High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons
Human induced pluripotent stem cell (iPSC)-derived neurons are being increasingly used for high content imaging and screening. However, iPSC-derived neuronal differentiation and maturation is time-intensive, often requiring >8 weeks. Unfortunately, the differentiating and maturing iPSC-derived ne...
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| Format: | Article |
| Language: | English |
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Elsevier
2023-09-01
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| Series: | SLAS Discovery |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2472555222137032 |
| _version_ | 1797682832004349952 |
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| author | Elizabeth R. Sharlow Danielle C. Llaneza William E. Grever Garnett A. Mingledorff Anna J. Mendelson George S. Bloom John S. Lazo |
| author_facet | Elizabeth R. Sharlow Danielle C. Llaneza William E. Grever Garnett A. Mingledorff Anna J. Mendelson George S. Bloom John S. Lazo |
| author_sort | Elizabeth R. Sharlow |
| collection | DOAJ |
| description | Human induced pluripotent stem cell (iPSC)-derived neurons are being increasingly used for high content imaging and screening. However, iPSC-derived neuronal differentiation and maturation is time-intensive, often requiring >8 weeks. Unfortunately, the differentiating and maturing iPSC-derived neuronal cultures also tend to migrate and coalesce into ganglion-like clusters making single-cell analysis challenging, especially in miniaturized formats. Using our defined extracellular matrix and low oxygen culturing conditions for the differentiation and maturation of human cortical neurons, we further modified neuronal progenitor cell seeding densities and feeder layer-free culturing conditions in miniaturized formats (i.e., 96 well) to decrease neuronal clustering, enhance single-cell identification and reduce edge effects usually observed after extended neuronal cell culture. Subsequent algorithm development refined capabilities to distinguish and identify single mature neurons, as identified by NeuN expression, from large cellular aggregates, which were excluded from image analysis. Incorporation of astrocyte conditioned medium during differentiation and maturation periods significantly increased the percentage (i.e., ∼10% to ∼30%) of mature neurons (i.e., NeuN+) detected at 4-weeks post-differentiation. Pilot, proof of concept studies using this optimized assay system yielded negligible edge effects and robust Z-factors in population-based as well as image-based neurotoxicity assay formats. Moreover, moxidectin, an FDA-approved drug with documented neurotoxic adverse effects, was identified as a hit using both screening formats. This miniaturized, feeder layer-free format and image analysis algorithm provides a foundational imaging and screening platform, which enables quantitative single-cell analysis of differentiated human neurons. |
| first_indexed | 2024-03-12T00:05:35Z |
| format | Article |
| id | doaj.art-d14cfbcac61844679a8e3bf159917758 |
| institution | Directory Open Access Journal |
| issn | 2472-5552 |
| language | English |
| last_indexed | 2024-03-12T00:05:35Z |
| publishDate | 2023-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | SLAS Discovery |
| spelling | doaj.art-d14cfbcac61844679a8e3bf1599177582023-09-17T04:57:14ZengElsevierSLAS Discovery2472-55522023-09-01286275283High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neuronsElizabeth R. Sharlow0Danielle C. Llaneza1William E. Grever2Garnett A. Mingledorff3Anna J. Mendelson4George S. Bloom5John S. Lazo6Department of Pharmacology, University of Virginia, 340 Jefferson Park Avenue, Pinn Hall, 5th Floor, P.O. Box 800735, Charlottesville, VA 22908-0735, USA; Corresponding author.Department of Pharmacology, University of Virginia, 340 Jefferson Park Avenue, Pinn Hall, 5th Floor, P.O. Box 800735, Charlottesville, VA 22908-0735, USAPerkin Elmer, 940 Winter St, Waltham, MA 02451, USADepartment of Pharmacology, University of Virginia, 340 Jefferson Park Avenue, Pinn Hall, 5th Floor, P.O. Box 800735, Charlottesville, VA 22908-0735, USADepartment of Pharmacology, University of Virginia, 340 Jefferson Park Avenue, Pinn Hall, 5th Floor, P.O. Box 800735, Charlottesville, VA 22908-0735, USADepartment of Biology, University of Virginia, 420 Gilmer Hall, 485 McCormick Road, P.O. Box 400328, Charlottesville VA 22904, USA; Department of Cell Biology, University of Virginia, 420 Gilmer Hall, 485 McCormick Road, P.O. Box 400328, Charlottesville VA 22904, USA; Department of Neuroscience, University of Virginia, 420 Gilmer Hall, 485 McCormick Road, P.O. Box 400328, Charlottesville VA 22904, USADepartment of Pharmacology, University of Virginia, 340 Jefferson Park Avenue, Pinn Hall, 5th Floor, P.O. Box 800735, Charlottesville, VA 22908-0735, USAHuman induced pluripotent stem cell (iPSC)-derived neurons are being increasingly used for high content imaging and screening. However, iPSC-derived neuronal differentiation and maturation is time-intensive, often requiring >8 weeks. Unfortunately, the differentiating and maturing iPSC-derived neuronal cultures also tend to migrate and coalesce into ganglion-like clusters making single-cell analysis challenging, especially in miniaturized formats. Using our defined extracellular matrix and low oxygen culturing conditions for the differentiation and maturation of human cortical neurons, we further modified neuronal progenitor cell seeding densities and feeder layer-free culturing conditions in miniaturized formats (i.e., 96 well) to decrease neuronal clustering, enhance single-cell identification and reduce edge effects usually observed after extended neuronal cell culture. Subsequent algorithm development refined capabilities to distinguish and identify single mature neurons, as identified by NeuN expression, from large cellular aggregates, which were excluded from image analysis. Incorporation of astrocyte conditioned medium during differentiation and maturation periods significantly increased the percentage (i.e., ∼10% to ∼30%) of mature neurons (i.e., NeuN+) detected at 4-weeks post-differentiation. Pilot, proof of concept studies using this optimized assay system yielded negligible edge effects and robust Z-factors in population-based as well as image-based neurotoxicity assay formats. Moreover, moxidectin, an FDA-approved drug with documented neurotoxic adverse effects, was identified as a hit using both screening formats. This miniaturized, feeder layer-free format and image analysis algorithm provides a foundational imaging and screening platform, which enables quantitative single-cell analysis of differentiated human neurons.http://www.sciencedirect.com/science/article/pii/S2472555222137032NeuronFeeder layer-freeAlgorithmNeuNEdge effectsMiniaturization |
| spellingShingle | Elizabeth R. Sharlow Danielle C. Llaneza William E. Grever Garnett A. Mingledorff Anna J. Mendelson George S. Bloom John S. Lazo High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons SLAS Discovery Neuron Feeder layer-free Algorithm NeuN Edge effects Miniaturization |
| title | High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons |
| title_full | High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons |
| title_fullStr | High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons |
| title_full_unstemmed | High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons |
| title_short | High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons |
| title_sort | high content screening miniaturization and single cell imaging of mature human feeder layer free ipsc derived neurons |
| topic | Neuron Feeder layer-free Algorithm NeuN Edge effects Miniaturization |
| url | http://www.sciencedirect.com/science/article/pii/S2472555222137032 |
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