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Molecular beacon–enabled purification of living cells by targeting cell type–specific mRNAs

Nature Protocols volume 9, pages 2411–2424 (2014)Cite this article

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Abstract

Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics, the MB-based method enables the isolation of a wide variety of cells. For example, the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design, validation and nucleofection into cells, we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol, we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ∼97% purity, as confirmed by electrophysiology and immunocytochemistry. After designing MBs, their ordering and validation requires 2 weeks, and the isolation process requires 3 h.

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Figure 1: MB structure and control molecular beacons.
Figure 2: Major elements of the PROCEDURE.
Figure 3: Example of control experiments required to demonstrate beacon specificity.
Figure 4: Anticipated results of MB validation, optimization and target cell type characterization.

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Acknowledgements

This work was supported in part by the National Heart, Lung, and Blood Institute of the US National Institutes of Health (NIH) as a Program of Excellence in Nanotechnology (HHSN268201000043C), by the NIH grant DP3DK094346, the National Science Foundation Science and Technology Centers (NTC) grant CBET-0939511 and the Atlanta Clinical and Translational Science Institute (ACTSI) pilot grant (Public Health Summit grant UL1 RR025008 from the Clinical and Translational Science Award program, the NIH, and the National Center for Research Resources). K.B. is a recipient of an American Heart Association postdoctoral fellowship. We thank G. Daley (Harvard University) for BJ1 cells.

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Author notes

  1. Brian M Wile and Kiwon Ban: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA

    Brian M Wile & Gang Bao

  2. Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA

    Kiwon Ban & Young-Sup Yoon

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  1. Brian M Wile
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Contributions

All authors contributed equally to this work. B.M.W. designed and validated the MBs, and K.B. differentiated stem cells into cardiac cells and carried out their subsequent characterization. All authors discussed the results and implications and commented on the manuscript at all stages.

Corresponding authors

Correspondence to Young-Sup Yoon or Gang Bao.

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The authors declare no competing financial interests.

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Wile, B., Ban, K., Yoon, YS. et al. Molecular beacon–enabled purification of living cells by targeting cell type–specific mRNAs. Nat Protoc 9, 2411–2424 (2014). https://doi.org/10.1038/nprot.2014.154

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