Abstract
High-content screening (HCS) permits simultaneous observation and analysis of multiple cellular variables including cell morphology, survival, and differentiation in live cells at the single-cell level, at the level of the culture well, and across the entire culture. By combining high-throughput technologies such as robotics, chemical libraries, and automated high-resolution microscopy, scientists are able to evaluate a much broader array of experimental conditions than can be studied using conventional cell biological techniques that study fewer parameters at any one time. Thus, HCS assays provide a means to vastly improve our basic understanding of stem cell biology. We have developed a HCS assay that allows the study of the effects of hundreds of small molecules in parallel. The protocol described in this chapter was developed to assess the effects of small molecules on the survival, proliferation, and expression of pluripotent markers following single-cell dissociation of human embryonic stem cells, but can be applied to the study of other types of stem cells including induced pluripotent stem cells. A detailed protocol for the setup of HCS assays and the parameters used to identify chemical modifiers of survival in human pluripotent stem cells, as well as secondary assays used to validate the small-molecule “hits” obtained during the high-content screen, are described.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Damoiseaux R, Sherman SP, Alva JA, Peterson C, Pyle AD (2009). Integrated chemical genomics reveals modifiers of survival in human embryonic stem cells. Stem Cells 27, 533–542.
Malo N, Hanley JA, Cerquozzi S, Pelletier J, Nadon R (2006). Statistical practice in high-throughput screening data analysis. Nat Biotechnol 24, 167–175.
Zhang JH, Chung TD, Oldenburg KR (1999). A high-throughput homogeneous assay for reverse transcriptase using generic reagents and time-resolved fluorescence detection. J Biomol Screen 4, 67.
Hohenstein KA, Pyle AD, Chern JY, Lock LF, Donovan PJ (2008). Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells. Stem Cells 26: 1436–1443.
Acknowledgments
S.P.S is supported by a CIRM training grant TG2-01169, A.D.P. is supported by an NIH R21 (1R21NS064855-01) from the Roadmap Initiative, CRCC, and JCCC Seed Grant. J.A. is supported by a UC Regent’s President’s Postdoctoral Fellowship. We would also like to acknowledge the UCLA MSSR, in particular Dr. Robert Damoiseaux for assistance with the HCS assay development. Thanks are due to Dr. Carrie Miceli and Dr. Stan Nelson and labs for use of the MetaDrug/GeneGo software. We would also like to thank Dr. Donald Kohn and Dr. Amander Clark for consultation on establishment of efficient lentiviral protocols for hESCs in our lab.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Sherman, S.P., Alva, J.A., Thakore-Shah, K., Pyle, A.D. (2011). Human Pluripotent Stem Cells: The Development of High-Content Screening Strategies. In: Schwartz, P., Wesselschmidt, R. (eds) Human Pluripotent Stem Cells. Methods in Molecular Biology, vol 767. Humana Press. https://doi.org/10.1007/978-1-61779-201-4_21
Download citation
DOI: https://doi.org/10.1007/978-1-61779-201-4_21
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-200-7
Online ISBN: 978-1-61779-201-4
eBook Packages: Springer Protocols