genes whose expression was altered by decreasing RBBP4 and RBBP9 expression. Affymetrix and real-time PCR studies of siRNA-treated human PS cells showed that reduced RBBP4 or RBBP9 expression concomitantly decreased expression of POU5F1,NANOG,SOX2,and/or FOXD3 plus certain cell cycle genes (e.g.,CCNA2,CCNB1),while increasing expression of genes involved in organogenesis (particularly neurogenesis). CONCLUSIONS: These results reveal new candidate positive regulators of human PS cells,providing evidence of their ability to regulate expression of pluripotency,cell cycle,and differentiation genes in human PS cells. These data provide valuable new leads for further elucidating mechanisms of human pluripotency. View Publication

We have tracked the fate of immature human B cells at a critical stage in their development when the mature B cell repertoire is shaped. We show that a major subset of bone marrow emigrant immature human B cells,the transitional 2 (T2) B cells,homes to gut-associated lymphoid tissue (GALT) and that most T2 B cells isolated from human GALT are activated. Activation in GALT is a previously unknown potential fate for immature human B cells. The process of maturation from immature transitional B cell through to mature naive B cell includes the removal of autoreactive cells from the developing repertoire,a process which is known to fail in systemic lupus erythematosus (SLE). We observe that immature B cells in SLE are poorly equipped to access the gut and that gut immune compartments are depleted in SLE. Thus,activation of immature B cells in GALT may function as a checkpoint that protects against autoimmunity. In healthy individuals,this pathway may be involved in generating the vast population of IgA plasma cells and also the enigmatic marginal zone B cell subset that is poorly understood in humans. View Publication

In this study,we developed a cell system to study TCF4 in human oligodendrocyte differentiation,showed that TCF4 regulates human oligodendroglial differentiation in a dose-dependent manner,and established a system to dissect TCF4 function in a human tissue–like context. Heterozygous mutations of TCF4 in humans cause Pitt–Hopkins syndrome,a neurodevelopmental disease associated with intellectual disability and brain malformations. Although most studies focus on the role of TCF4 in neural stem cells and neurons,we here set out to assess the implication of TCF4 for oligodendroglial differentiation. We discovered that both monoallelic and biallelic mutations in TCF4 result in a diminished capacity to differentiate human neural progenitor cells toward myelinating oligodendrocytes through the forced expression of the transcription factors SOX10,OLIG2,and NKX6.2. Using this experimental strategy,we established a novel organoid model,which generates oligodendroglial cells within a human neurogenic tissue–like context. Also,here we found a reduced ability of TCF4 heterozygous cells to differentiate toward oligodendroglial cells. In sum,we establish a role of human TCF4 in oligodendrocyte differentiation and provide a model system,which allows to dissect the disease etiology in a human tissue–like context. View Publication

Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene (FUT2) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme,while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals,while HIEs from secretors are permissive to infection. However,whether FUT2 expression alone is critical for infection remains unproven,since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication,we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies,these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status.IMPORTANCE Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however,previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GI.1 and several GII HuNoV strains. View Publication

Poor recovery of cryopreserved human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells is a significant impediment to progress with pluripotent stem cells. In this study,we demonstrate that Y-27632,a specific inhibitor of Rho kinase (ROCK) activity,significantly enhances recovery of hES cells from cryopreserved stocks when cultured with or without a growth inactivated feeder layer. Furthermore,treatment with the ROCK inhibitor for several days increased the number of colonies and colony size of hES cells compared to shorter exposures. Remarkably,hES cells that had formed relatively few colonies 5 days after thawing exhibited rapid growth upon addition of Y-27632. Additionally,we determined that Y-27632 significantly improves the recovery of cryopreserved human iPS cells and their growth upon subculture. Thus,Y-27632 provides a means to kick-start" slow-growing human pluripotent stem cells� View Publication

The present study describes and compares functional properties of Nuli-1 cells and primary human nasal epithelial cells (HNEC) including TLR expression and function. Differences in gene expression were identified for non-TLR genes that play a role in TLR response pathways. However,experiments comparing TLR gene expression for both Nuli-1 cells and HNECs indicated conserved expression in both cell types. Stimulation of the two cell types resulted in a conserved response to TLR3 agonists,but in differences in response to agonists for TLR5 and TLR6/2. HNECs were much more susceptible to infection with Staphylococcus aureus than NuLi-1 cells. Furthermore,when cultured at air-liquid interface (ALI),NuLi-1 cells possessed much lower trans-epithelial resistance than primary HNEC and did not exhibit maintenance of cell morphology or mucous production which was observed in HNECs. Nor did they produce the characteristic interconnecting pattern of tight junction complexes at the apicolateral margin of adjacent cells. Caution should therefore be exercised when selecting cell lines for immunological studies and a thorough screen of properties relevant to the study should always be carried out prior to commencement. View Publication

Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation,two hESCs lines were cultured on mixed feeder cells (MFCs,MEFs: HFFs = 1:1) and HFFs feeder,respectively,and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry,quantitative fluorescent real-time PCR,transmission and scanning electron microscopy,and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However,compared to hESCs line on MFCs feeder,hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2,PITX3,NURR1,and TH genes. In addition,the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion,HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons,but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore,feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines,but also electrophysiological properties of hESCs-derived DA neurons. View Publication

Differentiation approaches to obtain mesenchymal stem cells (MSCs) have gradually developed over the last few decades. The problem is that different protocols give different MSC types,making further research difficult. Here,we tried three different approaches to differentiate embryonic stem cells (ESCs) from early mesoderm to MSCs using serum-containing or xeno-free differentiation medium and observed differences in the cells’ morphology,doubling rate,ability to form colonies,surface marker analysis,and multilineage differentiation potential of the obtained cell lines. We concluded that the xeno-free medium best fits the criteria of MSCs’ morphology,growth kinetics,and surface marker characterization. In contrast,the serum-containing medium gives better potential for further MSC differentiation into osteogenic,chondrogenic,and adipogenic lineages. View Publication

Deamination of the nucleoside analogues ARA-C and 5-AZA-CdR by CR deaminase results in a loss of antileukemic activity. To prevent the inactivation of these analogues,inhibitors of CR deaminase may prove to be useful agents. In the present study we investigated the effects of the deaminase inhibitors Zebularine,5-F-Zebularine,and diazepinone riboside on the deamination of CR,ARA-C,and 5-AZA-CdR using highly purified human CR deaminase (EC 3.5.4.5). These inhibitors produced a competitive type of inhibition with each substrate,the potency of which followed the patterns diazepinone riboside greater than 5-F-Zebularine and THU greater than Zebularine. 5-AZA-CdR was more sensitive than ARA-C to the inhibition produced by these deaminase inhibitors. The inhibition constants for diazepinone riboside lay in the range of 5-15 nM,suggesting that this inhibitor could be an excellent candidate for use in combination chemotherapy with either ARA-C or 5-AZA-CdR in patients with leukemia. View Publication

Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs,especially for long-term studies. If 3D culture systems were to be used for such purposes,it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time,and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality,as demonstrated by improved urea production and hepatic marker expression. In addition,hPSC-HLCs in the scaffolds exhibit a more mature phenotype,as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays,making this system highly attractive for drug testing applications. View Publication