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High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress

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ABSTRACT

Using astrocytes differentiated from human embryonic stem cells, an assay was developed to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. The 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, 22 were identified that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress.

No MeSH data available.


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The high-content oxidative stress assay to identify cytoprotective compounds. (A): Human embryonic stem cell (hESC)-differentiated astrocytes are treated with compounds from a chemical library for 24 hours before treatment with 12 mM H2O2 for 1 hour. Treatment with 12 mM H2O2 induces cell death processes, characterized by cell and nuclear shrinkage and chromatin condensation. Compounds active in the assay prevent or reduce apoptotic events. Shown are images of astrocytes after a 24-hour treatment with 100 µM terfenadine, demonstrating nuclei of cells undergoing cell death, or DMSO, the vehicle control. Images were taken using the IN Cell Analyzer 2000 and 4′,6-diamidino-2-phenylindole (DAPI) filters. Magnification, ×20. (B, C): hESC-differentiated astrocytes in a 1,536-well plate format were incubated with H2O2 up to a concentration of 12 mM for 1 hour. Assay media treatment is the far left bar in the graphs, followed by the increasing concentrations of H2O2 tested: 400 nM to 12 mM. (B): Mean nuclear fluorescence intensity of the DNA dye Hoechst 33342 shows a H2O2 concentration-dependent increase, indicative of chromatin condensation that is a characteristic feature of apoptosis. (C): The shape of nuclei also changes with increasing concentrations of H2O2, as demonstrated by a concentration-dependent decrease in mean nuclear compactness. Abbreviation: RFU, relative fluorescence unit.
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Figure 2: The high-content oxidative stress assay to identify cytoprotective compounds. (A): Human embryonic stem cell (hESC)-differentiated astrocytes are treated with compounds from a chemical library for 24 hours before treatment with 12 mM H2O2 for 1 hour. Treatment with 12 mM H2O2 induces cell death processes, characterized by cell and nuclear shrinkage and chromatin condensation. Compounds active in the assay prevent or reduce apoptotic events. Shown are images of astrocytes after a 24-hour treatment with 100 µM terfenadine, demonstrating nuclei of cells undergoing cell death, or DMSO, the vehicle control. Images were taken using the IN Cell Analyzer 2000 and 4′,6-diamidino-2-phenylindole (DAPI) filters. Magnification, ×20. (B, C): hESC-differentiated astrocytes in a 1,536-well plate format were incubated with H2O2 up to a concentration of 12 mM for 1 hour. Assay media treatment is the far left bar in the graphs, followed by the increasing concentrations of H2O2 tested: 400 nM to 12 mM. (B): Mean nuclear fluorescence intensity of the DNA dye Hoechst 33342 shows a H2O2 concentration-dependent increase, indicative of chromatin condensation that is a characteristic feature of apoptosis. (C): The shape of nuclei also changes with increasing concentrations of H2O2, as demonstrated by a concentration-dependent decrease in mean nuclear compactness. Abbreviation: RFU, relative fluorescence unit.

Mentions: To develop a high-throughput screening assay and screen chemical libraries using hESC-differentiated astrocytes, culturing conditions in the 1,536-well format had to be optimized for these cells, and cell viability in this format confirmed (supplemental online data). A detailed protocol for the oxidative stress assay to identify potentially cytoprotective compounds by screening the LOPAC1280 and NPC compound libraries can be found in supplemental online Table 1. The supplemental online data contains additional information on the development and optimization of this assay. The optimal concentration of, and incubation time with, hydrogen peroxide (H2O2), which was used to induce oxidative stress, was experimentally determined to be 12 mM for 1 hour, which caused approximately 50%–80% of the hESC-differentiated astrocytes to display an apoptotic nuclear profile. Although this level of H2O2 is likely not physiologically relevant (postischemia concentrations of H2O2 are 50–100 μM [27]), treatment of astrocytes with more physiological concentrations of H2O2 did not induce levels of apoptosis significant enough to allow for the generation of a reliable and robust assay that is necessary for compound library screening. A compound tested in the assay that was found to reduce the number of apoptotic astrocytes after treatment with H2O2, as assessed by nuclear characteristics, was considered active in the assay and of interest (Fig. 2A).


