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Chronic treatment with antidepressants increases neurogenesis in the adult hippocampus. This increase in the production of new neurons may be required for the behavioral effects of antidepressants. However,it is not known which class of cells within the neuronal differentiation cascade is targeted by the drugs. We have generated a reporter mouse line,which allows identification and classification of early neuronal progenitors. It also allows accurate quantitation of changes induced by neurogenic agents in these distinct subclasses of neuronal precursors. We use this line to demonstrate that the selective serotonin reuptake inhibitor antidepressant fluoxetine does not affect division of stem-like cells in the dentate gyrus but increases symmetric divisions of an early progenitor cell class. We further demonstrate that these cells are the sole class of neuronal progenitors targeted by fluoxetine in the adult brain and suggest that the fluoxetine-induced increase in new neurons arises as a result of the expansion of this cell class. This finding defines a cellular target for antidepressant drug therapies. View Publication

OBJECTIVE: Studies in pulmonary arteries (PA) of patients with chronic obstructive pulmonary disease (COPD) suggest that bone marrow-derived endothelial progenitor cells (CD133(+)) may infiltrate the intima and differentiate into smooth muscle cells (SMC). This study aimed to evaluate the plasticity of CD133(+) cells to differentiate into SMC and endothelial cells (EC) in both cell culture and human isolated PA. METHODS: Plasticity of granulocyte-colony stimulator factor (G-CSF)-mobilized peripheral blood CD133(+) cells was assessed in co-cultures with primary lines of human PA endothelial cells (PAEC) or SMC (PASMC) and in isolated human PA. We also evaluated if the phenotype of differentiated progenitor cells was acquired by fusion or differentiation. RESULTS: The in vitro studies demonstrated CD133(+) cells may acquire the morphology and phenotype of the cells they were co-cultured with. CD133(+) cells co-incubated with human isolated PA were able to migrate into the intima and differentiate into SMC. Progenitor cell differentiation was produced without fusion with mature cells. CONCLUSIONS: We provide evidence of plasticity of CD133(+) cells to differentiate into both endothelial cells and SMC,reinforcing the idea of their potential role in the remodeling process of PA in COPD. This process was conducted by transdifferentiation and not by cell fusion. View Publication

Tumor-initiating cells (TICs) often survive therapy and give rise to second-line tumors. We tested the plausibility of sphere cultures as models of TICs. Microarray data and microRNA data analysis confirmed the validity of spheres as models of TICs for breast and prostate cancer as well as mesothelioma cell lines. Microarray data analysis revealed the Trp pathway as the only pathway upregulated significantly in all types of studied TICs,with increased levels of indoleamine-2,3-dioxygenase-1 (IDO1),the rate-limiting enzyme of Trp metabolism along the kynurenine pathway. All types of TICs also expressed higher levels of the Trp uptake system consisting of CD98 and LAT1 with functional consequences. IDO1 expression was regulated via both transcriptional and posttranscriptional mechanisms,depending on the cancer type. Serial transplantation of TICs in mice resulted in gradually increased IDO1. Mitocans,represented by α-tocopheryl succinate and mitochondrially targeted vitamin E succinate (MitoVES),suppressed IDO1 in TICs. MitoVES suppressed IDO1 in TICs with functional mitochondrial complex II,involving transcriptional and posttranscriptional mechanisms. IDO1 increase and its suppression by VE analogues were replicated in TICs from primary human glioblastomas. Our work indicates that IDO1 is increased in TICs and that mitocans suppress the protein. View Publication

Tumor-initiating cells are a subpopulation in aggressive cancers that exhibit traits shared with stem cells,including the ability to self-renew and differentiate,commonly referred to as stemness. In addition,such cells are resistant to chemo- and radiation therapy posing a therapeutic challenge. To uncover stemness-associated functions in glioma-initiating cells (GICs),transcriptome profiles were compared to neural stem cells (NSCs) and gene ontology analysis identified an enrichment of Ca2+ signaling genes in NSCs and the more stem-like (NSC-proximal) GICs. Functional analysis in a set of different GIC lines regarding sensitivity to disturbed homeostasis using A23187 and Thapsigargin,revealed that NSC-proximal GICs were more sensitive,corroborating the transcriptome data. Furthermore,Ca2+ drug sensitivity was reduced in GICs after differentiation,with most potent effect in the NSC-proximal GIC,supporting a stemness-associated Ca2+ sensitivity. NSCs and the NSC-proximal GIC line expressed a larger number of ion channels permeable to potassium,sodium and Ca2+. Conversely,a higher number of and higher expression levels of Ca2+ binding genes that may buffer Ca2+,were expressed in NSC-distal GICs. In particular,expression of the AMPA glutamate receptor subunit GRIA1,was found to associate with Ca2+ sensitive NSC-proximal GICs,and decreased as GICs differentiated along with reduced Ca2+ drug sensitivity. The correlation between high expression of Ca2+ channels (such as GRIA1) and sensitivity to Ca2+ drugs was confirmed in an additional nine novel GIC lines. Calcium drug sensitivity also correlated with expression of the NSC markers nestin (NES) and FABP7 (BLBP,brain lipid-binding protein) in this extended analysis. In summary,NSC-associated NES+/FABP7+/GRIA1+ GICs were selectively sensitive to disturbances in Ca2+ homeostasis,providing a potential target mechanism for eradication of an immature population of malignant cells. View Publication

