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

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

Differentiation of pluripotent stem cells to mature astrocytes and maintenance in 96-, 384-, and 1,536-well plates. (A): A differentiation timeline along with staining for nestin (an NSC marker), CD44 (marker of astrocyte precursor cells), and GFAP (a mature astrocyte marker) at critical time points in the differentiation protocol. The cells are proliferative, thus requiring passaging, and large stocks of cells can be cryopreserved after day 17. Later, when cells are brought up for use, differentiation into mature astrocytes is continued. (B): Human embryonic stem cell (hESC)-differentiated astrocytes adhered to, and were successfully maintained for >24-hours on PureCoat amine-coated microtiter plates. Astrocytes were healthy, stained positive for GFAP, and exhibited typical astrocyte morphology. Images from microtiter plates were acquired on an IN Cell Analyzer 2000. Magnification, ×40. Abbreviations: GFAP, glial fibrillary acidic protein; hNSC, human neural stem cell.
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Figure 1: Differentiation of pluripotent stem cells to mature astrocytes and maintenance in 96-, 384-, and 1,536-well plates. (A): A differentiation timeline along with staining for nestin (an NSC marker), CD44 (marker of astrocyte precursor cells), and GFAP (a mature astrocyte marker) at critical time points in the differentiation protocol. The cells are proliferative, thus requiring passaging, and large stocks of cells can be cryopreserved after day 17. Later, when cells are brought up for use, differentiation into mature astrocytes is continued. (B): Human embryonic stem cell (hESC)-differentiated astrocytes adhered to, and were successfully maintained for >24-hours on PureCoat amine-coated microtiter plates. Astrocytes were healthy, stained positive for GFAP, and exhibited typical astrocyte morphology. Images from microtiter plates were acquired on an IN Cell Analyzer 2000. Magnification, ×40. Abbreviations: GFAP, glial fibrillary acidic protein; hNSC, human neural stem cell.

Mentions: Human neural stem cells (hNSCs) derived from H9 (WA09) human embryonic stem cells (N7800-100; Thermo Fisher Scientific, Carlsbad, CA, https://www.thermofisher.com) were cultured on Geltrex substrate in StemPro NSC serum-free medium (SFM) (A10509-01; Thermo Fisher Scientific) consisting of KnockOut Dulbecco’s modified Eagle’s medium (DMEM)/F-12, StemPro Neural Supplement, GlutaMAX-I, basic fibroblast growth factor (20 ng/ml; PHG0021; Thermo Fisher Scientific), and epidermal growth factor (20 ng/ml; PHG0315; Thermo Fisher Scientific). The hNSCs were expanded for 1 week, and passaged every 2–3 days. After 1 week, the NSCs were differentiated into astrocytes as described recently [22, 23]. Briefly, the medium was changed to astrocyte differentiation medium consisting of StemPro hESC SFM (A1000701 [Thermo Fisher Scientific]; DMEM/F-12, GlutaMAX-I supplement, bovine serum albumin 25%, and StemPro hESC supplement), in addition to 1% fetal bovine serum, FGF2 (8 ng/ml; PHG0264; Thermo Fisher Scientific), activin A (10 ng/ml; 120-14; PeproTech, Quebec, Canada, https://www.peprotech.com), heregulin 1β (10 ng/ml; 100-03; PeproTech), and insulin-like growth factor-1 analog (200 ng/ml; 100-11; PeproTech) to promote astrocyte differentiation. The culture was designated as “Astro P0” at this stage and expanded by passaging (1:6 split) every 4–5 days for approximately 1 month (Fig. 1A). Cells were cryopreserved at various time points during the differentiation process. Cell viability upon thaw was generally >75%. Immunocytochemistry (ICC) was used to confirm the differentiation status of hESC-derived astrocytes and gene expression was analyzed using protocols described previously [23]; more detail is provided in the supplemental online data. All hESC-differentiated astrocytes used for experiments were from days 35–45 (Fig. 1A).