High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress
The high-content oxidative stress assay to identify cytoprotective compounds. (A): Human embryonic stem cell (hESC)-differentiated astrocytes are treated with compounds from a chemical library for 24 hours before treatment with 12 mM H2O2 for 1 hour. Treatment with 12 mM H2O2 induces cell death processes, characterized by cell and nuclear shrinkage and chromatin condensation. Compounds active in the assay prevent or reduce apoptotic events. Shown are images of astrocytes after a 24-hour treatment with 100 µM terfenadine, demonstrating nuclei of cells undergoing cell death, or DMSO, the vehicle control. Images were taken using the IN Cell Analyzer 2000 and 4′,6-diamidino-2-phenylindole (DAPI) filters. Magnification, ×20. (B, C): hESC-differentiated astrocytes in a 1,536-well plate format were incubated with H2O2 up to a concentration of 12 mM for 1 hour. Assay media treatment is the far left bar in the graphs, followed by the increasing concentrations of H2O2 tested: 400 nM to 12 mM. (B): Mean nuclear fluorescence intensity of the DNA dye Hoechst 33342 shows a H2O2 concentration-dependent increase, indicative of chromatin condensation that is a characteristic feature of apoptosis. (C): The shape of nuclei also changes with increasing concentrations of H2O2, as demonstrated by a concentration-dependent decrease in mean nuclear compactness. Abbreviation: RFU, relative fluorescence unit.
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Figure 2: The high-content oxidative stress assay to identify cytoprotective compounds. (A): Human embryonic stem cell (hESC)-differentiated astrocytes are treated with compounds from a chemical library for 24 hours before treatment with 12 mM H2O2 for 1 hour. Treatment with 12 mM H2O2 induces cell death processes, characterized by cell and nuclear shrinkage and chromatin condensation. Compounds active in the assay prevent or reduce apoptotic events. Shown are images of astrocytes after a 24-hour treatment with 100 µM terfenadine, demonstrating nuclei of cells undergoing cell death, or DMSO, the vehicle control. Images were taken using the IN Cell Analyzer 2000 and 4′,6-diamidino-2-phenylindole (DAPI) filters. Magnification, ×20. (B, C): hESC-differentiated astrocytes in a 1,536-well plate format were incubated with H2O2 up to a concentration of 12 mM for 1 hour. Assay media treatment is the far left bar in the graphs, followed by the increasing concentrations of H2O2 tested: 400 nM to 12 mM. (B): Mean nuclear fluorescence intensity of the DNA dye Hoechst 33342 shows a H2O2 concentration-dependent increase, indicative of chromatin condensation that is a characteristic feature of apoptosis. (C): The shape of nuclei also changes with increasing concentrations of H2O2, as demonstrated by a concentration-dependent decrease in mean nuclear compactness. Abbreviation: RFU, relative fluorescence unit.
Mentions: To develop a high-throughput screening assay and screen chemical libraries using hESC-differentiated astrocytes, culturing conditions in the 1,536-well format had to be optimized for these cells, and cell viability in this format confirmed (supplemental online data). A detailed protocol for the oxidative stress assay to identify potentially cytoprotective compounds by screening the LOPAC1280 and NPC compound libraries can be found in supplemental online Table 1. The supplemental online data contains additional information on the development and optimization of this assay. The optimal concentration of, and incubation time with, hydrogen peroxide (H2O2), which was used to induce oxidative stress, was experimentally determined to be 12 mM for 1 hour, which caused approximately 50%–80% of the hESC-differentiated astrocytes to display an apoptotic nuclear profile. Although this level of H2O2 is likely not physiologically relevant (postischemia concentrations of H2O2 are 50–100 μM [27]), treatment of astrocytes with more physiological concentrations of H2O2 did not induce levels of apoptosis significant enough to allow for the generation of a reliable and robust assay that is necessary for compound library screening. A compound tested in the assay that was found to reduce the number of apoptotic astrocytes after treatment with H2O2, as assessed by nuclear characteristics, was considered active in the assay and of interest (Fig. 2A).

View Article: PubMed Central - PubMed

ABSTRACT

Using astrocytes differentiated from human embryonic stem cells, an assay was developed to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. The 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, 22 were identified that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress.

No MeSH data available.


Related in: MedlinePlus