Long-lived multipotent stem cells (ISCs) at the base of intestinal crypts adjust their phenotypes to accommodate normal maintenance and post-injury regeneration of the epithelium. Their long life,lineage plasticity,and proliferative potential underlie the necessity for tight homeostatic regulation of the ISC compartment. In that context,the guanylate cyclase C (GUCY2C) receptor and its paracrine ligands regulate intestinal epithelial homeostasis,including proliferation,lineage commitment,and DNA damage repair. However,a role for this axis in maintaining ISCs remains unknown. Transgenic mice enabling analysis of ISCs (Lgr5-GFP) in the context of GUCY2C elimination (Gucy2c -/- ) were combined with immunodetection techniques and pharmacological treatments to define the role of the GUCY2C signaling axis in supporting ISCs. ISCs were reduced inGucy2c -/- mice,associated with loss of active Lgr5+cells but a reciprocal increase in reserve Bmi1+cells. GUCY2C was expressed in crypt base Lgr5+cells in which it mediates canonical cyclic (c) GMP-dependent signaling. Endoplasmic reticulum (ER) stress,typically absent from ISCs,was elevated throughout the crypt base inGucy2c -/- mice. The chemical chaperone tauroursodeoxycholic acid resolved this ER stress and restored the balance of ISCs,an effect mimicked by the GUCY2C effector 8Br-cGMP. Reduced ISCs inGucy2c -/- mice was associated with greater epithelial injury and impaired regeneration following sub-lethal doses of irradiation. These observations suggest that GUCY2C provides homeostatic signals that modulate ER stress and cell vulnerability as part of the machinery contributing to the integrity of ISCs. View Publication

Cryopreservation is an essential technique to preserve stem cells,semipermanently sustaining their potentials. There are two main approaches of cryopreservation for human pluripotent stem cells (hPSCs). The first is the vitrification,which involves instantaneous freeze and thaw of hPSCs. The second is the conventional slow-cooling method and a rapid thaw. Both cryopreservation protocols have been standardized and optimized to yield high survivability of hPSCs. View Publication

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However,it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here,we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days,at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis,thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional,morphological and functional signatures of differentiated neurons,with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons,suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types. View Publication

The lectin galectin-9 may help establish and maintain chronic hepatitis C virus infection. Galectin-9 is elevated in the liver and sera of hepatitis C virus patients,induces apoptosis of hepatitis C virus-specific T cells,and increases inhibitory regulatory T cells. Kupffer cells stain strongly for galectin-9 protein in hepatitis C virus patients. In the current study,we determined stimuli that induce galectin-9 production by monocytes and macrophages in hepatitis C virus infection. With the use of real-time PCR and flow cytometry,we analyzed galectin-9 mRNA and protein from human monocytes cocultured with hepatitis C virus-infected cells or noninfectious hepatitis C virus subgenomic replicon cells. We focused on finding the stimuli for galectin-9 production. Additionally,we measured galectin-9 during monocyte-to-macrophage maturation. Finally,we examined galectin-9 in peripheral monocytes from hepatitis C virus patients using flow cytometry. Galectin-9 mRNA increased 8-fold when primary monocytes were exposed to hepatitis C virus--infected cells. Maximum induction required proximity or contact and did not require IFN-γ or hepatitis C virus virions. Coculture of monocytes with subgenomic replicon cells increased galectin-9 5-fold,and purified exosomes from infected cells stimulated galectin-9 production. Stimulation of monocyte TLR3,-7,and -8 increased galectin-9 production. Differentiation of monocytes to macrophages increased galectin-9,and nonclassic monocytes from hepatitis C virus patients had the highest levels of galectin-9. Hepatitis C virus-infected cells stimulated monocytes to produce galectin-9 in close proximity,possibly,in part,as a result of exosomes and endosomal TLRs. Differentiation of monocytes to macrophages increased galectin-9. Nonclassic monocytes from hepatitis C virus patients express the highest galectin-9 levels,suggesting they may contribute to elevated galectin-9 and adaptive immune inhibition in hepatitis C virus infection. View Publication

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here,we show that rat ES cells can be efficiently derived,propagated,and genetically manipulated in the presence of small molecules that specifically inhibit GSK3,MEK,and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly,they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases. View Publication

Human embryonic stem cells (hESCs) share an identical genome with lineage-committed cells,yet possess the remarkable properties of self-renewal and pluripotency. The diverse cellular properties in different cells have been attributed to their distinct epigenomes,but how much epigenomes differ remains unclear. Here,we report that epigenomic landscapes in hESCs and lineage-committed cells are drastically different. By comparing the chromatin-modification profiles and DNA methylomes in hESCs and primary fibroblasts,we find that nearly one-third of the genome differs in chromatin structure. Most changes arise from dramatic redistributions of repressive H3K9me3 and H3K27me3 marks,which form blocks that significantly expand in fibroblasts. A large number of potential regulatory sequences also exhibit a high degree of dynamics in chromatin modifications and DNA methylation. Additionally,we observe novel,context-dependent relationships between DNA methylation and chromatin modifications. Our results provide new insights into epigenetic mechanisms underlying properties of pluripotency and cell fate commitment. textcopyright 2010 Elsevier Inc. View Publication

BACKGROUND: Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications,including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: We have quantitatively compared promoter activities of five commonly used constitutive promoters,including the human β-actin promoter (ACTB),cytomegalovirus (CMV),elongation factor-1α,(EF1α),phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies. CONCLUSION/SIGNIFICANCE: The ACTB,EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75-80% of the cells after 50 days in culture. During embryoid body (EB) differentiation,promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells,it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages,suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs. View Publication

Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor-initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines,we found that ALDH1A1 expression and activity was significantly higher in taxane- and platinum-resistant cell lines. In patient samples,72.9% of ovarian cancers had ALDH1A1 expression in which the percentage of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 vs. 13.81 months; P textless 0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor-initiating studies,where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly,tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations,but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer,ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy,significantly reducing tumor growth in mice compared with chemotherapy alone (a 74%-90% reduction; P textless 0.015). These data show that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients,and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced,but not absolute,tumorigenicity but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer. View Publication