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Adapting human pluripotent stem cells to high-throughput and high-content screening

Nature Protocols volume 8, pages 111–130 (2013)Cite this article

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Abstract

The increasing use of human pluripotent stem cells (hPSCs) as a source of cells for drug discovery, cytotoxicity assessment and disease modeling requires their adaptation to large-scale culture conditions and screening formats. Here, we describe a simple and robust protocol for the adaptation of human embryonic stem cells (hESCs) to high-throughput screening (HTS). This protocol can also be adapted to human induced pluripotent stem cells (hiPSCs) and high-content screening (HCS). We also describe a 7-d assay to identify compounds with an effect on hESC self-renewal and differentiation. This assay can be adapted to a variety of applications. The procedure involves the culture expansion of hESCs, their adaptation to 384-well plates, the addition of small molecules or other factors, and finally data acquisition and processing. In this protocol, the optimal number of hESCs plated in 384-well plates has been adapted to HTS/HCS assays of 7 d.

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Figure 1: Overall screening procedure.
Figure 2: hESCs at different stages of the assay.
Figure 3: Cell loss after suboptimal staining procedure.
Figure 4: Expected phenotypes for small-molecule regulators of hESC self-renewal, differentiation or death.

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Acknowledgements

We thank H. Djaballah, D. Placantonakis and A. Ciro for their technical help during the development of the assay. This project was supported by an R-21 grant (1R21NS053655) from the National Institute of Neurological Disorders and Stroke(NINDS)/NIH and by a grant from the Starr Foundation.

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Authors and Affiliations

  1. Center for Stem Cell Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Sabrina C Desbordes & Lorenz Studer

  2. Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Sabrina C Desbordes & Lorenz Studer

  3. Stem Cells in Neural Development and Disease Group, Institute of Developmental Genetics, HelmholtzZentrum München, German Research Center for Environmental Health, Munich-Neuherberg, Germany

    Sabrina C Desbordes

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  1. Sabrina C Desbordes
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Contributions

S.C.D. and L.S. conceived and designed the project. S.C.D. performed all the experiments and wrote the protocol. L.S. gave his comments on the writing.

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Correspondence to Sabrina C Desbordes.

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Desbordes, S., Studer, L. Adapting human pluripotent stem cells to high-throughput and high-content screening. Nat Protoc 8, 111–130 (2013). https://doi.org/10.1038/nprot.2012.139

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