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    ABSTRACT: Recent advances in human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) biology enable generation of dopaminergic neurons for potential therapy and drug screening. However, our current understanding of molecular and cellular signaling that controls human dopaminergic development and function is limited. Here, we report on a whole genome analysis of gene expression during dopaminergic differentiation of human ESC/iPSC using Illumina bead microarrays. We generated a transcriptome data set containing the expression levels of 28, 688 unique transcripts by profiling 5 lines (3 ESC and 2 iPSC lines) at four stages of differentiation: 1) undifferentiated ESC/iPSC, 2) neural stem cells (NSC), 3) dopaminergic precursors, and 4) dopaminergic neurons. This data set provides comprehensive information about genes expressed at each stage of differentiation. Our data indicate that distinct pathways are activated during neural and dopaminergic neuronal differentiation. For example, WNT, SHH, and cAMP signaling pathways were found overrepresented in dopaminergic populations by gene enrichment and pathway analysis, and their role was confirmed by perturbation analyses using RNAi (siRNA of SHH and WNT) or small molecule (dcAMP). In summary, whole genome profiling of dopaminergic differentiation enables systematic analysis of genes/pathways, networks, and cellular/molecular processes that control cell fate (dopaminergic) decisions. Such analyses will serve as the foundation for better understanding of dopaminergic development, function, and development of future stem cell-based therapies.
    Stem cells and development 09/2013; · 4.15 Impact Factor
  • Olga Momčilović, Justine Montoya-Sack, Xianmin Zeng
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    ABSTRACT: Parkinson's disease (PD) is the second most common neurodegenerative disorder. The motor symptoms of PD are caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta of mesencephalon. The causes for death of DA neurons are not well understood, but the strongest risk factor is increasing age. There is no cure currently available for PD, and treatment is limited to management of PD symptoms in patients. Primary DA neurons are virtually unobtainable from living patients and animal studies have proven inadequate for studying the mechanism of PD development. Pluripotent stem cells (PSC) are primary self-renewing cells capable of differentiating into all cell types of an organism, including DA neurons. PSCs represent an abundant source of cells that can be genetically modified or isolated from patients with complex diseases, enabling the production of large quantities of DA neurons for disease modeling, drug screening, and gene function studies. Furthermore, since PD arises as a result of deterioration of DA neurons in a specific brain region, it has been suggested that a relatively small number of cells could restore normal function. PSCs could provide a source of DA neurons for cell replacement therapy. In this Prospects article, we focus on the development and in vitro derivation of DA neurons from PSCs, as well as current applications of the technological advances, with the emphasis on future directions and efforts in the field. J. Cell. Biochem. 113: 3610-3619, 2012. © 2012 Wiley Periodicals, Inc.
    Journal of Cellular Biochemistry 07/2012; 113(12):3610-9. · 3.06 Impact Factor
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    ABSTRACT: BACKGROUND: Human pluripotent stem cells have the ability to generate all cell types present in the adult organism, therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly, many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines, namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells. METHODOLOGY/PRINCIPAL FINDINGS: We compared the energy metabolism of hESCs, IPSCs, and their somatic counterparts. Focusing on mitochondria, we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism, including glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer, as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism. CONCLUSIONS/FINDINGS: Our results demonstrate that, although the metabolic signature of IPSCs is not identical to that of hESCs, nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels, lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore, our work points to some of the strategies which human pluripotent stem cells may use to maintain high glycolytic rates, such as high levels of hexokinase II and inactive pyruvate dehydrogenase (PDH).
