Grantee Research Project Results
Final Report: Developmental Neurotox Assay using Scalable Neurons and Astrocytes in High Content Imaging
EPA Contract Number: EPD15002Title: Developmental Neurotox Assay using Scalable Neurons and Astrocytes in High Content Imaging
Investigators: Majumder, Anirban , Swetenburg, Raymond
Small Business: ArunA Biomedical, Inc.
EPA Contact: Richards, April
Phase: II
Project Period: November 1, 2014 through October 31, 2016 (Extended to October 31, 2017)
Project Amount: $300,000
RFA: Small Business Innovation Research (SBIR) - Phase II (2014) Recipients Lists
Research Category: SBIR - Innovation in Manufacturing , Small Business Innovation Research (SBIR)
Description:
Our objective was to establish a commercially viable and scalable method of differentiating human neural progenitor stem cells (hNP1TM) to astrocytes (hAstroProTM). In turn, the hAstroProTM were to be incorporated to the existing high content imaging (HCI) neurite outgrowth assay platform for neurotoxicology screening. Astrocytes have a significant role in maintaining the health and function of the central nervous system. During Phase I we successfully achieved our goals, demonstrating it is feasible to differentiate human neural progenitor cells to enriched human astrocytes cultures using a scalable process and established a co-culture system by combining these astrocytes with human neurons (hN2TM). We demonstrated specific detection and quantification of neurite outgrowth and neuronal viability in these co-cultures using conventional immunocytochemical techniques and a commercially available automated high throughput high content imaging (HCI) system. Further, we demonstrated quantification of endpoint specific effects of two known neurotoxins using the co-culture system and compared the results to a previously established neuron only assay to show astrocyte induced modulation of neurotoxicity, emphasizing the need replace or augment current neuronal assays with complex multi-cellular assays for more accurate prediction of toxicity outcomes in human neural tissue.
During Phase II we proposed to develop a multi-cellular HCI based assay for neurite outgrowth in a user friendly commercial kit format, to be followed by assays for other endpoints to meet the needs of a broader research community. Our key technical objectives in this phase were to:
- Develop a standard operating procedure for large scale production of astrocytes, and produce a single lot of cells to fulfill all cell requirements for assay development and testing at ArunA as well as at all beta test facilities.
- Develop a neurite outgrowth assay kit based on phase 1 results and test the assay using a training set of compounds recommended by the US-EPA for developing neurotoxin screening assays.
- Test ability to detect at least one functional endpoint other than neurite outgrowth using our co-culture system, to demonstrate versatility of the system.
Intuitively, ArunA's human astrocyte and neuron co-culture neurotoxicity kit would provide more accurate and informative endpoints for human chemical and drug toxicity and drug discovery assays than rodent cultures or human neurons alone.
After attempting to establishing standard operating protocols for industrial scale derivation of astrocytes, a neuron:astrocyte co-culture system recapitulating critical components of central nervous system is to be developed and validated with a suite of ToxCast compounds. Beyond production and validation of a novel neurotoxicology screening platform, successful completion of this project includes a demonstration scientific advantages and versatility of this platform.
Summary/Accomplishments (Outputs/Outcomes):
Building upon our experience bringing previous human embryonic stem cell derived cellular products into the scientific marketplace; we systematically optimized the derivation of astrocytes from our hNP1TM cells. Scale production batches of astrocytes were experientially evaluated to increase yield, homogeneity, and sterility of the product, all while minimizing reagent costs. Beyond production of the hAstroProTM cells, a series of experiments established a reproducible and sensitive co-culture system for hAstroProTM and human neurons (hN2TM) cells. Control compounds and ToxCast chemicals were evaluated in the co-culture system with the application of the Cellomics ArrayScan platform system. Additional efforts to identify alternative application for hAstroProTM cells in the context of electrophysiology and virology were undertaken as well.
An efficient and robust protocol for deriving, cryopreserving, and culturing hAstroProTM cells was developed. Some batches of hAstroProTM successfully meet our strict criteria of homogeneity as determined by protein marker expression, sterility, and viability after cryopreservation. Confident of our ability to provide a robust population of astrocytes, a neuron:astrocyte co-culture HCI assay platform was developed for neurotoxicant screening and a commercial assay kit generated. Our assay kit and original methodology successfully evaluated a series of eight ToxCast compounds and demonstrated the ability to highlight the metabolic effects of astrocytes. Lastly, efforts to broaden the commercial applications of our hAstroProTM cells were focused on developing a microelectrode array (MEA) assay system and evaluating our cells susceptibility to Zika virus infection. Despite encountering difficulties observing electrical activity on the MEA system, a series of experimental assays using hAstroProTM and hNP1TM cells provided the foundation of an in vitro model system of congenital Zika infection.
Conclusions:
We established more standardized methodologies for generating functional human astrocytes and developed a novel co-culture system that can be adopted for diverse use to address a range of commercial markets. Importantly, by providing a neuron and glia co-culture system, we provided a more faithful representation of human neural tissue. Potential applications/products include high content imaging (HCI) assays such as neurite outgrowth (as we demonstrated), and synaptogenesis assays, drug toxicity and drug development-related HCI, and neuro-developmental research. More generally, our co-culture system will provide a valuable and unique in vitro model to examine interactions between astrocytes and neurons. These assays should be commercially useful in the future when small sets of compounds of interest to drug and pesticide companies need to be tested in a relevant human cell based assay.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
| Other project views: | All 4 publications | 4 publications in selected types | All 4 journal articles |
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Wu X, Majumder A, Webb R, Stice SL. High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure. BMC Pharmacology and Toxicology 2016;17(1):62. |
EPD15002 (Final) R835551 (2016) R835551 (Final) |
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Goodfellow FT, Tesla B, Simchick G, Zhao Q, Hodge T, Brindley MA, Stice SL. Zika virus induced mortality and microcephaly in chicken embryos. Stem Cells and Development 2016;25(22):1691-1697. |
EPD15002 (Final) R835551 (2016) R835551 (Final) |
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Yang X, Wu X, Brown KA, Le T, Stice SL, Bartlett MG. Determination of chlorpyrifos and its metabolites in cells and culture media by liquid chromatography-electrospray ionization tandem mass spectrometry. Journal of Chromatography B 2017;1063:112-117. |
EPD15002 (Final) R835551 (Final) |
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Wu X, Yang X, Majumder A, Swetenburg R, Goodfellow F, Bartlett MG, Stice SL.Astrocytes are protective against chlorpyrifos developmental neurotoxicity in human pluripotent stem cell derived astrocyte-neuron co-cultures. Toxicol Sciences 2017;157(2):410-420. |
EPD15002 (Final) |
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Supplemental Keywords:
reagents, drug discovery assays, pluripotent stem cells assaySBIR Phase I:
Developmental Neurotox Assay Using Scalable Neurons and Astrocytes in High-Content Imaging | Final ReportThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.