High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress
Differentiation of pluripotent stem cells to mature astrocytes and maintenance in 96-, 384-, and 1,536-well plates. (A): A differentiation timeline along with staining for nestin (an NSC marker), CD44 (marker of astrocyte precursor cells), and GFAP (a mature astrocyte marker) at critical time points in the differentiation protocol. The cells are proliferative, thus requiring passaging, and large stocks of cells can be cryopreserved after day 17. Later, when cells are brought up for use, differentiation into mature astrocytes is continued. (B): Human embryonic stem cell (hESC)-differentiated astrocytes adhered to, and were successfully maintained for >24-hours on PureCoat amine-coated microtiter plates. Astrocytes were healthy, stained positive for GFAP, and exhibited typical astrocyte morphology. Images from microtiter plates were acquired on an IN Cell Analyzer 2000. Magnification, ×40. Abbreviations: GFAP, glial fibrillary acidic protein; hNSC, human neural stem cell.
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Related In: Results  -  Collection

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Figure 1: Differentiation of pluripotent stem cells to mature astrocytes and maintenance in 96-, 384-, and 1,536-well plates. (A): A differentiation timeline along with staining for nestin (an NSC marker), CD44 (marker of astrocyte precursor cells), and GFAP (a mature astrocyte marker) at critical time points in the differentiation protocol. The cells are proliferative, thus requiring passaging, and large stocks of cells can be cryopreserved after day 17. Later, when cells are brought up for use, differentiation into mature astrocytes is continued. (B): Human embryonic stem cell (hESC)-differentiated astrocytes adhered to, and were successfully maintained for >24-hours on PureCoat amine-coated microtiter plates. Astrocytes were healthy, stained positive for GFAP, and exhibited typical astrocyte morphology. Images from microtiter plates were acquired on an IN Cell Analyzer 2000. Magnification, ×40. Abbreviations: GFAP, glial fibrillary acidic protein; hNSC, human neural stem cell.
Mentions: Human neural stem cells (hNSCs) derived from H9 (WA09) human embryonic stem cells (N7800-100; Thermo Fisher Scientific, Carlsbad, CA, https://www.thermofisher.com) were cultured on Geltrex substrate in StemPro NSC serum-free medium (SFM) (A10509-01; Thermo Fisher Scientific) consisting of KnockOut Dulbecco’s modified Eagle’s medium (DMEM)/F-12, StemPro Neural Supplement, GlutaMAX-I, basic fibroblast growth factor (20 ng/ml; PHG0021; Thermo Fisher Scientific), and epidermal growth factor (20 ng/ml; PHG0315; Thermo Fisher Scientific). The hNSCs were expanded for 1 week, and passaged every 2–3 days. After 1 week, the NSCs were differentiated into astrocytes as described recently [22, 23]. Briefly, the medium was changed to astrocyte differentiation medium consisting of StemPro hESC SFM (A1000701 [Thermo Fisher Scientific]; DMEM/F-12, GlutaMAX-I supplement, bovine serum albumin 25%, and StemPro hESC supplement), in addition to 1% fetal bovine serum, FGF2 (8 ng/ml; PHG0264; Thermo Fisher Scientific), activin A (10 ng/ml; 120-14; PeproTech, Quebec, Canada, https://www.peprotech.com), heregulin 1β (10 ng/ml; 100-03; PeproTech), and insulin-like growth factor-1 analog (200 ng/ml; 100-11; PeproTech) to promote astrocyte differentiation. The culture was designated as “Astro P0” at this stage and expanded by passaging (1:6 split) every 4–5 days for approximately 1 month (Fig. 1A). Cells were cryopreserved at various time points during the differentiation process. Cell viability upon thaw was generally >75%. Immunocytochemistry (ICC) was used to confirm the differentiation status of hESC-derived astrocytes and gene expression was analyzed using protocols described previously [23]; more detail is provided in the supplemental online data. All hESC-differentiated astrocytes used for experiments were from days 35–45 (Fig. 1A).

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