    PLoS ONE 01/2011; 6(6):e20914. · 3.53 Impact Factor
  • Olga Momčilović, Christopher Navara, Gerald Schatten
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    ABSTRACT: Pluripotent stem cells have the capability to undergo unlimited self-renewal and differentiation into all somatic cell types. They have acquired specific adjustments in the cell cycle structure that allow them to rapidly proliferate, including cell cycle independent expression of cell cycle regulators and lax G(1) to S phase transition. However, due to the developmental role of embryonic stem cells (ES) it is essential to maintain genomic integrity and prevent acquisition of mutations that would be transmitted to multiple cell lineages. Several modifications in DNA damage response of ES cells accommodate dynamic cycling and preservation of genetic information. The absence of a G(1)/S cell cycle arrest promotes apoptotic response of damaged cells before DNA changes can be fixed in the form of mutation during the S phase, while G(2)/M cell cycle arrest allows repair of damaged DNA following replication. Furthermore, ES cells express higher level of DNA repair proteins, and exhibit enhanced repair of multiple types of DNA damage. Similarly to ES cells, induced pluripotent stem (iPS) cells are poised to proliferate and exhibit lack of G(1)/S cell cycle arrest, extreme sensitivity to DNA damage, and high level of expression of DNA repair genes. The fundamental mechanisms by which the cell cycle regulates genomic integrity in ES cells and iPS cells are similar, though not identical.
    Results and problems in cell differentiation 01/2011; 53:415-58.
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    ABSTRACT: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability. Here, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB), and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle, displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts. High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.
    PLoS ONE 01/2010; 5(10):e13410. · 3.53 Impact Factor
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    ABSTRACT: Human embryonic stem cell (hESC) pluripotency has been reported by several groups to be best maintained by culture under physiological oxygen conditions. Building on that finding, we inhibited complex III of the mitochondrial respiratory chain using antimycin A or myxothiazol to examine if specifically targeting the mitochondria would have a similar beneficial result for the maintenance of pluripotency. hESCs grown in the presence of 20 nM antimycin A maintained a compact morphology with high nuclear/cytoplasmic ratios. Furthermore, real-time PCR analysis demonstrated that the levels of Nanog mRNA were elevated 2-fold in antimycin A-treated cells. Strikingly, antimycin A was also able to replace bFGF in the media without compromising pluripotency, as long as autocrine bFGF signaling was maintained. Further analysis using low-density quantitative PCR arrays showed that antimycin A treatment reduced the expression of genes associated with differentiation, possibly acting through a ROS-mediated pathway. These results demonstrate that modulation of mitochondrial function results in increased pluripotency of the cell population, and sheds new light on the mechanisms and signaling pathways modulating hESC pluripotency.
    Stem Cell Research 09/2009; 3(2-3):142-56. · 4.47 Impact Factor
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    ABSTRACT: Human embryonic stem (ES) cells are highly sensitive to environmental insults including DNA damaging agents, responding with high levels of apoptosis. To understand the response of human ES cells to DNA damage, we investigated the function of the ataxia telangiectasia mutated (ATM) DNA damage signaling pathway in response to gamma-irradiation. Here, we demonstrate for the first time in human ES cells that ATM kinase is phosphorylated and properly localized to the sites of DNA double-strand breaks within 15 minutes of irradiation. Activation of ATM kinase resulted in phosphorylation of its downstream targets: Chk2, p53, and Nbs1. In contrast to murine ES cells, Chk2 and p53 were localized to the nucleus of irradiated human ES cells. We further show that irradiation resulted in a temporary arrest of the cell cycle at the G(2), but not G(1), phase. Human ES cells resumed cycling approximately 16 hours after irradiation, but had a fourfold higher incidence of aberrant mitotic figures compared to nonirradiated cells. Finally, we demonstrate an essential role of ATM in establishing G(2) arrest since inhibition with the ATM-specific inhibitor KU55933 resulted in abolishment of G(2) arrest, evidenced by an increase in the number of cycling cells 2 hours after irradiation. In summary, these results indicate that human ES cells activate the DNA damage checkpoint, resulting in an ATM-dependent G(2) arrest. However, these cells re-enter the cell cycle with prominent mitotic spindle defects.
    Stem Cells 06/2009; 27(8):1822-35. · 7.70 Impact Factor

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