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Identification and propagation of haploid human pluripotent stem cells

Nature Protocols volume 11, pages 2274–2286 (2016)Cite this article

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Abstract

Haploid human pluripotent stem cells (PSCs) integrate haploidy and pluripotency, providing a novel system for functional genomics and developmental research in humans. We have recently derived haploid human embryonic stem cells (ESCs) by parthenogenesis and demonstrated their wide differentiation potential and applicability for genetic screening. Because haploid cells can spontaneously become diploid, their enrichment at an early passage is key for successful derivation. In this protocol, we describe two methodologies, namely metaphase spread analysis and cell sorting, for the identification of haploid human cells within parthenogenetic ESC lines. The cell sorting approach also enables the isolation of haploid cells at low percentages, as well as the maintenance of highly enriched haploid ESC lines throughout passaging. The isolation of essentially pure populations of haploid human ESCs by this protocol requires basic PSC culture expertise and can be achieved within 4–6 weeks.

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Figure 1: Use of DNA content to distinguish between haploid and diploid human ESCs.
Figure 2: Schematic overview of the protocol.
Figure 3: Determination of the frequency of FACS-based enrichment rounds for maintaining a haploid human ESC line.
Figure 4: Gating of haploid cells in DNA-based FACS with Hoechst 33342 staining.

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Acknowledgements

We thank T. Golan-Lev and O. Yanuka for providing technical details for the protocol, and M. Peretz, A. Yilmaz and S. Bar for critical reading of the manuscript. I.S. is supported by the Adams Fellowships Program of the Israel Academy of Sciences and Humanities, D.E. is a NYSCF – Robertson Investigator, and N.B. is the Herbert Cohn Chair in Cancer Research. This work was partially supported by The Azrieli Foundation (N.B.), by the Russell Berrie Foundation Program in Cellular Therapies of Diabetes and by the New York Stem Cell Foundation (D.E.).

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Authors and Affiliations

  1. Department of Genetics, The Azrieli Center for Stem Cells and Genetic Research, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel

    Ido Sagi & Nissim Benvenisty

  2. Department of Pediatrics, Columbia University, New York, New York, USA

    Dieter Egli

  3. The New York Stem Cell Foundation Research Institute, New York, New York, USA

    Dieter Egli

Authors
  1. Ido Sagi
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  2. Dieter Egli
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I.S., D.E. and N.B. developed the techniques and wrote the manuscript.

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Correspondence to Dieter Egli or Nissim Benvenisty.

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The authors declare no competing financial interests.

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Sagi, I., Egli, D. & Benvenisty, N. Identification and propagation of haploid human pluripotent stem cells. Nat Protoc 11, 2274–2286 (2016). https://doi.org/10.1038/nprot.2016.145

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