title: High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress.
abstract: Astrocytes are the predominant cell type in the nervous system and play a significant role in maintaining neuronal health and homeostasis. Recently, astrocyte dysfunction has been implicated in the pathogenesis of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Astrocytes are thus an attractive new target for drug discovery for neurological disorders. Using astrocytes differentiated from human embryonic stem cells, we have developed an assay to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. This phenotypic oxidative stress assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, we identified a set of 22 that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress. Nine of these compounds were also found to be protective of induced pluripotent stem cell-differentiated astrocytes in a related assay. These compounds are thought to confer protection through hormesis, activating stress-response pathways and preconditioning astrocytes to handle subsequent exposure to hydrogen peroxide. In fact, four of these compounds were found to activate the antioxidant response element/nuclear factor-E2-related factor 2 pathway, a protective pathway induced by toxic insults. Our results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development.
  
  
  CONCLUSIONS
  Astrocytes play a key role in neurological diseases. Drug discovery efforts that target astrocytes can identify novel therapeutics. Human astrocytes are difficult to obtain and thus are challenging to use for high-throughput screening, which requires large numbers of cells. Using human embryonic stem cell-derived astrocytes and an optimized astrocyte differentiation protocol, it was possible to screen approximately 4,100 compounds in titration to identify 22 that are cytoprotective of astrocytes. This study is the largest-scale high-throughput screen conducted using human astrocytes, with a total of 17,536 data points collected in the primary screen. The results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development.
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland, USA Natasha.Thorne@fda.hhs.gov wzheng@mail.nih.gov.
authors.forename: Natasha
authors.initials: N
authors.lastname: Thorne
authors.affiliation: Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Nasir
authors.initials: N
authors.lastname: Malik
authors.affiliation: Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Sonia
authors.initials: S
authors.lastname: Shah
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Jean
authors.initials: J
authors.lastname: Zhao
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Bradley
authors.initials: B
authors.lastname: Class
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Francis
authors.initials: F
authors.lastname: Aguisanda
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Noel
authors.initials: N
authors.lastname: Southall
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Menghang
authors.initials: M
authors.lastname: Xia
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: McKew
authors.affiliation: NIH Center for Regenerative Medicine, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Mahendra
authors.initials: M
authors.lastname: Rao
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland, USA Natasha.Thorne@fda.hhs.gov wzheng@mail.nih.gov.
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
mesh_terms: D061745:Antioxidant Response Elements / Q000187:drug effects; D000975:Antioxidants / Q000494:pharmacology*; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism; D055495:Neurogenesis; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D010641:Phenotype; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
chemical_list: D000975:Antioxidants; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D016877:Oxidants; D054852:Small Molecule Libraries; D006861:Hydrogen Peroxide
keywords: Astrocytes; High-throughput screening; Neurodegenerative disease; Oxidative stress; Stem cells